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Sample records for fluorophore dual-view imaging

  1. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging.

    Science.gov (United States)

    Kumar, Abhishek; Wu, Yicong; Christensen, Ryan; Chandris, Panagiotis; Gandler, William; McCreedy, Evan; Bokinsky, Alexandra; Colón-Ramos, Daniel A; Bao, Zhirong; McAuliffe, Matthew; Rondeau, Gary; Shroff, Hari

    2014-11-01

    We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.

  2. Toxicity of Organic Fluorophores Used in Molecular Imaging: Literature Review

    Directory of Open Access Journals (Sweden)

    Raphael Alford

    2009-11-01

    Full Text Available Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of 19 widely used fluorophores was conducted by searching 26 comprehensive biomedical and chemical literature databases and analyzing the retrieved material. These fluorophores included Alexa Fluor 488 and 514, BODIPY FL, BODIPY R6G, Cy 5.5, Cy 7, cypate, fluorescein, indocyanine green, Oregon green, 8-phenyl BODIPY, rhodamine 110, rhodamine 6G, rhodamine X, rhodol, TAMRA, Texas red, and Tokyo green. Information regarding cytotoxicity, tissue toxicity, in vivo toxicity, and mutagenicity was included. Considerable toxicity-related information was available for the Food and Drug Administration (FDA-approved compounds indocyanine green and fluorescein, but published information on many of the non-FDA-approved fluorophores was limited. The information located was encouraging because the amounts of fluorophore used in molecular imaging probes are typically much lower than the toxic doses described in the literature. Ultimately, the most effective and appropriate probes for use in patients will be determined by their fluorescent characteristics and the safety of the conjugates.

  3. Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality

    Science.gov (United States)

    Bittel, Amy M.; Nickerson, Andrew; Saldivar, Isaac S.; Dolman, Nick J.; Nan, Xiaolin; Gibbs, Summer L.

    2016-07-01

    Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

  4. Toxicity of Organic Fluorophores Used in Molecular Imaging: Literature Review

    National Research Council Canada - National Science Library

    Alford, Raphael; Simpson, Haley M; Duberman, Josh; Hill, G. Craig; Ogawa, Mikako; Regino, Celeste; Kobayashi, Hisataka; Choyke, Peter L

    2009-01-01

    .... These fluorophores included Alexa Fluor 488 and 514, BODIPY FL, BODIPY R6G, Cy 5.5, Cy 7, cypate, fluorescein, indocyanine green, Oregon green, 8-phenyl BODIPY, rhodamine 110, rhodamine 6G, rhodamine X, rhodol, TAMRA, Texas red, and Tokyo green...

  5. Pancreas-targeted NIR fluorophores for dual-channel image-guided abdominal surgery.

    Science.gov (United States)

    Wada, Hideyuki; Hyun, Hoon; Vargas, Christina; Gravier, Julien; Park, GwangLi; Gioux, Sylvain; Frangioni, John V; Henary, Maged; Choi, Hak Soo

    2015-01-01

    Pancreas-related complications are some of the most serious ones in abdominal surgery. The goal of this study was to develop and validate novel near-infrared (NIR) fluorophores that would enable real-time pancreas imaging to avoid the intraoperative pancreatic injury. After initial screening of a large NIR fluorophore library, the performance of 3 selected pancreas-targeted 700 nm NIR fluorophores, T700-H, T700-F, and MB, were quantified in mice, rats, and pigs. Dose ranging using 25 and 100 nmol, and 2.5 µmol of T700-F, and its imaging kinetics over a 4 h period were tested in each species. Three different 800 nm NIR fluorophores were employed for dual-channel FLARE™ imaging in pigs: 2 μmol of ZW800-1 for vessels and kidney, 1 μmol of ZW800-3C for lymph nodes, and 2 μmol of ESNF31 for adrenal glands. T700-F demonstrated the highest signal to background ratio (SBR), with peak SBR at 4 h postinjection in mice. In pigs, T700-F produced an SBR≥2 against muscle, spleen, and lymph nodes for up to 8 h after a single intravenous injection. The combination of T700-F with each 800 nm NIR fluorophore provided simultaneous dual-channel intraoperative imaging of pancreas with surrounding organs in real time. Pancreas-targeted NIR fluorophores combined with the FLARE dual-channel imaging system enable the real-time intraoperative pancreas imaging which helps surgeons perform safer and more curative abdominal surgeries.

  6. Next generation NIR fluorophores for tumor imaging and fluorescence-guided surgery: A review.

    Science.gov (United States)

    Haque, Ashanul; Faizi, Md Serajul Haque; Rather, Jahangir Ahmad; Khan, Muhammad S

    2017-04-01

    Cancer is a group of diseases responsible for the major causes of mortality and morbidity among people of all ages. Even though medical sciences have made enormous growth, complete treatment of this deadly disease is still a challenging task. Last few decades witnessed an impressive growth in the design and development of near infrared (NIR) fluorophores with and without recognition moieties for molecular recognitions, imaging and image guided surgeries. The present article reviews recently reported NIR emitting organic/inorganic fluorophores that targets and accumulates in organelle/organs specifically for molecular imaging of cancerous cells. Near infrared (NIR probe) with or without a tumor-targeting warhead have been considered and discussed for their applications in the field of cancer imaging. In addition, challenges persist in this area are also delineated in this review.

  7. Photoacoustic pump-probe tomography of fluorophores in vivo using interleaved image acquisition for motion suppression

    Science.gov (United States)

    Märk, Julia; Wagener, Asja; Zhang, Edward; Laufer, Jan

    2017-01-01

    In fluorophores, the excited state lifetime can be modulated using pump-probe excitation. By generating photoacoustic (PA) signals using simultaneous and time-delayed pump and probe excitation pulses at fluences below the maximum permissible exposure, a modulation of the signal amplitude is observed in fluorophores but not in endogenous chromophores. This provides a highly specific contrast mechanism that can be used to recover the location of the fluorophore using difference imaging. The practical challenges in applying this method to in vivo PA tomography include the typically low concentrations of fluorescent contrast agents, and tissue motion. The former results in smaller PA signal amplitudes compared to those measured in blood, while the latter gives rise to difference image artefacts that compromise the unambiguous and potentially noise-limited detection of fluorescent contrast agents. To address this limitation, a method based on interleaved pump-probe image acquisition was developed. It relies on fast switching between simultaneous and time-delayed pump-probe excitation to acquire PA difference signals in quick succession, and to minimise the effects of tissue motion. The feasibility of this method is demonstrated in tissue phantoms and in initial experiments in vivo. PMID:28091571

  8. Photoacoustic pump-probe tomography of fluorophores in vivo using interleaved image acquisition for motion suppression

    Science.gov (United States)

    Märk, Julia; Wagener, Asja; Zhang, Edward; Laufer, Jan

    2017-01-01

    In fluorophores, the excited state lifetime can be modulated using pump-probe excitation. By generating photoacoustic (PA) signals using simultaneous and time-delayed pump and probe excitation pulses at fluences below the maximum permissible exposure, a modulation of the signal amplitude is observed in fluorophores but not in endogenous chromophores. This provides a highly specific contrast mechanism that can be used to recover the location of the fluorophore using difference imaging. The practical challenges in applying this method to in vivo PA tomography include the typically low concentrations of fluorescent contrast agents, and tissue motion. The former results in smaller PA signal amplitudes compared to those measured in blood, while the latter gives rise to difference image artefacts that compromise the unambiguous and potentially noise-limited detection of fluorescent contrast agents. To address this limitation, a method based on interleaved pump-probe image acquisition was developed. It relies on fast switching between simultaneous and time-delayed pump-probe excitation to acquire PA difference signals in quick succession, and to minimise the effects of tissue motion. The feasibility of this method is demonstrated in tissue phantoms and in initial experiments in vivo.

  9. Differentiation of ocular fundus fluorophores by fluorescence lifetime imaging using multiple excitation and emission wavelengths

    Science.gov (United States)

    Hammer, M.; Schweitzer, D.; Schenke, S.; Becker, W.; Bergmann, A.

    2006-10-01

    Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH II provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

  10. Preventing Radiobleaching of Cyanine Fluorophores Enhances Stability of Nuclear/NIRF Multimodality Imaging Agents

    Science.gov (United States)

    Hernandez, Reinier; Heskamp, Sandra; Rijpkema, Mark; Bos, Desirée L.; Goldenberg, David M.; McBride, William J.; Morgenstern, Alfred; Bruchertseifer, Frank; Cai, Weibo; Boerman, Otto C.

    2017-01-01

    Despite the large interest in nuclear/optical multimodality imaging, the effect of radiation on the fluorescence of fluorophores remains largely unexplored. Herein, we report on the radiobleaching of cyanine fluorophores and describe conditions to provide robust radioprotection under practical (pre)clinical settings. We determined the radiosensitivity of several cyanine fluorescent compounds, including IRDye 800CW (800CW) and a dual modality imaging tetrapeptide containing DOTA as chelator and Dylight 800 as fluorophore, exposed to increasing activities of 111In, 68Ga, or 213Bi (γ, EC/β, and α emitter, respectively). An activity and type of radiation-dependent radiation-induced loss of fluorescence, radiobleaching, of 800CW was observed upon incubation with escalating activities of 111In, 68Ga, or 213Bi. 68Ga showed the largest radiolytic effect, followed by 111In and 213Bi. The addition of oxygen radical scavengers including ethanol, gentisic acid, and ascorbic acid (AA), provided a concentration dependent radioprotective effect. These results supported the hypothesis of a free radical-mediated radiobleaching mechanism. AA provided the most robust radioprotection over a wide range of concentrations and preserved fluorescence at much higher radioactivity levels. Overall, both near-infrared fluorescent compounds displayed similar sensitivity, except for 213Bi-irradiated solutions, where the dual modality construct exhibited enhanced radiolysis, presumably due to direct radiation damage from α particles. Concurrently, AA was not able to preserve fluorescence of the dual-modality molecule labeled with 213Bi. Our findings have important consequences for several research areas including ROS sensing, radiation-mediated drug release (uncaging), fluorescent dosimetry, and in the preparation of dual-modality radiopharmaceuticals. PMID:28042311

  11. Alkynyl-naphthalimide Fluorophores: Gold Coordination Chemistry and Cellular Imaging Applications.

    Science.gov (United States)

    Langdon-Jones, Emily E; Lloyd, David; Hayes, Anthony J; Wainwright, Shane D; Mottram, Huw J; Coles, Simon J; Horton, Peter N; Pope, Simon J A

    2015-07-01

    A range of fluorescent alkynyl-naphthalimide fluorophores has been synthesized and their photophysical properties examined. The fluorescent ligands are based upon a 4-substituted 1,8-naphthalimide core and incorporate structural variations (at the 4-position) to tune the amphiphilic character: chloro (L1), 4-[2-(2-aminoethoxy)ethanol] (L2), 4-[2-(2-methoxyethoxy)ethylamino] (L3), piperidine (L4), morpholine (L5), 4-methylpiperidine (L6), and 4-piperidone ethylene ketal (L7) variants. The amino-substituted species (L2-L7) are fluorescent in the visible region at around 517-535 nm through a naphthalimide-localized intramolecular charge transfer (ICT), with appreciable Stokes' shifts of ca. 6500 cm(-1) and lifetimes up to 10.4 ns. Corresponding two-coordinate Au(I) complexes [Au(L)(PPh3)] were isolated, with X-ray structural studies revealing the expected coordination mode via the alkyne donor. The Au(I) complexes retain the visible fluorescence associated with the coordinated alkynyl-naphthalimide ligand. The ligands and complexes were investigated for their cytotoxicity across a range of cell lines (LOVO, MCF-7, A549, PC3, HEK) and their potential as cell imaging agents for HEK (human embryonic kidney) cells and Spironucleus vortens using confocal fluorescence microscopy. The images reveal that these fluorophores are highly compatible with fluorescence microscopy and show some clear intracellular localization patterns that are dependent upon the specific nature of the naphthalimide substituent.

  12. Heteroaromatic donors in donor-acceptor-donor based fluorophores facilitate zinc ion sensing and cell imaging.

    Science.gov (United States)

    Sreejith, Sivaramapanicker; Divya, Kizhumuri P; Jayamurthy, Purushothaman; Mathew, Jomon; Anupama, V N; Philips, Divya Susan; Anees, Palappuravan; Ajayaghosh, Ayyappanpillai

    2012-11-01

    The excited state intra molecular charge transfer (ICT) property of fluorophores has been extensively used for the design of fluorescent chemosensors. Herein, we report the synthesis and properties of three donor–π-acceptor–π-donor (D–π-A–π-D) based molecular probes BP, BT and BA. Two heteroaromatic rings, pyrrole (BP), and thiophene (BT) and a non-heteroaromatic ring N-alkoxy aniline (BA) were selected as donor moieties which were linked to a bipyridine binding site through a vinylic linkage. The heteroaromatic systems BP and BT perform selective and ratiometric emission signalling for zinc ions whereas the non-heteroaromatic probe BA does not. The advantages of the D–π-A–π-D design strategy in the design of ICT based probes for the selective fluorescent ratiometric signalling of zinc ions in biological media is discussed. Further, the use of BP, BT and BA for imaging Zn(2+) ions from MCF-7 cell lines is demonstrated.

  13. A laser scanner for imaging fluorophore labeled molecules in electrophoretic gels

    Energy Technology Data Exchange (ETDEWEB)

    Fisk, D.J.; Sutherland, J.C. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.

    1995-08-01

    A laser scanner for imaging electrophoretic gels was constructed and tested. The scanner incorporates a green helium-neon (HeNe) laser (543.5nm wavelength) and can achieve a spatial resolution of 19{micro}m. The instrument can function in two modes : snap-shot and finish-line. In snapshot mode, all samples are electrophoresed for the same time and the gel is scanned after completion of electrophoresis, while in finish-line mode, fluorophore labeled samples are electrophoresed for a constant distance and the image is formed as the samples pass under the detector. The resolving power of the finish-line mode of imaging is found to be greater than that of the snapshot mode of imaging. This laser scanner is also compared with a Charge Coupled Device (CCD) camera and in terms of resolving power is found to be superior. Sensitivity of the instrument is presented in terms of the minimum amount of DNA that can be detected verses its molecular length.

  14. Multimodal fluorescence molecular imaging for in vivo characterization of skin cancer using endogenous and exogenous fluorophores

    Science.gov (United States)

    Miller, Jessica P.; Habimana-Griffin, LeMoyne; Edwards, Tracy S.; Achilefu, Samuel

    2017-06-01

    Similarity of skin cancer with many benign skin pathologies requires reliable methods to detect and differentiate the different types of these lesions. Previous studies have explored the use of disparate optical techniques to identify and estimate the invasive nature of melanoma and basal cell carcinoma with varying outcomes. Here, we used a concerted approach that provides complementary information for rapid screening and characterization of tumors, focusing on squamous cell carcinoma (SCC) of the skin. Assessment of in vivo autofluorescence lifetime (FLT) imaging of endogenous fluorophores that are excitable at longer wavelengths (480 nm) than conventional NADH and FAD revealed a decrease in the short FLT component for SCC compared to normal skin, with mean values of 0.57±0.026 ns and 0.61±0.021 ns, respectively (p=0.004). Subsequent systemic administration of a near-infrared fluorescent molecular probe in SCC bearing mice, followed by the implementation of image processing methods on data acquired from two-dimensional and three-dimensional fluorescence molecular imaging, allowed us to estimate the tumor volume and depth, as well as quantify the fluorescent probe in the tumor. The result suggests the involvement of lipofuscin-like lipopigments and riboflavin in SCC metabolism and serves as a model for staging SCC.

  15. Silicon-on-glass pore network micromodels with oxygen-sensing fluorophore films for chemical imaging and defined spatial structure

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Kelly, Ryan T.; Suter, Jonathan D.; Anheier, Norman C.

    2012-11-21

    Pore network microfluidic models were fabricated by a silicon-on-glass technique that provides the precision advantage of dry etched silicon while creating a structure that is transparent across all microfluidic channels and pores, and can be imaged from either side. A silicon layer is bonded to an underlying borosilicate glass substrate and thinned to the desired height of the microfluidic channels and pores. The silicon is then patterned and through-etched by deep reactive ion etching (DRIE), with the underlying glass serving as an etch stop. After bonding on a transparent glass cover plate, one obtains a micromodel in oxygen impermeable materials with water wet surfaces where the microfluidic channels are transparent and structural elements such as the pillars creating the pore network are opaque. The micromodel can be imaged from either side. The advantageous features of this approach in a chemical imaging application are demonstrated by incorporating a Pt porphyrin fluorophore in a PDMS film serving as the oxygen sensing layer and a bonding surface, or in a polystyrene film coated with a PDMS layer for bonding. The sensing of a dissolved oxygen gradient was demonstrated using fluorescence lifetime imaging, and it is shown that different matrix polymers lead to optimal use in different ranges dissolved oxygen concentration. Imaging with the opaque pillars in between the observation direction and the continuous fluorophore film yields images that retain spatial information in the sensor image.

  16. Steady state anisotropy two-photon microscopy resolves multiple, spectrally similar fluorophores, enabling in vivo multilabel imaging.

    Science.gov (United States)

    Dubach, J Matthew; Vinegoni, Claudio; Weissleder, Ralph

    2014-08-01

    The use of spectrally distinguishable fluorescent dyes enables imaging of multiple targets. However, in two-photon microscopy, the number of fluorescent labels with distinct emission spectra that can be effectively excited and resolved is constrained by the confined tuning range of the excitation laser and the broad and overlapping nature of fluorophore two-photon absorption spectra. This limitation effectively reduces the number of available imaging channels. Here, we demonstrate that two-photon steady state anisotropy imaging (2PSSA) offers the capability to resolve otherwise unresolvable fluorescent tracers both in live cells and in mouse tumor models. This approach expands the number of biological targets that can be imaged simultaneously, increasing the total amount of information that can be obtained through imaging.

  17. Fluorophore-tagged pharmacophores for antitumor cytotoxicity: Modified chiral lipidic dialkynylcarbinols for cell imaging.

    Science.gov (United States)

    Listunov, Dymytrii; Mazères, Serge; Volovenko, Yulian; Joly, Etienne; Génisson, Yves; Maraval, Valérie; Chauvin, Remi

    2015-10-15

    Chiral lipidic dialkynylcarbinols (DACs), recently highlighted as antitumoral pharmacophores, have been conjugated to difluoroboron-dipyrromethene (bodipy), 7-hydroxy-coumarine, and 7-nitro-benzoxadiazole (NBD) fluorophore motifs through triazole clips. The labeled lipids preserve cytotoxic activity against HCT116 cells, and fluorescence microscopy of the stained cells showed clear signals in the intra-cellular membrane system. While the bodipy conjugate also labels lipid droplets very brightly, as expected, the coumarine and NBD probes appear as promising specific tools for the identification of the intra-cellular targets of DACs' cytotoxicity.

  18. Epicocconone-Hemicyanine Hybrids: Near Infrared Fluorophores for Protein Staining and Cell Imaging.

    Science.gov (United States)

    Karuso, Peter; Loa Kum Cheung, Wendy; Peixoto, Philippe A; Boulangé, Agathe; Franck, Xavier

    2017-02-03

    The development of new near infrared (NIR) dyes is crucial for diverse applications and especially bioimaging, as they absorb and emit light in the "therapeutic window" (650-950 nm). We report here a new family of NIR fluorophores that has been obtained by hybridising hemicyanines with epicocconone. Emission wavelengths of these hybrid dyes is in the range 715-795 nm and is combined with large Stokes' shifts (75-95 nm). The absorption and emission wavelength can be modulated according to the hemicyanine moiety and adding sulfonic acid moieties enhances water solubility. We demonstrate their application in the sensitive detection of proteins in gel electrophoresis and the staining of specific cellular organelles in confocal microscopy. These results are particularly encouraging and bring forward a new fluorescent skeleton for chemical biology.

  19. Ultra-Fast Fluorescence Imaging in Vivo with Conjugated Polymer Fluorophores in the Second Near-Infrared Window

    CERN Document Server

    Hong, Guosong; Antaris, Alexander L; Diao, Shuo; Wu, Di; Cheng, Kai; Zhang, Xiaodong; Chen, Changxin; Liu, Bo; He, Yuehui; Wu, Justin Z; Yuan, Jun; Zhang, Bo; Tao, Zhimin; Fukunaga, Chihiro; Dai, Hongjie

    2014-01-01

    In vivo fluorescence imaging in the second near-infrared window (1.0-1.7 microns) can afford deep tissue penetration and high spatial resolution, owing to the reduced scattering of long-wavelength photons. Here, we synthesize a series of low-bandgap donor/acceptor copolymers with tunable emission wavelengths of 1050-1350 nm in this window. Non-covalent functionalization with phospholipid-polyethylene glycol results in water-soluble and biocompatible polymeric nanoparticles, allowing for live cell molecular imaging at > 1000 nm with polymer fluorophores for the first time. Importantly, the high quantum yield of the polymer allows for in vivo, deep-tissue and ultrafast imaging of mouse arterial blood flow with an unprecedented frame rate of > 25 frames per second. The high time resolution results in spatially and time resolved imaging of the blood flow pattern in cardiogram waveform over a single cardiac cycle (~ 200 ms) of a mouse, which has not been observed with fluorescence imaging in this window before.

  20. A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid-based imaging technology.

    Science.gov (United States)

    Kimoto, Michiko; Mitsui, Tsuneo; Yamashige, Rie; Sato, Akira; Yokoyama, Shigeyuki; Hirao, Ichiro

    2010-11-03

    In the development of orthogonal extra base pairs for expanding the genetic alphabet, we created novel, unnatural base pairs between fluorophore and quencher nucleobase analogues. We found that the nucleobase analogue, 2-nitropyrrole (denoted by Pn), and its 4-substitutions, such as 2-nitro-4-propynylpyrrole (Px) and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (NH(2)-hx-Px), act as fluorescence quenchers. The Pn and Px bases specifically pair with their pairing partner, 7-(2,2'-bithien-5-yl)imidazo[4,5-b]pyridine (Dss), which is strongly fluorescent. Thus, these unnatural Dss-Pn and Dss-Px base pairs function as reporter-quencher base pairs, and are complementarily incorporated into DNA by polymerase reactions as a third base pair in combination with the natural A-T and G-C pairs. Due to the static contact quenching, the Pn and Px quencher bases significantly decreased the fluorescence intensity of Dss by the unnatural base pairings in DNA duplexes. In addition, the Dss-Px pair exhibited high efficiency and selectivity in PCR amplification. Thus, this new unnatural base pair system would be suitable for detection methods of target nucleic acid sequences, and here we demonstrated the applications of the Dss-Pn and Dss-Px pairs as molecular beacons and in real-time PCR. The genetic alphabet expansion system with the replicable, unnatural fluorophore-quencher base pair will be a useful tool for sensing and diagnostic applications, as well as an imaging tool for basic research.

  1. A Three-Photon Active Organic Fluorophore for Deep Tissue Ratiometric Imaging of Intracellular Divalent Zinc.

    Science.gov (United States)

    Philips, Divya Susan; Sreejith, Sivaramapanicker; He, Tingchao; Menon, Nishanth Venugopal; Anees, Palapuravan; Mathew, Jomon; Sajikumar, Sreedharan; Kang, Yuejun; Stuparu, Mihaiela Corina; Sun, Handong; Zhao, Yanli; Ajayaghosh, Ayyappanpillai

    2016-05-20

    Deep tissue bioimaging with three-photon (3P) excitation using near-infrared (NIR) light in the second IR window (1.0-1.4 μm) could provide high resolution images with an improved signal-to-noise ratio. Herein, we report a photostable and nontoxic 3P excitable donor-π-acceptor system (GMP) having 3P cross-section (σ3 ) of 1.78×10(-80)  cm(6)  s(2)  photon(-2) and action cross-section (σ3 η3 ) of 2.31×10(-81)  cm(6)  s(2)  photon(-2) , which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn(2+) binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ3 of 1.85×10(-80)  cm(6)  s(2)  photon(-2) and σ3 η3 of 3.33×10(-81)  cm(6)  s(2)  photon(-2) . The application of this probe is demonstrated for ratiometric 3P imaging of Zn(2+) in vitro using HuH-7 cell lines. Furthermore, the Zn(2+) concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn(2+) ion imaging.

  2. Methods for effective fluorophore injection and imaging of lymphatics in small animals

    Science.gov (United States)

    DSouza, Alisha V.; Marra, Kayla A.; Gunn, Jason R.; Samkoe, Kimberley S.; Tichauer, Kenneth M.; Pogue, Brian W.

    2016-03-01

    Morbidity and complexity involved in lymph node staging via surgical resection and biopsy calls for staging techniques that are less invasive. While visible blue dyes are commonly used in locating sentinel lymph nodes, since they follow tumor-draining lymphatic vessels, they do not provide a metric to evaluate presence of cancer. An area of active research is to use fluorescent dyes to assess tumor burden of sentinel and secondary lymph nodes. The goal of this work was to successfully perform fluorescence imaging of IRDye®680RD in the lymphatics, in a repeatable manner.

  3. Self-Assembled Polyelectrolyte Nanoparticles as Fluorophore-Free Contrast Agents for Multicolor Optical Imaging

    Directory of Open Access Journals (Sweden)

    Da Hye Shin

    2015-03-01

    Full Text Available In this work, we describe the fabrication of self-assembled polyelectrolyte nanoparticles that provide a multicolor optical imaging modality. Poly(γ-glutamic acid(γ-PGA formed self-assembled nanoparticles through electrostatic interactions with two different cationic polymers: poly(L-lysine(PLL and chitosan. The self-assembled γ-PGA/PLL and γ-PGA/chitosan nanoparticles were crosslinked by glutaraldehyde. Crosslinking of the ionic self-assembled nanoparticles with glutaraldehyde not only stabilized the nanoparticles but also generated a strong autofluorescence signal. Fluorescent Schiff base bonds (C=N and double bonds (C=C were generated simultaneously by crosslinking of the amine moiety of the cationic polyelectrolytes with monomeric glutaraldehyde or with polymeric glutaraldehyde. The unique optical properties of the nanoparticles that resulted from the crosslinking by glutaraldehyde were analyzed using UV/Vis and fluorescence spectroscopy. We observed that the fluorescence intensity of the nanoparticles could be regulated by adjusting the crosslinker concentration and the reaction time. The nanoparticles also exhibited high performance in the labeling and monitoring of therapeutic immune cells (macrophages and dendritic cells. These self-assembled nanoparticles are expected to be a promising multicolor optical imaging contrast agent for the labeling, detection, and monitoring of cells.

  4. In vivo and in vitro investigations of retinal fluorophores in age-related macular degeneration by fluorescence lifetime imaging

    Science.gov (United States)

    Hammer, M.; Quick, S.; Klemm, M.; Schenke, S.; Mata, N.; Eitner, A.; Schweitzer, D.

    2009-02-01

    Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently for the observation of the age pigment lipofuscin, a precursor of age-related macular degeneration (AMD). However, a deeper understanding of the generation of single compounds contributing to the lipofuscin as well as of the role of other fluorophores such as FAD, glycated proteins, and collagen needs their discrimination by fluorescence lifetime imaging (FLIM). FLIM at the ocular fundus is performed using a scanning laser ophthalmoscope equipped with a picosecond laser source (448nm or 468nm respectively, 100ps, 80 MHz repetition rate) and dual wavelength (490-560nm and 560-7600nm) time-correlated single photon counting. A three-exponential fit of the fluorescence decay revealed associations of decay times to anatomical structures. Disease-related features are identified from alterations in decay times and-amplitudes. The in-vivo investigations in patients were paralleled by experiments in an organ culture of the porcine ocular fundus. Photo-oxidative stress was induced by exposure to blue light (467nm, 0.41 mW/mm2). Subsequent analysis (fluorescence microscopy, HPLC, LC-MS) indicated the accumulation of the pyridinium bis-retinoid A2E and its oxidation products as well as oxidized phospholipids. These compounds contribute to the tissue auto-fluorescence and may play a key role in the pathogenesis of AMD. Thus, FLIM observation at the ocular fundus in vivo enhances our knowledge on the etiology of AMD and may become a diagnostic tool.

  5. The Exploration of Stereoscopic Imaging Technique in Dual View 60 Co Container Inspection System%双视角60Co集装箱检测系统中立体成像技术

    Institute of Scientific and Technical Information of China (English)

    薛岳; 苗积臣; 吴志芳; 邢桂来

    2014-01-01

    为了克服传统集装箱检测系统难以辨别前后重叠在一起的物体的缺点,双视角60 Co集装箱检测系统将立体成像技术应用在传统的检测系统中。双视角立体成像与分析是系统的核心技术之一,通过研究与比对不同的立体成像方式,针对系统的不同终端,分别应用色分法和时差分像法将系统从不同视角获得的图像分别传送至检测人员的左右眼,使其在大脑中融合成一幅立体图像。论文设计的双视角60 Co集装箱检测系统实现了对集装箱等大型客体的立体成像检测,不仅可以对前后重叠的物体进行有效区分,而且可以生成客体的三维影像,提高了系统的检测效果。%In traditional container inspection system, projection of different objects will overlap, making it diffi-cult to distinguish between them.In order to overcome this shortcoming, stereoscopic imaging is applied in the Binocular 60 Co container inspection system.The binocular stereoscopic imaging is one of the core technologies in the system.By studying and comparing different ways of stereoscopic imaging, according to different termi-nals of the system, anaglyph method and time-sequential method are respectively applied to transmit the image gained from different perspective to the corresponding eye of the inspector, generating stereoscopic image in the brain.The Binocular 60 Co container inspection system designed in this paper implements the stereoscopic image inspection for the containers and other large objects.This system is able to distinguish overlapping objects effec-tively, and detection effectiveness is improved through the stereoscopic image of the objects.

  6. Dual view x-ray inspection system for foreign objects detection in canned food

    Science.gov (United States)

    Lu, Zhiwen; Peng, Ningsong

    2013-04-01

    X-ray inspection technique for foreign objects in food products can determine and mark the presence of contaminants within the product by using image processing and pattern recognition technique on the X-ray transmission images. This paper presents the dual view X-ray inspection technique for foreign objects in food jar via analyzing the weak points of the traditional single view X-ray inspection technique. In addition, a prototype with the new technique is developed in accordance with glass splinters detection within the food jar (glass jar especially) which is a typical tickler. Some algorithms such as: adaptive image segmentation based on contour tracking, nonlinear arctan function transform and etc., are applied to improve image quality and achieve effective inspection results. The false recognition rate is effectively reduced and the detection sensitivity is highly enhanced. Finally the actual test results of this prototype are given.

  7. A coupled radiative transfer and diffusion approximation model for the solution of the forward problem and the a-priori fluorophore distribution estimation in fluorescence imaging

    Science.gov (United States)

    Gorpas, D.; Yova, D.; Politopoulos, K.

    2009-02-01

    Although fluorescence imaging has been applied in tumour diagnosis from the early 90s, just the last few years it has met an increasing scientific interest due to the advances in the biophotonics field and the combined technological progress of the acquisition and computational systems. In addition there are expectations that fluorescence imaging will be further developed and applied in deep tumour diagnosis in the years to come. However, this evolving field of imaging sciences has still to encounter important challenges. Among them is the expression of an accurate forward model for the solution of the reconstruction problem. The scope of this work is to introduce a three dimensional coupled radiative transfer and diffusion approximation model, applicable on the fluorescence imaging. Furthermore, the solver incorporates the super-ellipsoid models and sophisticated image processing algorithms to additionally provide a-priori estimation about the fluorophores distribution, information that is very important for the solution of the inverse problem. Simulation experiments have proven that the proposed methodology preserves the accuracy levels of the radiative transfer equation and the time efficacy of the diffusion approximation, while in the same time shows extended success on the registration between acquired and simulated images.

  8. In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore

    Directory of Open Access Journals (Sweden)

    Haibiao Gong

    2010-02-01

    Full Text Available Overexpression of epidermal growth factor receptor (EGFR is associated with many types of cancers. It is of great interest to noninvasively image the EGFR expression in vivo. In this study, we labeled an EGFR-specific Affibody molecule (Eaff with a near-infrared (NIR dye IRDye800CW maleimide and tested the binding of this labeled molecule (Eaff800 in cell culture and xenograft mouse tumor models. Unlike EGF, Eaff did not activate the EGFR signaling pathway. Results showed that Eaff800 was bound and taken up specifically by EGFR-overexpressing A431 cells. When Eaff800 was intravenously injected into nude mice bearing A431 xenograft tumors, the tumor could be identified 1 hour after injection and it became most prominent after 1 day. Images of dissected tissue sections demonstrated that the accumulation of Eaff800 was highest in the liver, followed by the tumor and kidney. Moreover, in combination with a human EGFR type 2 (HER2-specific probe Haff682, Eaff800 could be used to distinguish between EGFR- and HER2-overexpressing tumors. Interestingly, the organ distribution pattern and the clearance rate of Eaff800 were different from those of Haff682. In conclusion, Eaff molecule labeled with a NIR fluorophore is a promising molecular imaging agent for EGFR-overexpressing tumors.

  9. Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

    Science.gov (United States)

    Podgorski, Kaspar; Terpetschnig, Ewald; Klochko, Oleksii P; Obukhova, Olena M; Haas, Kurt

    2012-01-01

    Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM) at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647) by imaging dye-filled neurons in vivo over 5 days, and utility for sensitive subcellular imaging by synthesizing a specific peptide-conjugate label for the synaptic protein PSD-95.

  10. Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

    Directory of Open Access Journals (Sweden)

    Kaspar Podgorski

    Full Text Available Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647 by imaging dye-filled neurons in vivo over 5 days, and utility for sensitive subcellular imaging by synthesizing a specific peptide-conjugate label for the synaptic protein PSD-95.

  11. Evaluation of a Triple-Helical Peptide with Quenched Fluorophores for Optical Imaging of MMP-2 and MMP-9 Proteolytic Activity

    Directory of Open Access Journals (Sweden)

    Xuan Zhang

    2014-06-01

    Full Text Available Matrix metalloproteinases (MMP 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP, incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M−1 s−1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.

  12. Mechanism of Intramolecular Photostabilization in Self-Healing Cyanine Fluorophores

    NARCIS (Netherlands)

    van der Velde, Jasper H. M.; Ploetz, Evelyn; Hiermaier, Matthias; Oelerich, Jens; de Vries, Jan; Roelfes, Johannes; Cordes, Thorben

    2013-01-01

    Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark-states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However,

  13. Super-resolution fluorescence imaging of nanoimprinted polymer patterns by selective fluorophore adsorption combined with redox switching

    KAUST Repository

    Yabiku, Y.

    2013-10-22

    We applied a super-resolution fluorescence imaging based on selective adsorption and redox switching of the fluorescent dye molecules for studying polymer nanostructures. We demonstrate that nano-scale structures of polymer thin films can be visualized with the image resolution better than 80 nm. The method was applied to image 100 nm-wide polymer nanopatterns fabricated by thermal nanoimprinting. The results point to the applicability of the method for evaluating residual polymer thin films and dewetting defect of the polymer resist patterns which are important for the quality control of the fine nanoimprinted patterns. 2013 Author(s).

  14. Fluorescence-quenching of a liposomal-encapsulated near-infrared fluorophore as a tool for in vivo optical imaging.

    Science.gov (United States)

    Tansi, Felista L; Rüger, Ronny; Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Hilger, Ingrid

    2015-01-05

    Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

  15. Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

    Science.gov (United States)

    Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Hilger, Ingrid

    2015-01-01

    Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials. PMID:25591069

  16. Deep-Tissue Anatomical Imaging of Mice Using Carbon Nanotube Fluorophores in the Second Near Infrared Window

    CERN Document Server

    Welsher, Kevin; Dai, Hongjie

    2011-01-01

    Fluorescent imaging in the second near infrared window (NIR II, 1-1.4 {\\mu}m) holds much promise due to minimal autofluorescence and tissue scattering. Here, using well functionalized biocompatible single-walled carbon nanotubes (SWNTs) as NIR II fluorescent imaging agents, we performed high frame rate video imaging of mice during intravenous injection of SWNTs and investigated the path of SWNTs through the mouse anatomy. We observed in real-time SWNT circulation through the lungs and kidneys several seconds post-injection, and spleen and liver at slightly later time points. Dynamic contrast enhanced imaging through principal component analysis (PCA) was performed and found to greatly increase the anatomical resolution of organs as a function of time post-injection. Importantly, PCA was able to discriminate organs such as the pancreas which could not be resolved from real-time raw images. Tissue phantom studies were performed to compare imaging in the NIR II region to the traditional NIR I biological transpar...

  17. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations.

    Science.gov (United States)

    Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M; Pereira, Pedro Matos; Henriques, Ricardo

    2016-08-12

    Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours.

  18. A rapid SNAP-tag fluorogenic probe based on an environment-sensitive fluorophore for no-wash live cell imaging.

    Science.gov (United States)

    Liu, Tao-Kai; Hsieh, Pei-Ying; Zhuang, Yu-De; Hsia, Chi-Yang; Huang, Chi-Ling; Lai, Hsiu-Ping; Lin, Hung-Sheung; Chen, I-Chia; Hsu, Hsin-Yun; Tan, Kui-Thong

    2014-10-17

    One major limitation of labeling proteins with synthetic fluorophores is the high fluorescence background, which necessitates extensive washing steps to remove unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on an environment-sensitive fluorophore for labeling with SNAP-tag proteins. The probe exhibits dramatic fluorescence turn-on of 280-fold upon being labeled to SNAP-tag. The major advantages of our fluorogenic probe are the dramatic fluorescence turn-on, ease of synthesis, high selectivity, and rapid labeling with SNAP-tag. No-wash labeling of both intracellular and cell surface proteins was successfully achieved in living cells, and the localization of these proteins was specifically visualized.

  19. Mechanism of intramolecular photostabilization in self-healing cyanine fluorophores.

    Science.gov (United States)

    van der Velde, Jasper H M; Ploetz, Evelyn; Hiermaier, Matthias; Oelerich, Jens; de Vries, Jan Willem; Roelfes, Gerard; Cordes, Thorben

    2013-12-16

    Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark-states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood. In this contribution, we investigate the mechanism of intramolecular photostabilization in self-healing cyanines, in which photodamage is automatically repaired. Experimental evidence is provided to demonstrate that a single photostabilizer, that is, the vitamin E derivative Trolox, efficiently heals the cyanine fluorophore Cy5 in the absence of any photostabilizers in solution. A plausible mechanism is that Trolox interacts with the fluorophore through intramolecular quenching of triplet-related dark-states, which is a mechanism that appears to be common for both triplet-state quenchers (cyclooctatetraene) and redox-active compounds (Trolox, ascorbic acid, methylviologen). Additionally, the influence of solution-additives, such as cysteamine and procatechuic acid, on the self-healing process are studied. The results suggest the potential applicability of self-healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super-resolution. The presented data contributes to an improved understanding of the mechanism involved in intramolecular photostabilization and has high relevance for the future development of self-healing fluorophores, including their applications in various research fields.

  20. Excitation-wavelength dependence of multiphoton excitation of fluorophores of human skin in vivo

    Science.gov (United States)

    Breunig, Hans Georg; Studier, Hauke; König, Karsten

    2010-02-01

    We present in vivo measurements of the excitation-wavelength dependence of the autofluorescence of major endogenous fluorophores of human skin with a multiphoton tomograph. For the investigation high-resolution multiphoton images at different depths inside the skin were recorded and the main fluorophores identified. In particular, for the autofluorescence of the fluorophores keratin, NAD(P)H, elastin and for the second-harmonic-generation light induced by collagen fibers clear trends are shown.

  1. Determination of land surface reflectance using the AATSR dual-view capability

    OpenAIRE

    Sogacheva, L.; Kolmonen, P; T. H. Virtanen; Rodriguez, E.; A.-M. Sundström; G. de Leeuw

    2014-01-01

    In this study, a method is presented to retrieve the surface reflectance using reflectance measured at the top of the atmosphere for the two views provided by the Along-Track Scanning Radiometer (AATSR). In the first step, the aerosol optical depth (AOD) is obtained using the AATSR dual view algorithm (ADV) by eliminating the effect of the surface on the measured radiances. Hence the AOD is independent of surface properties and can thus be used in the second step to p...

  2. Determination of land surface reflectance using the AATSR dual-view capability

    OpenAIRE

    Sogacheva, L.; Kolmonen, P; T. H. Virtanen; Rodriguez, E.; Sundström, A.-M.; G. de Leeuw

    2015-01-01

    In this study, a method is presented to retrieve the surface reflectance using the radiances measured at the top of the atmosphere for the two views provided by the Advanced Along-Track Scanning Radiometer (AATSR). In the first step, the aerosol optical depth (AOD) is obtained using the AATSR dual-view algorithm (ADV) by eliminating the effect of the surface on the measured radiances. Hence the AOD is independent of surface properties and can thus be used in the second step ...

  3. Determination of land surface reflectance using the AATSR dual-view capability

    Science.gov (United States)

    Sogacheva, L.; Kolmonen, P.; Virtanen, T. H.; Rodriguez, E.; Sundström, A.-M.; de Leeuw, G.

    2015-02-01

    In this study, a method is presented to retrieve the surface reflectance using the radiances measured at the top of the atmosphere for the two views provided by the Advanced Along-Track Scanning Radiometer (AATSR). In the first step, the aerosol optical depth (AOD) is obtained using the AATSR dual-view algorithm (ADV) by eliminating the effect of the surface on the measured radiances. Hence the AOD is independent of surface properties and can thus be used in the second step to provide the aerosol part of the atmospheric correction which is needed for the surface reflectance retrieval. The method is applied to provide monthly maps of both AOD and surface reflectance at two wavelengths (555 and 659 nm) for the whole year of 2007. The results are validated versus surface reflectance provided by the AERONET-based Surface Reflectance Validation Network (ASRVN). Correlation coefficients are 0.8 and 0.9 for 555 and 659 nm, respectively. The standard deviation is 0.001 for both wavelengths and the absolute error is less than 0.02. Pixel-by-pixel comparison with MODIS (Moderate Resolution Imaging Spectrometer) monthly averaged surface reflectances show a good correlation (0.91 and 0.89 for 555 and 659 nm, respectively) with somewhat higher values (up to 0.05) obtained by ADV over bright surfaces. The difference between the ADV- and MODIS-retrieved surface reflectances is smaller than ±0.025 for 68.3% of the collocated pixels at 555 nm and 79.9% of the collocated pixels at 659 nm. An application of the results over Australia illustrates the variation in the surface reflectances for different land cover types. The validation and comparison results suggest that the algorithm can be successfully used for both the AATSR and ATSR-2 (which has characteristics similar to AATSR) missions, which together cover a 17-year period of measurements (1995-2012), as well as a prototype for the Sea and Land Surface Temperature Radiometer (SLSTR) planned to be launched in the fall of 2015

  4. Determination of land surface reflectance using the AATSR dual-view capability

    Directory of Open Access Journals (Sweden)

    L. Sogacheva

    2014-07-01

    Full Text Available In this study, a method is presented to retrieve the surface reflectance using reflectance measured at the top of the atmosphere for the two views provided by the Along-Track Scanning Radiometer (AATSR. In the first step, the aerosol optical depth (AOD is obtained using the AATSR dual view algorithm (ADV by eliminating the effect of the surface on the measured radiances. Hence the AOD is independent of surface properties and can thus be used in the second step to provide the aerosol part of the atmospheric correction which is needed for the surface reflectance retrieval. The method is applied to provide monthly maps of both AOD and surface reflectance at two wavelengths (555 and 659 nm for the whole year of 2007. The results are validated vs. surface reflectance provided by the AERONET-based Surface Reflectance Validation Network (ASRVN. Correlation coefficients are 0.8 and 0.9 for 555 and 659 nm, respectively. The standard deviation is 0.001 for both wavelengths and the absolute error is less than 0.02. Pixel-by-pixel comparison with MODIS (MODerate resolution Imaging Spectrometer monthly averaged surface reflectances show a good correlation (0.91 and 0.89 for 555 and 659 nm, respectively with some (up to 0.05 overestimation by ADV over bright surfaces. The difference between the ADV and MODIS retrieved surface reflectance is smaller than ±0.025 for 68.3% of the collocated pixels at 555 nm and 79.9% of the collocated pixels at 659 nm. An application of the results over Australia illustrates the variation of the surface reflectances for different land cover types. The validation and comparison results suggest that the algorithm can be successfully used for the both AATSR and ATSR-2 (which has characteristics similar to AATSR missions, which together cover 17 years period of measurements (1995–2012, as well as a prototype for The Sea and Land Surface Temperature Radiometer (SLSTR to be launched in 2015 onboard the Sentinel-3 satellite.

  5. Electrophile-integrating Smiles rearrangement provides previously inaccessible C4'-O-alkyl heptamethine cyanine fluorophores.

    Science.gov (United States)

    Nani, Roger R; Shaum, James B; Gorka, Alexander P; Schnermann, Martin J

    2015-01-16

    New synthetic methods to rapidly access useful fluorophores are needed to advance modern molecular imaging techniques. A new variant of the classical Smiles rearrangement is reported that enables the efficient synthesis of previously inaccessible C4'-O-alkyl heptamethine cyanines. The key reaction involves N- to O-transposition with selective electrophile incorporation on nitrogen. A representative fluorophore exhibits excellent resistance to thiol nucleophiles, undergoes productive bioconjugation, and can be used in near-IR fluorescence imaging applications.

  6. The 'Visualized' macrocycles: Chemistry and application of fluorophore tagged cyclodextrins.

    Science.gov (United States)

    Benkovics, Gábor; Malanga, Milo; Fenyvesi, Éva

    2017-10-15

    Cyclodextrins are macrocyclic molecules able to form host-guest complexes due to their hydrophobic cavity. Because of their carbohydrate nature they do not absorb light in the UV-vis region (200-800nm), but they can be converted into spectroscopically active compounds via modification with a chromophore unit. Among the chromophores, the group of fluorophores can provide high sensitivity in analytical applications (chemosensing) and low detection limit in optical imaging methods (fluorescent microscopy). Fluorophore-tagged cyclodextrins therefore combine interesting spectroscopic properties with promising supramolecular features which make these conjugates widely applicable in various pharmaceutical fields. The aim of this work is to review the various types of fluorophores which have been used for cyclodextrin tagging, to discuss the synthetic strategies used for the conjugation and to summarize the pharmaceutical applications of these 'visualized' macrocycles including their use in photodynamic therapy. The recent achievements in studying how the fluorophore-appended cyclodextrin derivatives cross biological barriers are also reviewed. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. In Vivo Optical Imaging of Acute Myeloid Leukemia by Green Fluorescent Protein: Time-Domain Autofluorescence Decoupling, Fluorophore Quantification, and Localization

    Directory of Open Access Journals (Sweden)

    Emmet McCormack

    2007-05-01

    Full Text Available Human xenografts of acute myeloid leukemia (AML in nonobese diabetic/severe combined immunodeficient (NOD/SCID mice result in disease states of diffuse, nonpalpable tissue infiltrates exhibiting a variable disease course, with some animals not developing a disease phenotype. Thus, disease staging and, more critically, quantification of preclinical therapeutic effect in these models are particularly difficult. In this study, we present the generation of a green fluorescent protein (GFP-labeled human leukemic cell line, NB4, and validate the potential of a time-domain imager fitted with a 470 nm picosecond pulsed laser diode to decouple GFP fluorescence from autofluorescence on the basis of fluorescence lifetime and thus determine the depth and relative concentration of GFP inclusions in phantoms of homogeneous and heterogeneous optical properties. Subsequently, we developed an optical imageable human xenograft model of NB4-GFP AML and illustrate early disease detection, depth discrimination of leukemic infiltrates, and longitudinal monitoring of disease course employing time-domain optical imaging. We conclude that early disease detection through use of time-domain imaging in this initially slowly progressing AML xenograft model permits accurate disease staging and should aid in future preclinical development of therapeutics for AML.

  8. Liposomal encapsulation of a near-infrared fluorophore enhances fluorescence quenching and reliable whole body optical imaging upon activation in vivo.

    Science.gov (United States)

    Tansi, Felista L; Rüger, Ronny; Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kaiser, Werner A; Hilger, Ingrid

    2013-11-11

    In the past decade, there has been significant progress in the development of water soluble near-infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650-900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto-fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY-676-COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY-676-COOH reveal strong fluorescence quenching. It is demonstrated that the non-targeted PEGylated fluorescence-activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan-induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY-676-COOH, prolonged stability and retention of liposomal-DY-676-COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY-676-COOH-loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases.

  9. Monoalkoxy BODIPYs—A Fluorophore Class for Bioimaging

    OpenAIRE

    Courtis, Alexandra M.; Santos, Sofia A.; Guan, Yinghua; Hendricks, J. Adam; Ghosh, Balaram; Szantai-Kis, D. Miklos; Reis, Surya A.; Shah, Jagesh V.; Mazitschek, Ralph

    2014-01-01

    Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, ...

  10. Functionalization of a viscosity-sensitive fluorophore for probing of biological systems.

    Science.gov (United States)

    Petric, A; Jacobson, A F; Barrio, J R

    1998-06-16

    Functionalization of a viscosity-sensitive visible wavelength fluorophore 2-(1,1-dicyanopropenyl-2)-6-dimethylaminonaphthalene (DDNP), with the intent to incorporate its favorable optical properties into a probe for structural and functional imaging by fluorescence microscopy, is described. Spiperone, a highly potent ligand for the dopamine D2 receptors, was conjugated via an ethylpiperazine moiety to the fluorophore giving fluorescent probes that can be excited in the UV and Vis range.

  11. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

    Science.gov (United States)

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-11-19

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

  12. Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

    Science.gov (United States)

    Courtis, Alexandra M; Santos, Sofia A; Guan, Yinghua; Hendricks, J Adam; Ghosh, Balaram; Szantai-Kis, D Miklos; Reis, Surya A; Shah, Jagesh V; Mazitschek, Ralph

    2014-06-18

    Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

  13. Electrophile-Integrating Smiles Rearrangement Provides Previously Inaccessible C4′-O-Alkyl Heptamethine Cyanine Fluorophores

    Science.gov (United States)

    2016-01-01

    New synthetic methods to rapidly access useful fluorophores are needed to advance modern molecular imaging techniques. A new variant of the classical Smiles rearrangement is reported that enables the efficient synthesis of previously inaccessible C4′-O-alkyl heptamethine cyanines. The key reaction involves N- to O- transposition with selective electrophile incorporation on nitrogen. A representative fluorophore exhibits excellent resistance to thiol nucleophiles, undergoes productive bioconjugation, and can be used in near-IR fluorescence imaging applications. PMID:25562683

  14. Buffer controlled photoswitching microscopy using standard organic fluorophores

    Science.gov (United States)

    Buschmann, Volker; Fore, Samantha; van de Linde, Sebastian; Sauer, Markus; Wolter, Steve; Heilemann, Mike; Koberling, Felix; Erdmann, Rainer

    2011-03-01

    The interest in super-resolution microscopy techniques has dramatically increased in the last years due to the unprecedented insight into cellular structure which has become possible [1]. In all widefield-based techniques, such as Stochastical Optical Reconstruction Microscopy (STORM) or Photo-activation localization microscopy (PALM), the dye-sensor-molecules are switched between a bright and a dark state. Many organic fluorophores exhibit intrinsic dark states with a lifetime that can be tuned by adjusting the level of oxidants and reductants in the buffer, thereby allowing to reversibly switch individual fluorophores between an on- and off-state [2]. This behavior is used in the dSTORM method. We exploited this redox-level adjusted photoswitching behaviour based on addition of millimolar amounts of reducing thiols for high-resolution imaging on a setup based on an inverse microscope coupled with ultrasensitive CCD camera detection. In order to quickly control the quality of the measurement, we used real-time computation of the subdiffraction-resolution image [3]. This greatly increases the applicability of the method, as image analysis times are greatly reduced.

  15. Multiphoton excitation characteristics of cellular fluorophores of human skin in vivo.

    Science.gov (United States)

    Breunig, Hans Georg; Studier, Hauke; König, Karsten

    2010-04-12

    In vivo multiphoton tomography with a wavelength-tunable femtosecond laser has been performed to investigate the autofluorescence intensity of major endogenous fluorophores of human skin in dependence on the excitation wavelength. In high-resolution multiphoton images of different skin layers, clear trends were found for fluorophores like keratin, NAD(P)H, melanin as well as for the elastin and collagen networks. The analysis of the measurements is supplemented by additional measurements of fluorescence lifetime imaging and signal-decay curves by time-correlated single-photon counting.

  16. Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm

    Science.gov (United States)

    Baird, Fiona J.; Wadsworth, Marilyn P.; Hill, Jane E.

    2015-01-01

    Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores – ranging in emission from 435 to 665 nm – applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent. PMID:22587931

  17. Rational design of fluorophores for in vivo applications.

    Science.gov (United States)

    Ptaszek, Marcin

    2013-01-01

    Several classes of small organic molecules exhibit properties that make them suitable for fluorescence in vivo imaging. The most promising candidates are cyanines, squaraines, boron dipyrromethenes, porphyrin derivatives, hydroporphyrins, and phthalocyanines. The recent designing and synthetic efforts have been dedicated to improving their optical properties (shift the absorption and emission maxima toward longer wavelengths and increase the brightness) as well as increasing their stability and water solubility. The most notable advances include development of encapsulated cyanine dyes with increased stability and water solubility, squaraine rotaxanes with increased stability, long-wavelength-absorbing boron dipyrromethenes, long-wavelength-absorbing porphyrin and hydroporphyrin derivatives, and water-soluble phthalocyanines. Recent advances in luminescence and bioluminescence have made self-illuminating fluorophores available for in vivo applications. Development of new types of hydroporphyrin energy-transfer dyads gives the promise for further advances in in vivo multicolor imaging.

  18. Conventional fluorescence microscopy below the diffraction limit with simultaneous capture of two fluorophores in DNA origami

    Science.gov (United States)

    Glasgow, Ben J.

    2016-02-01

    A conventional fluorescence microscope was previously constructed for simultaneous imaging of two colors to gain sub-diffraction localization. The system is predicated on color separation of overlapping Airy discs, construction of matrices of Cartesian coordinates to determine locations as well as centers of the point spread functions of fluorophores. Quantum dots that are separated by as little as 10 nm were resolved in the x-y coordinates. Inter-fluorophore distances that vary by 10 nm could also be distinguished. Quantum dots are bright point light source emitters that excite with a single laser and can serve as a label for many biomolecules. Here, alterations in the method are described to test the ability to resolve Atto 488 and Atto 647 dyes attached to DNA origami at ~40 nm spacing intervals. Dual laser excitation is used in tandem with multi-wavelength bandpass filters. Notwithstanding challenges from reduced intensity in Atto labeled DNA origami helical bundles compared to quantum dots, preliminary data show a mean inter-fluorophore distance of 56 nm with a range (14-148 nm). The range closely matches published results with DNA origami with other methods of subdiffraction microscopy. Sub-diffraction simultaneous two-color imaging fluorescence microscopy acronymically christened (SSTIFM) is a simple, readily accessible, technique for measurement of inter-fluorophore distances in compartments less than 40 nm. Preliminary results with so called nanorulers are encouraging for use with other biomolecules.

  19. Fluorophore-cored dendrimers for patterns in metalloprotein sensing.

    Science.gov (United States)

    Jiwpanich, Siriporn; Sandanaraj, Britto S; Thayumanavan, S

    2009-02-21

    In fluorophore-cored dendrimers with peripheral binding functionalities, the effect of generation upon protein binding-induced fluorescence quenching can be unpredictable; this is because the increase in fluorophore-binding functionality distance with generation is also accompanied by an increase in the number of binding moieties and the interplay between the two features is utilized to create patterns for metalloprotein sensing.

  20. 78 FR 18999 - Prospective Grant of Start-Up Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates...

    Science.gov (United States)

    2013-03-28

    ...: Photosensitizing Antibody-Fluorophore Conjugates for Photoimmunotherapy AGENCY: National Institutes of Health...-01), and entitled ``Photosensitizing Antibody- Fluorophore Conjugates,'' to Aspyrian Therapeutics.... The field of use may be limited to ``use of photosensitizing antibody-fluorophore conjugate by...

  1. Using two dyes with the same fluorophore to monitor cellular calcium concentration in an extended range.

    Directory of Open Access Journals (Sweden)

    Lourdes Figueroa

    Full Text Available We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca(2+] at equilibrium and an approximate analytic expression that does not require the equilibrium assumption. The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark.

  2. Quantitative detection of multiple fluorophore sites as a tool for diagnosis and monitoring disease progression in salivary glands

    Science.gov (United States)

    Gannot, Israel; Bonner, Robert F.; Gannot, Gallya; Fox, Philip C.; You, Joon S.; Waynant, Ronald W.; Gandjbakhche, Amir H.

    1997-08-01

    A series of fluorescent surface images were obtained from physical models of localized fluorophores embedded at various depths and separations in tissue phantoms. Our random walk theory was applied to create an analytical model of multiple flurophores embedded in tissue-like phantom. Using this model, from acquired set of surface images, the location of the fluorophores was reconstructed and compared it to their known 3-D distributions. A good correlation was found, and the ability to resolve fluorophores as a function of depth and separation was determined. In parallel in in-vitro study, specific coloring of sections of minor salivary glands was also demonstrated. These results demonstrate the possibility of using inverse methods to reconstruct unknown locations and concentrations of optical probes specifically bound to infiltrating lymphocytes in minor salivary glands of patients with Sjogren's syndrome.

  3. Optical tomography of fluorophores in dense scattering media based on ultrasound-enhanced chemiluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Masaki, E-mail: masaki@tohtech.ac.jp; Kikuchi, Naoto; Sato, Akihiro [Department of Electronics and Intelligent Systems, Tohoku Institute of Technology, Sendai 982-8577 (Japan)

    2015-01-12

    This letter proposes and demonstrates ultrasound-combined optical imaging in dense scattering media. A peroxyoxalate chemiluminescence system that includes fluorophores to chemically excite the pigment is stimulated by ultrasound irradiation with power of less than 0.14 W/cm{sup 2}. Using focused ultrasound, the chemiluminescence is selectively spatially enhanced, which leads to imaging of the pigment when embedded in a light-scattering medium via scanning of the focal point. The ultrasonically enhanced intensity of the chemiluminescence depends on the base intensity of the chemiluminescence without the applied ultrasound irradiation, which thereby enables quantitative determination of the fluorophore concentration. The authors demonstrate the potential of this method to resolve chemiluminescent targets in a dense scattering medium that is comparable to biological tissue. An image was acquired of a chemiluminescent target that included indocyanine green as the fluorophore embedded at a depth of 20 mm in an Intralipid-10% 200 ml/l solution scattering medium (the reduced scattering coefficient was estimated to be approximately 1.3 mm{sup −1}), indicating the potential for expansion of this technique for use in biological applications.

  4. Water-soluble pyrrolopyrrole cyanine (PPCy) NIR fluorophores.

    Science.gov (United States)

    Wiktorowski, Simon; Rosazza, Christelle; Winterhalder, Martin J; Daltrozzo, Ewald; Zumbusch, Andreas

    2014-05-11

    Water-soluble derivatives of pyrrolopyrrole cyanines (PPCys) have been synthesized by a post-synthetic modification route. In highly polar media, these dyes are excellent NIR fluorophores. Labeling experiments show how these novel dyes are internalized into mammalian cells.

  5. Photo-physical characterization of fluorophore Ru(bpy32+ for optical biosensing applications

    Directory of Open Access Journals (Sweden)

    E.L. Sciuto

    2015-12-01

    Full Text Available We studied absorption, emission and lifetime of the coordination compound tris(2,2′-bipyridylruthenium(II fluorophore (Ru(bpy32+ both dissolved in water solutions and dried. Lifetime measurements were carried out using a new detector, the Silicon Photomultiplier (SiPM, which is more sensitive and physically much smaller than conventional optical detectors, such as imager and scanner. Through these analyses and a morphological characterization with transmission electron microscopy, revealed its usability for sensor applications, in particular, as dye in optical DNA-chip technology, a viable alternative to the conventional CY5 fluorophore. The use of Ru(bpy32+ would solve some of the typical disadvantages related to Cy5’s application, such as self-absorption of fluorescence and photobleaching. In addition, the Ru(bpy32+ longer lifetime may play a key role in the definition of new optical DNA-chip.

  6. Gold nanoparticle-fluorophore complex for conditionally fluorescing signal mediator

    Energy Technology Data Exchange (ETDEWEB)

    Wang Jianting [Department of Chemical Engineering University of, Louisville, KY 40292 (United States); Achilefu, Samuel [Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Nantz, Michael [Department of Chemistry, University of Louisville, Louisville, KY 40292 (United States); Kang, Kyung A., E-mail: kyung.kang@louisville.edu [Department of Chemical Engineering University of, Louisville, KY 40292 (United States)

    2011-06-10

    Fluorescent contrast agents with high specificity and sensitivity are valuable for accurate disease detection and diagnosis. Spherical gold nanoparticles (GNPs) can be smartly utilized for developing highly effective agents. The strong electromagnetic (plasmon) field on their surface can be very effective in influencing the electrons of fluorophores and, thus, manipulating the fluorescence output (i.e., either quenching or enhancement). Fluorescence quenching can be used for negative sensing, or for conditional de-quenching to increase the specificity. Fluorescence enhancement allows sensing to be more sensitive. The level of fluorescence alteration depends on the GNP size, the excitation and emission wavelengths and quantum yield of the fluorophore, and the distance between the GNP and the fluorophore. To understand the mechanisms of the fluorescence change by GNP, we have theoretically analyzed the parameters involved in the fluorescence alteration for commonly used fluorophores, with an emphasis on quenching. The results showed that the fluorescence of fluorophores with the excitation (Ex) and emission (Ex) wavelengths close to the GNP resonance peak tended to be significantly quenched by GNPs. For those fluorophores emitting fluorescence in red or near infrared, to achieve quenching, the distance between GNP and the fluorophore was required to be very short. In general, a shorter distance resulted in more quenching. Bigger GNPs require a shorter distance to achieve the same level of quenching. The fluorescence of a fluorophore with a lower quantum yield (especially the one with emission in far-red or near-infrared) is more difficult to be quenched by GNPs (requires very short distance). Instead, it can be enhanced. Based on the theoretical study, we have developed a near-infrared contrast agent, i.e., Cypate conjugated GNP via a short peptide spacer. Normally the fluorescence of Cypate was quenched. The spacer has a motif of a substrate for urokinase type

  7. Synthetic Biomimetic Fluorophores for Micro/Nanosensor

    Science.gov (United States)

    2006-11-01

    Transport proteins in blood plasma bind and carry specific molecules or ions from one organ to another. Other proteins are specialized for nutrients ...cytoplasmic fluorescent proteins for live-cell imaging of Magnaporthe grisea in planta . Mycologia 94: 280-289. Davenport, DN, 1955: JAC: Luminescence in

  8. L-shaped benzimidazole fluorophores: synthesis, characterization and optical response to bases, acids and anions.

    Science.gov (United States)

    Lirag, Rio Carlo; Le, Ha T M; Miljanić, Ognjen Š

    2013-05-14

    Nine L-shaped benzimidazole fluorophores have been synthesized, computationally evaluated and spectroscopically characterized. These "half-cruciform" fluorophores respond to bases, acids and anions through changes in fluorescence that vary from moderate to dramatic.

  9. Fluorescence in nanobiotechnology: sophisticated fluorophores for novel applications.

    Science.gov (United States)

    Hötzer, Benjamin; Medintz, Igor L; Hildebrandt, Niko

    2012-08-06

    Nanobiotechnology is one of the fastest growing and broadest-ranged interdisciplinary subfields of the nanosciences. Countless hybrid bio-inorganic composites are currently being pursued for various uses, including sensors for medical and diagnostic applications, light- and energy-harvesting devices, along with multifunctional architectures for electronics and advanced drug-delivery. Although many disparate biological and nanoscale materials will ultimately be utilized as the functional building blocks to create these devices, a common element found among a large proportion is that they exert or interact with light. Clearly continuing development will rely heavily on incorporating many different types of fluorophores into these composite materials. This review covers the growing utility of different classes of fluorophores in nanobiotechnology, from both a photophysical and a chemical perspective. For each major structural or functional class of fluorescent probe, several representative applications are provided, and the necessary technological background for acquiring the desired nano-bioanalytical information are presented.

  10. Studies on the Fluorescence Properties of Chloromethyl Polystyrene Supported Fluorophore

    Institute of Scientific and Technical Information of China (English)

    REN; HongXia

    2001-01-01

    The basic materials used in making photoluminescence materials are all synthesized polymers at present, such as PVC, etc [1]. Red and green LEDs are easy to be prepared, but the blue LED is difficult to be done. In this paper, we used chloromethyl polystyrene as supporter, leading into the known fluorophore by polymer chemical reaction, so as to obtaining blue luminous material.  The PS-CH2Cl supported fluorophore are prepared by PS-CH2Cl and benzidine (B), dimethylbenzidine (MA), 4,4'-diamino-diphenyl ether (DAPE), 4,4'-diamino-diphenyl sulfone (DAPS), respectively. They were characterized by IR and elemental analysis. The stead state fluorescence properties of these supported polymers were investigated.The results were summarized in table 1.  ……

  11. Studies on the Fluorescence Properties of Chloromethyl Polystyrene Supported Fluorophore

    Institute of Scientific and Technical Information of China (English)

    REN HongXia; LEI ZiQiang; WANG SongBai; YANG ZhiWang; WANG YunPu

    2001-01-01

    @@ The basic materials used in making photoluminescence materials are all synthesized polymers at present, such as PVC, etc [1]. Red and green LEDs are easy to be prepared, but the blue LED is difficult to be done. In this paper, we used chloromethyl polystyrene as supporter, leading into the known fluorophore by polymer chemical reaction, so as to obtaining blue luminous material. The PS-CH2Cl supported fluorophore are prepared by PS-CH2Cl and benzidine (B), dimethylbenzidine (MA), 4,4'-diamino-diphenyl ether (DAPE), 4,4'-diamino-diphenyl sulfone (DAPS), respectively. They were characterized by IR and elemental analysis. The stead state fluorescence properties of these supported polymers were investigated.The results were summarized in table 1.

  12. NIR FRET Fluorophores for Use as an Implantable Glucose Biosensor

    Directory of Open Access Journals (Sweden)

    Majed DWEIK

    2008-12-01

    Full Text Available Development of an in vivo optical sensor requires the utilization of Near Infra Red (NIR fluorophores due to their ability to operate within the biological tissue window. Alexa Fluor 750 (AF750 and Alexa Fluor 680 (AF680 were examined as potential NIR fluorophores for an in vivo fluorescence resonance energy transfer (FRET glucose biosensor. AF680 and AF750 found to be a FRET pair and percent energy transfer was calculated. Next, the tested dye pair was utilized in a competitive binding assay in order to detect glucose. Concanavalin A (Con A and dextran have binding affinity, but in the presence of glucose, glucose displaces dextran due to its higher affinity to Con A than dextran. Finally, the percent signal transfer through porcine skin was examined. The results showed with approximately 4.0 mm porcine skin thickness, 1.98 % of the fluorescence was transmitted and captured by the detector.

  13. Photophysical Behaviors of Single Fluorophores Localized on Zinc Oxide Nanostructures

    Science.gov (United States)

    Fu, Yi; Zhang, Jian; Lakowicz, Joseph R.

    2012-01-01

    Single-molecule fluorescence spectroscopy has now been widely used to investigate complex dynamic processes which would normally be obscured in an ensemble-averaged measurement. In this report we studied photophysical behaviors of single fluorophores in proximity to zinc oxide nanostructures by single-molecule fluorescence spectroscopy and time-correlated single-photon counting (TCSPC). Single fluorophores on ZnO surfaces showed enhanced fluorescence brightness to various extents compared with those on glass; the single-molecule time trajectories also illustrated pronounced fluctuations of emission intensities, with time periods distributed from milliseconds to seconds. We attribute fluorescence fluctuations to the interfacial electron transfer (ET) events. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial electron transfer reaction processes. PMID:23109903

  14. Structure-activity relationship of nerve-highlighting fluorophores.

    Directory of Open Access Journals (Sweden)

    Summer L Gibbs

    Full Text Available Nerve damage is a major morbidity associated with numerous surgical interventions. Yet, nerve visualization continues to challenge even the most experienced surgeons. A nerve-specific fluorescent contrast agent, especially one with near-infrared (NIR absorption and emission, would be of immediate benefit to patients and surgeons. Currently, there are only three classes of small molecule organic fluorophores that penetrate the blood nerve barrier and bind to nerve tissue when administered systemically. Of these three classes, the distyrylbenzenes (DSBs are particularly attractive for further study. Although not presently in the NIR range, DSB fluorophores highlight all nerve tissue in mice, rats, and pigs after intravenous administration. The purpose of the current study was to define the pharmacophore responsible for nerve-specific uptake and retention, which would enable future molecules to be optimized for NIR optical properties. Structural analogs of the DSB class of small molecules were synthesized using combinatorial solid phase synthesis and commercially available building blocks, which yielded more than 200 unique DSB fluorophores. The nerve-specific properties of all DSB analogs were quantified using an ex vivo nerve-specific fluorescence assay on pig and human sciatic nerve. Results were used to perform quantitative structure-activity relationship (QSAR modeling and to define the nerve-specific pharmacophore. All DSB analogs with positive ex vivo fluorescence were tested for in vivo nerve specificity in mice to assess the effect of biodistribution and clearance on nerve fluorescence signal. Two new DSB fluorophores with the highest nerve to muscle ratio were tested in pigs to confirm scalability.

  15. Design of NIR Chromenylium-Cyanine Fluorophore Library for "Switch-ON" and Ratiometric Detection of Bio-Active Species In Vivo.

    Science.gov (United States)

    Wei, Yanfen; Cheng, Dan; Ren, Tianbing; Li, Yinhui; Zeng, Zebing; Yuan, Lin

    2016-02-02

    The real-time monitoring of key biospecies in the living systems has received thrusting attention during the past decades. Specifically, fluorescent detection based on near-infrared (NIR) fluorescent probes is highly favorable for live cells, live tissues, and even animal imaging, owing to the substantial merits of the NIR window, such as minimal phototoxicity, deep penetration into tissues, and low autofluorescence background. Nevertheless, developing potent NIR fluorescent probes still poses serious challenges to the chemists because traditional NIR fluorophores are less tunable than visible-wavelength fluorophores. To address this issue, here we report a set of novel NIR hybrid fluorophores, namely, the hybrid chromenylium-cyanine fluorophore (CC-Fluor), in which both the fluorescence intensity and the emission wavelength can be easily adjusted by the conformational changes and substitution groups. Compared to known NIR fluorophores, the new CC-Fluors are substantially advantageous for NIR probe development: (1) CC-Fluors display tunable and moderate Stokes shifts and quantum yields; (2) the fluorophores are stable at physiological conditions after long-term incubation; (3) the absorption maxima of CC-Fluors coincide with the common laser spectral lines in mainstream in vivo imaging systems; (4) most importantly, CC-Fluors can be easily modified to prepare NIR probes targeting various biospecies. To fully demonstrate the practical utility of CC-Fluors, we report two innovative NIR probes, a ratiometric pH probe and a turn-on Hg(2+) probe, both are successfully employed in live animal imaging. Hence, the detailed studies allow us to confirm that CC-Fluors can work as an excellent platform for developing NIR probes for the detection of species in living systems.

  16. Photoluminescence properties of polystyrene-hosted fluorophore thin films

    Directory of Open Access Journals (Sweden)

    Subha Chakraborty

    2016-12-01

    Full Text Available We report on a photo-luminescence study of four different fluorophores: Coumarin 6, 2,5-Diphenyloxazole (PPO, 1,4-Bis(5-phenyl-2-oxazolylbenzene (POPOP and Para-terpehnyl (PTP, doped in a polystyrene-based thin film. All of the samples are prepared by spin coating from a non-polar polymer solution at various concentrations. Their emission spectra and transient properties are characterized by photoluminescence measurements. Red-shifts in the emission spectra are observed for all four types of fluorophores as their concentration increases. We explain this phenomenon based on concentration dependence of solvatochromic effects and the results show good agreement with existing literature. We also show that the singlet-singlet annihilation processes are possibly a prevalent mechanism in the high concentration regime that affects the steady state and transient emission characteristics of the fluors. With the exception of PTP, photoluminescence quenching occurs as the fluorophore concentration in the polymer is increased. Rate equations for excited state decay mechanisms are analysed by considering different radiative and non-radiative energy transfer mechanisms. The results show consistency with our experimental observations. PTP shows the best photoluminescence results as an efficient fluor in the thin film, whereas PPO shows the strongest concentration dependent quenching and an anomalous lifetime distribution.

  17. Photoluminescence properties of polystyrene-hosted fluorophore thin films

    Science.gov (United States)

    Chakraborty, Subha; Harris, Katherine; Huang, Mengbing

    2016-12-01

    We report on a photo-luminescence study of four different fluorophores: Coumarin 6, 2,5-Diphenyloxazole (PPO), 1,4-Bis(5-phenyl-2-oxazolyl)benzene (POPOP) and Para-terpehnyl (PTP), doped in a polystyrene-based thin film. All of the samples are prepared by spin coating from a non-polar polymer solution at various concentrations. Their emission spectra and transient properties are characterized by photoluminescence measurements. Red-shifts in the emission spectra are observed for all four types of fluorophores as their concentration increases. We explain this phenomenon based on concentration dependence of solvatochromic effects and the results show good agreement with existing literature. We also show that the singlet-singlet annihilation processes are possibly a prevalent mechanism in the high concentration regime that affects the steady state and transient emission characteristics of the fluors. With the exception of PTP, photoluminescence quenching occurs as the fluorophore concentration in the polymer is increased. Rate equations for excited state decay mechanisms are analysed by considering different radiative and non-radiative energy transfer mechanisms. The results show consistency with our experimental observations. PTP shows the best photoluminescence results as an efficient fluor in the thin film, whereas PPO shows the strongest concentration dependent quenching and an anomalous lifetime distribution.

  18. Fluorophore-based sensor for oxygen radicals in processing plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Faraz A.; Shohet, J. Leon, E-mail: shohet@engr.wisc.edu [Plasma Processing and Technology Laboratory and Department of Electrical and Computer Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Sabat, Grzegorz; Sussman, Michael R. [Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Nishi, Yoshio [Department of Electrical Engineering, Stanford University, Stanford, California 94305 (United States)

    2015-11-15

    A high concentration of radicals is present in many processing plasmas, which affects the processing conditions and the properties of materials exposed to the plasma. Determining the types and concentrations of free radicals present in the plasma is critical in order to determine their effects on the materials being processed. Current methods for detecting free radicals in a plasma require multiple expensive and bulky instruments, complex setups, and often, modifications to the plasma reactor. This work presents a simple technique that detects reactive-oxygen radicals incident on a surface from a plasma. The measurements are made using a fluorophore dye that is commonly used in biological and cellular systems for assay labeling in liquids. Using fluorometric analysis, it was found that the fluorophore reacts with oxygen radicals incident from the plasma, which is indicated by degradation of its fluorescence. As plasma power was increased, the quenching of the fluorescence significantly increased. Both immobilized and nonimmobilized fluorophore dyes were used and the results indicate that both states function effectively under vacuum conditions. The reaction mechanism is very similar to that of the liquid dye.

  19. Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds.

    Science.gov (United States)

    Schrand, Amanda M; Lin, Jonathan B; Hens, Suzanne Ciftan; Hussain, Saber M

    2011-02-01

    Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

  20. NIR fluorophore-hollow gold nanosphere complex for cancer enzyme-triggered detection and hyperthermia.

    Science.gov (United States)

    Wang, Jianting; Wheeler, Damon; Zhang, Jin Z; Achilefu, Samuel; Kang, Kyung A

    2013-01-01

    Hollow gold nanospheres (HGN) may be delicately tuned to absorb near infrared light (NIR) by tailoring the diameter-to-shell ratio. This unique property can be utilized for enhancing the contrast for the NIR and X-ray/CT imaging, and also noninvasive and local, photothermal hyperthermia by conjugating cancer-targeting molecules on the particle surface. In addition, when an NIR fluorophore is placed on the surface of the NIR-tuned HGNs, the fluorescence can be significantly quenched due to the emitted light absorption by the HGNs. Combining the NIR fluorescence quenching property of HGNs and the enzyme secreting nature of cancer, we have developed a novel enzyme-triggered NIR contrast agent for cancer detection with high specificity. NIR fluorophore Cypate (Indocyanine Green based) was conjugated to HGN via a short spacer for fluorescence quenching. The spacer contains an enzyme-substrate-motif (G-G-R) that can be cleaved by urokinase-type plasminogen activator (uPA, a breast cancer enzyme). The nano-complex normally does not emit fluorescence but, in the presence of uPA, the fluorescence was restored, providing high specificity. The enzyme-specific emission allows us to characterize the nature of the cancer (e.g., invasive, metastatic, etc.). Once the cancer is detected, the same HGNs can be used to deliver heat to the cancer site for cancer-specific hyperthermia.

  1. Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection.

    Science.gov (United States)

    Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H; Brown, Tom

    2016-09-30

    A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Single Molecule Study of Photoconversion and Spectral Heterogeneities of Fluorophores

    DEFF Research Database (Denmark)

    Liao, Zhiyu

    and mass spectrometry. Upon illumination, A3-TOTA+ degrades in a step-wise manner by de-ethylation on the periphery. The unusual red-shifted fluorescence from the photoproducts is not as intense as the original emission, but the photostability is improved. The acquired knowledge about photoconversion can...... stimulate new pathways in engineering and designing photoconvertible fluorophores, based on the reaction with oxygen or other chemicals. Besides, this results show that dyes that convert into other emissive species could give problems when interpreting single molecule FRET systems. The revealed mechanism...

  3. Fluorophore-conjugated iron oxide nanoparticle labeling and analysis of engrafting human hematopoietic stem cells

    DEFF Research Database (Denmark)

    Maxwell, Dustin J; Bonde, Jesper; Hess, David A

    2008-01-01

    The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enables dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined...... culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores...... or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34(+) cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles...

  4. Discovery, understanding, and bioapplication of organic fluorophore: a case study with an indolizine-based novel fluorophore, Seoul-Fluor.

    Science.gov (United States)

    Kim, Eunha; Lee, Youngjun; Lee, Sanghee; Park, Seung Bum

    2015-03-17

    Owing to its high sensitivity and great applicability, the fluorescence phenomenon has been considered as an inevitable research tool in the modern scientific fields of chemistry, biology, materials science, biomedical science, and their interfaces. Many strategies have been pursued to understand and manipulate the photophysical properties of fluorescent materials, but the scientific community has been focused on the repeated application of existing organic fluorophores or the identification of unique fluorescence properties in a trial-and-error basis without systematic studies. Moreover, recent studies are emphasizing the necessity of deeper understanding about the structure-photophysical property relationship of organic fluorophores for the development of better fluorescent probes. Herein, we provide an overview of a novel fluorescent molecular framework, Seoul-Fluor, which can be rationally engineered to furnish a wide variety of fluorophores in terms of the photophysical properties. Seoul-Fluor is built on an indolizine-based fluorescent platform with three different positions to introduce various substituents: R(1) and R(2) substituents for electronic perturbation; R(3) substituent as a functional handle for bioconjugation. Over the past decade, we have demonstrated that the Seoul-Fluor system has (i) tunable and predictable emission wavelength covering a full visible-color range; (ii) controllable quantum yield via photoinduced electron transfer phenomenon; and (iii) environment-sensitive fluorogenic properties that can be modified through intramolecular charge transfer processes. We convincingly demonstrated the prediction of photophysical properties, that is, emission wavelength and quantum yield, through the construction of a systematic set of analogues and the subsequent analysis of their photophysical properties without the highly sophisticated theoretical support. Guided by quantifiable parameters such as the Hammett substituent constants or energy

  5. Comparative study of the three different fluorophore antibody conjugation strategies.

    Science.gov (United States)

    Shrestha, Dilip; Bagosi, Adrienn; Szöllősi, János; Jenei, Attila

    2012-09-01

    The progression in bioconjugational chemistry has significantly contributed to the evolution and success of protein biology. Mainly, antibody chemistry has been a subject of intensive study owing to the expansion of research areas warranted by using various derivatives of conjugated antibodies. Three reactive moieties (amine, sulfhydryl and carbohydrate) in the antibodies are chiefly favored for the conjugational purpose. This feature is known for decades, nevertheless, amine based conjugation is still the most preferred strategy despite the appreciation the other two methods receive in conserving the antigen binding affinity (ABA). No single report has been published, according to our knowledge, where these three conjugation strategies were applied to the same fluorophore antibody systems. In this study, we evaluated conjugation yield, time demand and cost efficiency of these conjugation procedures. Our results showed that amine based conjugations was by far the best technique due to its simplicity, rapidity, ease of operation, higher conjugate yield, cheaper cost and potential for larger fluorophore/protein labeling ratio without having much effect in ABA. Furthermore, sulfhydryl labeling clearly excelled in terms of reduced non-specific binding and mild effect in ABA but was usually complicated by an asymmetric antibody reduction due to mercaptoethylamine while carbohydrate oxidation based strategy performed the worst during our experiment.

  6. Benzimidazolylquinoxalines: novel fluorophores with tuneable sensitivity to solvent effects.

    Science.gov (United States)

    Burganov, Timur I; Zhukova, Nataliya A; Mamedov, Vakhid A; Bannwarth, Christoph; Grimme, Stefan; Katsyuba, Sergey A

    2017-02-22

    We report on the photophysical properties, conjugation, conformational behavior, intra- and intermolecular hydrogen bonds (HBs) of a series of novel fluorophores, consisting of 3-arylquinoxaline and benzimidazole moieties linked by a single CC bond. Computations employing density functional theory (DFT) reveal that conjugation between these moieties stabilizes syn-conformers with two HB centers located on the same side of the molecule. Anti-conformers form stronger intermolecular HBs with DMSO and DMF than syn-conformers, and this influences the energy gap between syn- and anti-forms, especially upon excitation of the molecules to the S1 state. Substituents introduced in various positions of the molecules modify their conformational behavior, and mutual disposition of excited singlet states relative to the ground states. Various substitution patterns produce very different effects on relative quantum yield of luminescence: from a moderate increase in polar DMSO and DMF relative to 1,2-dichloroethane solutions to complete quenching of emission which is observable in polar media. The observed behavior is understood with the aid of DFT and time-dependent DFT calculations. The tuneability of the spectroscopic range of the luminescence and especially of its sensitivity to environmental effects allows rational design of the novel fluorophores of this family for various applications.

  7. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

    NARCIS (Netherlands)

    van der Velde, Jasper H M; Oelerich, Jens; Huang, Jingyi; Smit, Jochem H; Aminian Jazi, Atieh; Galiani, Silvia; Kolmakov, Kirill; Guoridis, Giorgos; Eggeling, Christian; Herrmann, Andreas; Roelfes, Gerard; Cordes, Thorben

    2016-01-01

    Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with 'self-healing' properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general str

  8. Fluorophore and dye-assisted dispersion of carbon nanotubes in aqueous solution.

    Science.gov (United States)

    Koh, Byumseok; Kim, Gwangseong; Yoon, Hyung Ki; Park, Jong Bae; Kopelman, Raoul; Cheng, Wei

    2012-08-14

    DNA short oligo, surfactant, peptides, and polymer-assisted dispersion of single-walled carbon nanotube (SWCNTs) in aqueous solution have been intensively studied. It has been suggested that van der Waals interaction, π-π stacking, and hydrophobic interaction are major factors that account for the SWCNTs dispersion. Fluorophore and dye molecules such as Rhodamine B and fluorescein have both hydrophilic and hydrophobic moieties. These molecules also contain π-conjugated systems that can potentially interact with SWCNTs to induce its dispersion. Through a systematic study, here we show that SWCNTs can be dispersed in aqueous solution in the presence of various fluorophore or dye molecules. However, the ability of a fluorophore or dye molecule to disperse SWCNTs is not correlated with the stability of the fluorophore/dye-SWCNT complex, suggesting that the on-rate of fluorophore/dye binding to SWCNTs may dominate the efficiency of this process. We also examined the uptake of fluorophore molecules by mammalian cells when these molecules formed complexes with SWCNTs. The results can have potential applications in the delivery of poor cell-penetrating fluorophore molecules.

  9. Three-dimensional spatial localization of thin fluorophore-filled capillaries in thick scattering media

    Science.gov (United States)

    Desrochers, Johanne; Vermette, Patrick; Fontaine, Réjean; Bérubé-Lauzière, Yves

    2008-06-01

    Fluorescence optical diffuse tomography (fDOT) is of much interest in molecular imaging to retrieve information from fluorescence signals emitted from specifically targeted bioprocesses deep within living tissues. An exciting application of fDOT is in the growing field of tissue engineering, where 3D non-invasive imaging techniques are required to ultimately grow 3D engineered tissues. Via appropriate labelling strategies and fluorescent probes, fDOT has the potential to monitor culture environment and cells viability non-destructively directly within the bioreactor environment where tissues are to be grown. Our ultimate objective is to image the formation of blood vessels in bioreactor conditions. Herein, we use a non-contact setup for small animal fDOT imaging designed for 3D light collection around the sample. We previously presented a time of flight approach using a numerical constant fraction discrimination technique to assign an early photons arrival time to every fluorescence time point-spread function collected around the sample. Towards bioreactor in-situ imaging, we have shown the capability of our approach to localize a fluorophore-filled 500 μm capillary immersed coaxially in a cylindrically shaped bioreactor phantom containing an absorbing/scattering medium representative of experiments on real tissue cultures. Here, we go one step further, and present results for the 3D localization of thinner indocyanine green labelled capillaries (250 μm and 360 μm inner diameter) immersed in the same phantom conditions and geometry but with different spatial configurations (10° and 30° capillary inclination).

  10. Single Benzene Green Fluorophore: Solid-State Emissive, Water-Soluble, and Solvent- and pH-Independent Fluorescence with Large Stokes Shifts.

    Science.gov (United States)

    Beppu, Teruo; Tomiguchi, Kosuke; Masuhara, Akito; Pu, Yong-Jin; Katagiri, Hiroshi

    2015-06-15

    Benzene is the simplest aromatic hydrocarbon with a six-membered ring. It is one of the most basic structural units for the construction of π conjugated systems, which are widely used as fluorescent dyes and other luminescent materials for imaging applications and displays because of their enhanced spectroscopic signal. Presented herein is 2,5-bis(methylsulfonyl)-1,4-diaminobenzene as a novel architecture for green fluorophores, established based on an effective push-pull system supported by intramolecular hydrogen bonding. This compound demonstrates high fluorescence emission and photostability and is solid-state emissive, water-soluble, and solvent- and pH-independent with quantum yields of Φ=0.67 and Stokes shift of 140 nm (in water). This architecture is a significant departure from conventional extended π-conjugated systems based on a flat and rigid molecular design and provides a minimum requirement for green fluorophores comprising a single benzene ring.

  11. Genotype-independent transmission of transgenic fluorophore protein by boar spermatozoa.

    Directory of Open Access Journals (Sweden)

    Wiebke Garrels

    Full Text Available Recently, we generated transposon-transgenic boars (Sus scrofa, which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring. The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.

  12. Small-molecule fluorophores with large Stokes shifts: 9-iminopyronin analogues as clickable tags.

    Science.gov (United States)

    Horváth, Peter; Šebej, Peter; Šolomek, Tomáš; Klán, Petr

    2015-02-06

    The design, synthesis, and both experimental and theoretical studies of several novel 9-(acylimino)- and 9-(sulfonylimino)pyronin derivatives containing either an oxygen or a silicon atom at position 10 are reported. These compounds, especially the Si analogues, exhibit remarkably large Stokes shifts (around 200 nm) while still possessing a high fluorophore brightness, absorption bands in the near-UV and visible part of the spectrum, and high thermal and photochemical stabilities in protic solvents. The reason for the observed large Stokes shifts is an intramolecular charge-transfer excitation of an electron from the HOMO to the LUMO of the chromophore, accompanied by elongation of the C9-N bond and considerable solvent reorganization due to hydrogen bonding to the solvent. Due to the photophysical properties of the studied compounds and their facile and high-yielding synthesis, as well as a simple protocol for their bioorthogonal ligation to a model saccharide using a Huisgen alkyne-azide cycloaddition, they represent excellent candidates for biochemical and biological applications as fluorescent tags and indicators for multichannel imaging. 9-(Acylimino)pyronins alter their optical properties upon protonation and may also be used as pH sensors.

  13. Modulation sensing of fluorophores in tissue: a new approach to drug compliance monitoring

    Science.gov (United States)

    Abugo, Omoefe O.; Gryczynski, Zygmunt; Lakowicz, Joseph R.

    1999-10-01

    We describe a method to detect the presence of fluorophores in scattering media, including intralipid suspensions and chicken muscle covered with skin. The fluorophores were rhodamine 800 (Rb800) and indocyanine green (IcG), both of which can be excited at long wavelengths where there is minimal absorption by tissues. These fluorophores were dissolved in intralipid or in chicken muscle under skin. A method to approximate the fluorophore concentration in such samples was developed using a long lifetime reference fluorophores in a polymer film placed immediately on the illuminated surface of the sample. Because of the long lifetime of the reference film, the modulation of its emission at low frequencies near 2 MHz is near zero. Since the lifetime of Rh800 and IcG are below 2 ns the modulation of the combined emission is a measure of the intensity of the fluorophore (Rh800 or IcG) relative to the long lifetime reference. Using this method we were able to measure the concentration-dependent intensities of Rh800 and IcG in an intralipid suspension. Additionally, micromolar concentrations of these probes could be detected in chicken muscles, even when the muscle was covered with a layer of chicken skin. The presence of an India ink absorber in the intralipid had only a moderate effect on the modulation values. We suggest the use of this transdermal detection of long-wavelength fluorophores as a noninvasive method to monitor patient compliance when taking medicines used for treatment of chronic diseases such as AIDS or tuberculosis.

  14. Dual changes in conformation and optical properties of fluorophores within a metal-organic framework during framework construction and associated sensing event.

    Science.gov (United States)

    Cho, Won; Lee, Hee Jung; Choi, Goeun; Choi, Sora; Oh, Moonhyun

    2014-09-03

    Microsized chemosensor particle (CPP-16, CPP means coordination polymer particle), which is made from a metal-organic framework (MOF), is synthesized using pyrene-functionalized organic building block. This building block contains three important parts, a framework construction part, a Cu(2+) detection part, and a fluorophore part. PXRD studies have revealed that CPP-16 has a 3D cubic structure of MOF-5. During both MOF formation and sensing event, fluorophores within CPP-16 undergo dual changes in conformation and optical properties. After MOF construction, pyrene moieties experience an unusual complete conversion from monomer to excimer form. This conversion takes place due to a confinement effect induced by space limitations within the MOF structure. The selective sensing ability of CPP-16 on Cu(2+) over many other metal ions is verified by emission spectra and is also visually identified by fluorescence microscopy images. Specific interaction of Cu(2+) with binding sites within CPP-16 causes a second conformational change of the fluorophores, where they change from stacked excimer (CPP-16) to quenched excimer states (CPP-16·Cu(2+)).

  15. Fluorescein Derivatives in Intravital Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Michael S. Roberts

    2013-08-01

    Full Text Available Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples.

  16. Resonance energy transfer between fluorescent BSA protected Au nanoclusters and organic fluorophores.

    Science.gov (United States)

    Raut, Sangram; Rich, Ryan; Fudala, Rafal; Butler, Susan; Kokate, Rutika; Gryczynski, Zygmunt; Luchowski, Rafal; Gryczynski, Ignacy

    2014-01-01

    Bovine serum albumin (BSA) protected nanoclusters (Au and Ag) represent a group of nanomaterials that holds great promise in biophysical applications due to their unique fluorescence properties and lack of toxicity. These metal nanoclusters have utility in a variety of disciplines including catalysis, biosensing, photonics, imaging and molecular electronics. However, they suffer from several disadvantages such as low fluorescence quantum efficiency (typically near 6%) and broad emission spectrum (540 nm to 800 nm). We describe an approach to enhance the apparent brightness of BSA Au clusters by linking them with a high extinction donor organic dye pacific blue (PB). In this conjugate PB acts as a donor to BSA Au clusters and enhances its brightness by resonance energy transfer (RET). We found that the emission of BSA Au clusters can be enhanced by a magnitude of two-fold by resonance energy transfer (RET) from the high extinction donor PB, and BSA Au clusters can act as an acceptor to nanosecond lifetime organic dyes. By pumping the BSA Au clusters using a high extinction donor, one can increase the effective brightness of less bright fluorophores like BSA Au clusters. Moreover, we prepared another conjugate of BSA Au clusters with the near infrared (NIR) dye Dylight 750 (Dy750), where BSA Au clusters act as a donor to Dy750. We observed that BSA Au clusters can function as a donor, showing 46% transfer efficiency to the NIR dye Dy750 with a long lifetime component in the acceptor decay through RET. Such RET-based probes can be used to prevent the problems of a broad emission spectrum associated with the BSA Au clusters. Moreover, transferring energy from BSA Au clusters to Dy750 will result in a RET probe with a narrow emission spectrum and long lifetime component which can be utilized in imaging applications.

  17. A fluorescence ratiometric sensor for hypochlorite based on a novel dual-fluorophore response approach.

    Science.gov (United States)

    Long, Lingliang; Zhang, Dongdong; Li, Xiufen; Zhang, Jinfang; Zhang, Chi; Zhou, Liping

    2013-05-02

    A fluorescence ratiometric sensor for OCl(-) has been developed based on a novel dual fluorophore response approach. The sensor molecule contains a coumarin fluorophore and a rhodamine fluorophore, and the two fluorophores are directly linked to an OCl(-) recognition group. The structure of the sensor was characterized by ESI-MS, NMR, and X-ray crystallographic analysis. Upon treatment with OCl(-), both fluorophores in the sensor responded simultaneously at two separate optical windows, with large enhancement of the fluorescence ratio (I578/I501) from 0.01 to 39.55. The fluorescence ratios for the sensor showed a good linearity with the concentration of OCl(-) in the range of 0.2-40 μM and the detection limits is 0.024 μM (SN(-1)=3). Investigation of reaction products indicated that the sensor reaction with OCl(-) produced two new fluorescent molecules, which were responsible for the fluorescence changes in two optical windows. In addition, the sensor showed high selectivity to OCl(-) over other reactive oxygen species, reactive nitrogen species, cations, and anions. The sensor has also been successfully applied to detection of OCl(-) in natural water samples with satisfactory recovery.

  18. Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides.

    Science.gov (United States)

    Sayers, E J; Cleal, K; Eissa, N G; Watson, P; Jones, A T

    2014-12-10

    For cell penetrating peptides (CPPs) to fulfil their promise as effective delivery vectors we need a better understanding of their mechanisms of cell binding and uptake. This is especially the case when they are linked to different types of cargo. Here we describe new studies based on our previous findings suggesting that, for peptide-CPP chimeras, distal hydrophobic residues upstream of the CPP sequence can have profound effects on the way they interact with cells. We studied peptides bearing an N-terminal Glycine or Phenylalanine linked via a neutral and flexible bridging group, SGSGSGSG, to three well-studied CPPs: octaarginine, penetratin and TP10. Using a combination of flow cytometry, live-cell imaging and image analysis we examined the effects of this single amino acid change on binding and uptake of Alexa488-fluorophore, bovine serum albumin and quantum dot cargoes. The influence of the glycine-phenylalanine switch for fluorophore delivery was most dramatic in TP10, increasing cellular uptake by 4.4 and 9.9 fold in non-adherent and adherent cells, respectively. Only penetratin showed effective uptake of bovine serum albumin with the phenylalanine variant showing an increase of 1.6 fold over the glycine variant. The uptake of quantum dots was most efficiently demonstrated by octaarginine, with the glycine variant increasing uptake 4.8 fold and the phenylalanine variant increasing uptake 9.5 fold over quantum dots alone. Overall the data demonstrate that hydrophobicity distal to the CPP could be utilised to enhance their capacity to bind to the cell membrane and deliver a range of macromolecules to the insides of cells.

  19. 双视点3D视频文件的裸眼立体组合投影实时显示算法%Real-Time Tiled Multi-projector Autostereoscopic Display Algorithm for Dual-view 3D Video Files

    Institute of Scientific and Technical Information of China (English)

    郭华源; 秦开怀; 毛苗; 孙丰

    2015-01-01

    基于Client/Server结构和sort-last并行绘制策略, 提出双视点3D视频文件的裸眼立体组合投影实时显示算法. 首先在服务端全屏播放左右或上下格式的3D视频文件, 以不低于25帧/s的速率在线截屏并按JPEG格式压缩后转发给12个Client PC.每个Client PC接收每一帧截屏图像后,利用Fragment Shader和多渲染目标(MRT)通过一遍绘制完成2个单视点子图像的裁剪、缩放、奇偶条纹倾斜绘制, 经几何和亮度校正, 并将2个子图像交织后再向前投影到光栅显示屏幕.该屏幕的投影表面为3.6 m×1.6 m,单台投影仪分辨率为1024×768,投影系统的分辨率为3584× 1536. 实验结果表明, 该算法的显示帧率≥24帧/s, 且当条纹倾斜角度为10°时裸眼立体显示效果最好.%To display the dual-view 3D video files, this paper presents a real-time tiled multi-projector autostereoscopic display algorithm based on the Client/Server structure and the sort-last distributed render-ing scheme. First, the dual-view 3D video is displayed in a full screen mode on the server. Second, the du-al-view images are screen captured at frames per second of more than 25 and distributed to all rendering clients. Then, a GPU-based image processing technique is utilized to split each HSBS or Half-OU image into two single-view sub-images according to the tiled parameters, and to resize the resolution by bilinear inter-polation and generate slanted stripe images through a single-pass rendering process. Lastly, after performing the geometric calibration and luminance correction, these images are interleaved to provide the autostereo-scopic vision on the optical display screen. The projection system, which is equipped with a front-projection screen that covers an area of 360?160 square centimeters and 24 projectors with a two-dimensional projec-tion resolution of 3584?1536 pixels, can provide glasses-free stereoscopic vision at 24 frames per second. Furthermore, it is

  20. Detection of fluorophore-labeled antibodies by surface-enhanced fluorescence on metal nanoislands

    Science.gov (United States)

    Schalkhammer, Thomas G. M.; Aussenegg, Franz R.; Leitner, Alfred; Brunner, Harald; Hawa, Gerhard; Lobmaier, Christina; Pittner, Fritz

    1997-06-01

    In fluorescence labelled immunosensing the discrimination of labels remaining in the bulk solution from labels bound to the analyte at the sensor surface is a basic optical problem. It is shown that application of surface enhanced fluorescence at a layer of noble metal nano-particles can increase the surface-to-background signal ratio. We explain the enhancement mechanism by an electrodynamic model and discuss the interaction between metal particle and fluorophore for the excitation and emission process. We show the principal guidelines for optimization of that processes. We find that the obtained discrimination power increases with decreasing intrinsic quantum efficiency of the fluorophore, suggesting the application of new classes of labels, namely low-quantum efficiency fluorophores. This theoretical finding is shown by a practical model experiment.

  1. Flexible and fluorophore-doped luminescent solar concentrators based on polydimethylsiloxane.

    Science.gov (United States)

    Tummeltshammer, Clemens; Taylor, Alaric; Kenyon, Anthony J; Papakonstantinou, Ioannis

    2016-02-15

    We demonstrate a simple and inexpensive method to fabricate flexible and fluorophore-doped luminescent solar concentrators (LSCs). Polydimethylsiloxane (PDMS) serves as a host material which additionally offers the potential to cast LSCs in arbitrary shapes. The laser dye Pyrromethene 567 is used as a prototype fluorophore, and it is shown that it has a high quantum yield of 93% over the concentration range investigated. The optical efficiency and loss channels of the flexible LSCs are investigated; it is also demonstrated that the efficiency remains high while bending the LSC which is essential for flexible LSCs to make an impact on solar energy.

  2. Photoactivatable Drug-Caged Fluorophore Conjugate Allows Direct Quantification of Intracellular Drug Transport

    Science.gov (United States)

    Kohler, Rainer H.; Weissleder, Ralph

    2013-01-01

    We report here a method that utilizes photoactivatable drug-caged fluorophore conjugate to quantify intracellular drug trafficking processes at single cell resolution. Photoactivation is performed in labeled cellular compartments to visualize intracellular drug exchange at physiologic conditions, without the need for washing, facilitating its translation to in vivo cancer models. PMID:24135896

  3. Interactions of carbohydrates and proteins by fluorophore-assisted carbohydrate electrophoresis

    Indian Academy of Sciences (India)

    Gang-Liang Huang; Xin-Ya Mei; Peng-George Wang

    2006-06-01

    A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.

  4. Synthesis, structures, redox and photophysical properties of benzodifuran-functionalised pyrene and anthracene fluorophores.

    Science.gov (United States)

    Keller, Stephan; Yi, Chenyi; Li, Chen; Liu, Shi-Xia; Blum, Carmen; Frei, Gabriela; Sereda, Olha; Neels, Antonia; Wandlowski, Thomas; Decurtins, Silvio

    2011-09-21

    Benzodifuran-functionalised pyrene and anthracene fluorophores 1 and 2 were obtained in reasonable yields. Their single crystal structures, electrochemical, optical absorption, and fluorescence characteristics have been described. They show strong luminescence with high quantum yields of 0.53 for 1 and 0.48 for 2.

  5. A whole new way of looking at things: the use of Dark Reader technology to detect fluorophors.

    Science.gov (United States)

    Seville, M

    2001-03-01

    The Dark Reader optical system (Clare Chemical Research, Denver, CO, USA) uses relatively low intensity broad-band visible blue light in combination with broad-band optical filters to detect fluorescence with a level of sensitivity that often surpasses that of UV transilluminators and can rival that of laser-based scanners. Applications of DR (Clare Chemical Research) devices include the detection of DNA and SYBR-stained protein samples following, and also during, electrophoresis. Unlike laser-based imaging systems, the fluorescence is directly visible to the user as well as being fully compatible with charge-coupled device (CCD) and Polaroid camera-based detection and imaging. Additionally, the DR optical system functions well in multicolor fluorophor environments. Because the Dark Reader does not emit any UV light, the extent of DNA damage incurred when visualizing DNA samples is drastically reduced compared to the damage produced by a UV device and this can have a significant benefit on downstream cloning protocols. Furthermore, dye photobleaching is minimal, extending the length of time that a fluorescent sample is visible. The inherent flexibility of the DR optical system allows many different configurations of the Dark Reader to be constructed such as transilluminators, hand lamps and integrated transilluminator-electrophoresis units.

  6. Surface plasmon enhanced energy transfer between gold nanorods and fluorophores: application to endocytosis study and RNA detection.

    Science.gov (United States)

    Zhang, Yinan; Wei, Guoke; Yu, Jun; Birch, David J S; Chen, Yu

    2015-01-01

    Previously we have demonstrated surface plasmon enhanced energy transfer between fluorophores and gold nanorods under two-photon excitation using fluorescence lifetime imaging microscopy (FLIM) in both solution and intracellular phases. These studies demonstrated that gold nanoparticle-dye energy transfer combinations are appealing, not only in Förster resonance energy transfer (FRET) imaging, but also energy transfer-based fluorescence lifetime sensing of bio-analytes. Here, we apply this approach to study the internalization of gold nanorods (GNRs) in HeLa cells using the early endosome labeling marker GFP. The observed energy transfer between GFP and the GNRs indicates the involvement of endocytosis in GNR uptake. Moreover, a novel nanoprobe based on oligonucleotide functionalized gold nanorods for nucleic acid sensing via dye-GNRs energy transfer is demonstrated, potentially opening up new possibilities in cancer diagnosis and prognosis. The influence of oligonucleotide design on such nanoprobe performance was studied for the first time using time-resolved fluorescence spectroscopy, bringing new insights to the optimization of the nanoprobe.

  7. Photostable and photoswitching fluorescent dyes for super-resolution imaging.

    Science.gov (United States)

    Minoshima, Masafumi; Kikuchi, Kazuya

    2017-01-12

    Super-resolution fluorescence microscopy is a recently developed imaging tool for biological researches. Several methods have been developed for detection of fluorescence signals from molecules in a subdiffraction-limited area, breaking the diffraction limit of the conventional optical microscopies and allowing visualization of detailed macromolecular structures in cells. As objectives are exposed to intense laser in the optical systems, fluorophores for super-resolution microscopy must be tolerated even under severe light irradiation conditions. The fluorophores must also be photoactivatable and photoswitchable for single-molecule localization-based super-resolution microscopy, because the number of active fluorophores must be controlled by light irradiation. This has led to growing interest in these properties in the development of fluorophores. In this mini-review, we focus on the development of photostable and photoswitching fluorescent dyes for super-resolution microscopy. We introduce recent efforts, including improvement of fluorophore photostability and control of photoswitching behaviors of fluorophores based on photochemical and photophysical processes. Understanding and manipulation of chemical reactions in excited fluorophores can develop highly photostable and efficiently photoswitchable fluorophores that are suitable for super-resolution imaging applications.

  8. Multiplexed Spectral Imaging of 120 Different Fluorescent Labels.

    Directory of Open Access Journals (Sweden)

    Alex M Valm

    Full Text Available The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image.

  9. Fluorescent Ensemble Based on Bispyrene Fluorophore and Surfactant Assemblies: Sensing and Discriminating Proteins in Aqueous Solution.

    Science.gov (United States)

    Fan, Junmei; Ding, Liping; Bo, Yu; Fang, Yu

    2015-10-14

    A particular bispyrene fluorophore (1) with two pyrene moieties covalently linked via a hydrophilic spacer was synthesized. Fluorescence measurements reveal that the fluorescence emission of 1 could be well modulated by a cationic surfactant, dodecyltrimethylammonium bromide (DTAB). Protein sensing studies illustrate that the selected ensemble based on 1/DTAB assemblies exhibits ratiometric responses to nonmetalloproteins and turn-off responses to metalloproteins, which can be used to differentiate the two types of proteins. Moreover, negatively charged nonmetalloproteins can be discriminated from the positively charged ones according to the difference in ratiometric responses. Fluorescence sensing studies with control bispyrenes indicate that the polarity of the spacer connecting two pyrene moieties plays an important role in locating bispyrene fluorophore in DTAB assemblies, which further influences its sensing behaviors to noncovalent interacting proteins. This study sheds light on the influence of the probe structure on the sensing performance of a fluorescent ensemble based on probe and surfactant assemblies.

  10. Long wave fluorophore sensor compounds and other fluorescent sensor compounds in polymers

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Joseph C.; Heiss, Aaron M.; Noronha, Glenn; Vachon, David J.; Lane, Stephen M.; Satcher, Jr., Joe H.; Peyser, Thomas A.; Van Antwerp, William Peter; Mastrototaro, John Joseph

    2004-07-20

    Fluorescent biosensor molecules, fluorescent biosensors and systems, as well as methods of making and using these biosensor molecules and systems are described. Embodiments of these biosensor molecules exhibit fluorescence emission at wavelengths greater than about 650 nm. Typical biosensor molecules include a fluorophore that includes an iminium ion, a linker moiety that includes a group that is an anilinic type of relationship to the fluorophore and a boronate substrate recognition/binding moiety, which binds glucose. The fluorescence molecules modulated by the presence or absence of polyhydroxylated analytes such as glucose. This property of these molecules of the invention, as well as their ability to emit fluorescent light at greater than about 650 nm, renders these biosensor molecules particularly well-suited for detecting and measuring in-vivo glucose concentrations.

  11. A comparative study of three different synthesis routes for hydrophilic fluorophore-doped silica nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Shahabi, Shakiba [University of Bremen, Advanced Ceramics (Germany); Treccani, Laura, E-mail: treccani@petroceramics.com [Petroceramics S.p.A., Kilometro Rosso Science Park (Italy); Rezwan, Kurosch [University of Bremen, Advanced Ceramics (Germany)

    2016-01-15

    The synthesis of fluorophore-doped silica nanoparticles (FDS NPs) with two conventional approaches, Stöber and microemulsion, as well as a novel amino acid-catalyzed seeds regrowth technique (ACSRT) is presented. The efficiency of each applied synthesis route toward incorporation of selected hydrophilic fluorophores, including rhodamine B isothiocyanate and fluorescein isothiocyanate, without and with an amine-containing crosslinker, into silica matrix was systematically studied. Our results clearly highlight the advantages of ACSRT to obtain FDS NPs with a remarkable encapsulation efficiency, high quantum yield, and enhanced stability against bleaching and dye leaking due to efficient embedding of the dyes inside silica network even without the amine-containing silane reagent. Moreover, evaluation of photostability of FDNPs internalized in human bone cells demonstrates the merits of ACSRT.

  12. Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction.

    Science.gov (United States)

    Ochocki, Joshua D; Mullen, Daniel G; Wattenberg, Elizabeth V; Distefano, Mark D

    2011-09-01

    Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.

  13. The use of nanocrystal quantum dot as fluorophore reporters in molecular beacon-based assays

    Science.gov (United States)

    Adegoke, Oluwasesan; Park, Enoch Y.

    2016-12-01

    The utilization of molecular beacon (MB) biosensor probes to detect nucleic acid targets has received enormous interest within the scientific community. This interest has been stimulated by the operational qualities of MB-based probes with respect to their unique sensitivity and specificity. The design of MB biosensors entails not only optimizing the sequence of the loop to hybridize with the nucleic acid target or optimization of the length of the stem to tune the sensitivity but also the selection of the appropriate fluorophore reporter to generate the signal transduction read-out upon hybridization of the probe with the target sequence. Traditional organic fluorescent dyes are mostly used for signal reporting in MB assays but their optical properties in comparison to semiconductor fluorescent quantum dot (Qdot) nanocrystals are at a disadvantage. This review highlights the progress made in exploiting Qdot as fluorophore reporters in MB-based assays with the aim of instigating further development in the field of Qdot-MB technology. The development reported to date indicates that unparalleled fluorescence signal reporting in MB-based assays can be achieved using well-constructed Qdot fluorophores.

  14. Sequence-Specific Biosensing of DNA Target through Relay PCR with Small-Molecule Fluorophore.

    Science.gov (United States)

    Yasmeen, Afshan; Du, Feng; Zhao, Yongyun; Dong, Juan; Chen, Haodong; Huang, Xin; Cui, Xin; Tang, Zhuo

    2016-07-15

    Polymerase chain reaction coupled with signal generation offers sensitive recognition of target DNA sequence; however, these procedures require fluorophore-labeled oligonucleotide probes and high-tech equipment to achieve high specificity. Therefore, intensive research has been conducted to develop reliable, convenient, and economical DNA detection methods. The relay PCR described here is the first sequence-specific detection method using a small-molecule fluorophore as a sensor and combines the classic 5'-3' exonuclease activity of Taq polymerase with an RNA mimic of GFP to build a label-free DNA detection platform. Primarily, Taq polymerase cleaves the 5' noncomplementary overhang of the target specific probe during extension of the leading primer to release a relay oligo to initiate tandem PCR of the reporting template, which encodes the sequence of RNA aptamer. Afterward, the PCR product is transcribed to mRNA, which could generate a fluorescent signal in the presence of corresponding fluorophore. In addition to high sensitivity and specificity, the flexibility of choosing different fluorescent reporting signals makes this method versatile in either single or multiple target detection.

  15. Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E

    2008-03-01

    The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

  16. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

    Science.gov (United States)

    van der Velde, Jasper H. M.; Oelerich, Jens; Huang, Jingyi; Smit, Jochem H.; Aminian Jazi, Atieh; Galiani, Silvia; Kolmakov, Kirill; Guoridis, Giorgos; Eggeling, Christian; Herrmann, Andreas; Roelfes, Gerard; Cordes, Thorben

    2016-01-01

    Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with `self-healing' properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer-dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

  17. Photoluminescence from amino-containing polymer in the presence of CO2: carbamato anion formed as a fluorophore.

    Science.gov (United States)

    Pan, Xiaoyong; Wang, Guan; Lay, Chee Leng; Tan, Beng Hong; He, Chaobin; Liu, Ye

    2013-09-26

    Organic photoluminescent materials are important to many applications especially for diagnosis and detection, and most of organic photoluminescent materials contain fluorophores with extended conjugated structures. Recently some of amino-containing polymers without fluorophores with extended conjugated structure are observed to be photoluminescent, and one possible cause of the photoluminescence is oxidation of the amines. Here we show that photoluminescence can be produced by exposing a typical amino-containing polymer, polyethylenimine, to carbon dioxide. We demonstrate that carbamato anion formed via the reaction between the amine and carbon dioxide is a fluorophore; and the loosely-bound protonated water molecule can increase UV absorption but reduce the photoluminescence emission. Also carbamato anion shows solvent- and excitation wavelength-dependent emission of photoluminescence. The photoluminescence profile of carbamoto anion was discussed. These results will facilitate the understanding of photoluminescence observed from amino-containing materials and the design of new fluorophores.

  18. A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation.

    Science.gov (United States)

    Jeon, Jongho; Lee, Kyung Hyun; Rao, Jianghong

    2012-10-14

    Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

  19. A method for analysis of lipid vesicle domain structure from confocal image data

    DEFF Research Database (Denmark)

    Husen, Peter Rasmussen; Fidorra, Matthias; Hartel, Steffen

    2012-01-01

    Quantitative characterization of the lateral structure of curved membranes based on fluorescence microscopy requires knowledge of the fluorophore distribution on the surface. We present an image analysis approach for extraction of the fluorophore distribution on a spherical lipid vesicle from....... The analysis allows for investigation of the morphology and size distribution of domains on the surface....

  20. Fluorescence signalling of the transition metal ions: Design strategy based on the choice of the fluorophore component

    Indian Academy of Sciences (India)

    N B Sankaran; S Banthia; A Samanta

    2002-12-01

    Transition metal ions are notorious for their fluorescence quenching abilities. In this paper, we discuss the design strategies for the development of efficient off-on fluorescence signalling systems for the transition metal ions. It is shown that even simple fluorophore-spacer-receptor systems can display excellent off-on fluorescence signalling towards the quenching metal ions when the fluorophore component is chosen judiciously.

  1. Growth and Assembly of Gold Nanorods and Their Interactions with Fluorophores and Photochromic Molecules

    Science.gov (United States)

    Ming, Tian

    Noble metal nanocrystals have drawn great attention in a wide range of research fields due to their extraordinary localized surface plasmon resonances, which are essentially collective charge density oscillations confined in metallic nanostructures. Their applications range from bioimaging, sensing and therapy in life sciences to plasmonic circuits and optical data storage in micro-optoelectronics. More attractively, they can be used to enhance light harvesting in solar energy conversion systems. In this thesis, I will systematically describe the preparation and assembly of gold nanorods and their interactions with fluorophores and photochromic molecules, both experimentally and theoretically. I will first introduce my studies on high-index-faceted gold nanocrystals. Elongated tetrahexahedral (THH) gold nanocrystals have been prepared in high yields using a seed-mediated growth method. Structural characterizations reveal that they are single crystals enclosed by 24 high-index {037} facets. Electrochemical measurements have proven that these THH Au nanocrystals are more chemically active than octahedral Au nanocrystals that are enclosed by low-index {1111} facets. Next, I will demonstrate the formation of large-area, 3D ordered assemblies of Au nanostructures that have different sizes and shapes, including nanorods, polyhedra, nanocubes, and bipyramids, by droplet evaporation. The nature of the resultant assemblies is strongly dependent on the shape of Au nanostructures for single-component systems; while the assembly of binary nanorod mixtures is dependent on the relative diameters of two nanorod samples for the nanorods used in our experiments. Most applications of plasmonic nanostructures are based on their interactions with other chemical/physical species. In my research work, gold nanorods interacting with photochromic molecules and fluorophores are extensively studied. For the case of photochromic molecules, I have demonstrated a plasmonic switch on the basis

  2. In vivo determination of optical properties and fluorophore characteristics of non-melanoma skin cancer

    Science.gov (United States)

    Rajaram, Narasimhan; Kovacic, Dianne; Migden, Michael F.; Reichenberg, Jason S.; Nguyen, Tri H.; Tunnell, James W.

    2009-02-01

    Diffuse optical spectroscopy (DOS) and laser-induced fluorescence (LIF) techniques have widely been used as noninvasive tools for early cancer detection in several organs including the cervix, oral cavity and gastrointestinal tract. Using a combined DOS/LIF approach, one can simultaneously measure the morphology and biochemical composition of tissue and use these features to diagnose malignancy. We report for the first time to our knowledge both the optical properties and native fluorophore characteristics of non-melanoma skin cancer in the UV-visible range. We collected in vivo diffuse reflectance and intrinsic fluorescence measurements from 44 skin lesions on 37 patients. The skin sites were further categorized into three groups of non-melanoma skin cancer according to histopathology: 1) pre-cancerous actinic keratosis 2) malignant squamous cell carcinoma (SCC) and 3) basal cell carcinoma (BCC). We used a custom-built probe-based clinical system that collects both white light reflectance and laser-induced fluorescence in the wavelength range of 350-700 nm. We extracted the blood volume fraction, oxygen saturation, blood vessel size, tissue microarchitecture and melanin content from diffuse reflectance measurements. In addition, we determined the native fluorophore contributions of NADH, collagen and FAD from laser-induced fluorescence for all groups. The scattering from tissue decreased with progression from clinically normal to precancerous actinic keratosis to malignant SCC. A similar trend was observed for clinically normal skin and malignant BCC. Statistically significant differences were observed in the collagen contributions, which were lower in malignant SCC and BCC as compared to normal skin. Our data demonstrates that the mean optical properties and fluorophore contributions of normal, benign and malignant nonmelanoma cancers are significantly different from each other and can potentially be used as biomarkers for the early detection of skin cancer.

  3. High-pressure studies on the calcium-ion-sensitive fluorophore Fluo-4

    Science.gov (United States)

    Frey, Eric W.; Urayama, Paul

    2007-10-01

    Fluorescence-based methods for intracellular calcium ion sensing are well established at ambient pressure. Because calcium ions play a ubiquitous role in cellular signaling, extending techniques of intracellular calcium-sensing to high pressures would play an important role in understanding the large variety of piezophysiologic effects. Here, we characterize the intracellular calcium-ion-sensitive fluorophore Fluo-4 under hydrostatic pressures up to 500 atm (50 MPa). Using an EGTA/MOPS solution as a calcium-buffer reference, we investigate the pressure dependence of the reaction pK and determine the thermodynamic volume change associated with the Fluo-4 calcium-binding reaction.

  4. Spectrofluorometric Determination of Putrescine Using Orthophthaldehyde as a Fluorophore: A Novel Method.

    Science.gov (United States)

    Oyelakin, Oladele; Traoré, Moumouny; Mbaye, El Hadji Babacar; Konté, Abdourahmane; Cissé, Lamine; Faye, Abdoulaye N; Faye, Ousman; Mbaye, Moussa; Kital, Khemesse; Seye, Gaye; Coly, Atanasse; Tine, Alphonse; Delattre, François

    2016-01-01

    This paper concerns spectrofluorometric analysis of putrescine using orthophthaladehyde as a fluorophore in aqueous alkaline medium. Wavelengths of excitation and emission in acid, neutral, and alkaline media were different. There is a maximum intensity of fluorescence in alkaline medium 24 h after complexation compared with other media. Putrescine and orthophthaladehyde are used at an equimolar ratio, and the product is kinetically stable in alkaline medium. Calibration curves obtained gave limits of detection and quantification of 39 and 65 ng/mL, respectively. The correlation coefficient obtained in alkaline medium was 0.992 at pH 12. Results obtained largely showed a good reproducibility of our method.

  5. Efficient electroluminescence from a perylenediimide fluorophore obtained from a simple solution processed OLED

    Energy Technology Data Exchange (ETDEWEB)

    Cespedes-Guirao, F J; Fernandez-Lazaro, F; Sastre-Santos, A [Division de Quimica Organica, Instituto de Bioingenieria, Universidad Miguel Hernandez, Avda. de la Universidad, s/n, Elche 03202 (Spain); Garcia-Santamaria, S; Bolink, H J [Instituto de Ciencia Molecular, Universidad de Valencia, PO Box 22085, ES-46071 Valencia (Spain)], E-mail: henk.bolink@uv.es, E-mail: asastre@umh.es, E-mail: fdofdez@umh.es

    2009-05-21

    Simple solution processed organic light emitting diodes are used to screen the performance of two types of highly efficient, narrow band red emitting fluorescent perylenediimides (PDIs). PDIs substituted at the diimide positions seem to form aggregates in the thin film architecture as evidenced by the shifted electroluminescent spectrum. When substituted on the bay position and when used both as the emitting and the electron transporting specie, bright electroluminescence with a narrow width around 610 nm reaching 500 cd m{sup -2} at moderate voltages was observed, demonstrating the usefulness of these fluorophores for OLED applications.

  6. Rational Design of in Vivo Tau Tangle-Selective Near-Infrared Fluorophores: Expanding the BODIPY Universe.

    Science.gov (United States)

    Verwilst, Peter; Kim, Hye-Ri; Seo, Jinho; Sohn, Nak-Won; Cha, Seung-Yun; Kim, Yeongmin; Maeng, Sungho; Shin, Jung-Won; Kwak, Jong Hwan; Kang, Chulhun; Kim, Jong Seung

    2017-09-15

    The elucidation of the cause of Alzheimer's disease remains one of the greatest questions in neurodegenerative research. The lack of highly reliable low-cost sensors to study the structural changes in key proteins during the progression of the disease is a contributing factor to this lack of insight. In the current work, we describe the rational design and synthesis of two fluorescent BODIPY-based probes, named Tau 1 and Tau 2. The probes were evaluated on the molecular surface formed by a fibril of the PHF6 ((306)VQIVYK(311)) tau fragment using molecular docking studies to provide a potential molecular model to rationalize the selectivity of the new probes as compared to a homologous Aβ-selective probe. The probes were synthesized in a few steps from commercially available starting products and could thus prove to be highly cost-effective. We demonstrated the excellent photophysical properties of the dyes, such as a large Stokes shift and emission in the near-infrared window of the electromagnetic spectrum. The probes demonstrated a high selectivity for self-assembled microtubule-associated protein tau (Tau protein), in both solution and cell-based experiments. Moreover, the administration to an acute murine model of tauopathy clearly revealed the staining of self-assembled hyperphosphorylated tau protein in pathologically relevant hippocampal brain regions. Tau 1 demonstrated efficient blood-brain barrier penetrability and demonstrated a clear selectivity for tau tangles over Aβ plaques, as well as the capacity for in vivo imaging in a transgenic mouse model. The current work could open up avenues for the cost-effective monitoring of the tau protein aggregation state in animal models as well as tissue staining. Furthermore, these fluorophores could serve as the basis for the development of clinically relevant sensors, for example based on PET imaging.

  7. Single-fluorophore membrane transport activity sensors with dual-emission read-out.

    Science.gov (United States)

    Ast, Cindy; De Michele, Roberto; Kumke, Michael U; Frommer, Wolf B

    2015-06-19

    We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer is important for the successful generation of single-fluorophore-based dual-emission sensors.

  8. Characterization of fluorescent-dissolved organic matter and identification of specific fluorophores in textile effluents.

    Science.gov (United States)

    Li, Wentao; Xu, Zixiao; Wu, Qian; Li, Yan; Shuang, Chendong; Li, Aimin

    2015-03-01

    This study focused on the characterization of fluorescent-dissolved organic matter and identification of specific fluorophores in textile effluents. Samples from different textile wastewater treatment plants were characterized by high-performance liquid chromatography and size exclusion chromatography as well as fluorescence excitation-emission matrix spectra. Despite the highly heterogeneous textile effluents, the fluorescent components and their physicochemical properties were found relatively invariable, which is beneficial for the combination of biological and physicochemical treatment processes. The humic-like substance with triple-excitation peaks (excitation (Ex) 250, 310, 365/emission (Em) 460 nm) presented as the specific fluorescence indicator in textile effluents. It was also the major contributor to UV absorbance at 254 nm and resulted in the brown color of biologically treated textile effluents. By spectral comparison, the specific fluorophore in textile effluents could be attributed to the intermediate structure of azo dyes 1-amino-2-naphthol, which was transferred into the special humic-like substances during biological treatment.

  9. Nitrogen doped carbon dots derived from Sargassum fluitans as fluorophore for DNA detection.

    Science.gov (United States)

    Godavarthi, S; Mohan Kumar, K; Vázquez Vélez, E; Hernandez-Eligio, A; Mahendhiran, M; Hernandez-Como, N; Aleman, M; Martinez Gomez, L

    2017-07-01

    This work focused on the use of waste seaweed Sargassum fluitans (S. fluitans) as carbon source precursor to prepare nitrogen doped carbon dots (NCDs) by hydrothermal method. High resolution transmission electron microscopic (HR-TEM) studies revealed that the synthesized water soluble NCDs are in the size range of 2-8nm and exhibits excellent fluorescent properties with a quantum yield of 18.2%. Elemental nitrogen in NCDs was evidenced by X-ray photoelectron spectroscopy (XPS) and Fourier transformed infrared spectrum (FT-IR). The phytochemical analysis of S. fluitans using (1)H NMR and (13)C NMR revealed the presence of few amino acids which act as nitrogen source in the preparation of NCDs. Application of NCDs as fluorophore for double stranded DNA, single stranded DNA and RNA detection was highlighted in this study. Excellent fluorescent tagging abilities of NCDs with the biological nucleic acids were evidenced using gel electrophoresis. Significant increase in fluorescence was observed upon tagging of NCDs with nucleic acids and this particular phenomenon helps better in visualizing the nucleic acids. All three nucleic acids i.e. double stranded DNA, single stranded DNA and RNA showed similar phenomenon upon tagging with NCDs. Thus synthesized NCDs may be used as an alternate fluorophore for commercial toxic organic staining agents to visualize nucleic acids. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Dual view FIDA measurements on MAST

    CERN Document Server

    Michael, C A; Crowley, B; Jones, O; Heidbrink, W W; Pinches, S; Braeken, E; Akers, R; Challis, C; Turnyanskiy, M; Patel, A; Muir, D; Gaffka, R; Bailey, S

    2013-01-01

    A Fast Ion Deuterium Alpha (FIDA) spectrometer was installed on MAST to measure radially resolved information about the fast ion density and its distribution in energy and pitch angle. Toroidally and vertically-directed collection lenses are employed, to detect both passing and trapped particle dynamics, and reference views are installed to subtract the background. This background is found to contain a substantial amount of passive FIDA emission driven by edge neutrals, and to depend delicately on viewing geometry. Results are compared with theoretical expectations based on the codes NUBEAM (for fast ion distributions) and FIDASIM. Calibrating via the measured beam emission peaks, the toroidal FIDA signal profile agrees with classical simulations in MHD quiescent discharges where the neutron rate is also classical. Long-lived modes (LLM) and chirping modes decrease the core FIDA signal significantly, and the profile can be matched closely to simulations using anomalous diffusive transport; a spatially uniform...

  11. Environment-sensitive fluorophores with benzothiadiazole and benzoselenadiazole structures as candidate components of a fluorescent polymeric thermometer.

    Science.gov (United States)

    Uchiyama, Seiichi; Kimura, Kohki; Gota, Chie; Okabe, Kohki; Kawamoto, Kyoko; Inada, Noriko; Yoshihara, Toshitada; Tobita, Seiji

    2012-07-27

    An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.

  12. How nanoparticles encapsulating fluorophores allow a double detection of biomolecules by localized surface plasmon resonance and luminescence

    Energy Technology Data Exchange (ETDEWEB)

    Barbillon, G; Faure, A C; Kork, N El; Moretti, P [Universite de Lyon, Universite Lyon 1, CNRS UMR 5620, Laboratoire de Physico-Chimie des Materiaux Luminescents (LPCML), Domaine Scientifique de La Doua, Bat Kastler, 10 rue Andre Marie Ampere 69622 Villeurbanne Cedex (France); Roux, S; Tillement, O [Universite de Lyon, Universite Lyon 1, CNRS UMR 5620, Laboratoire de Physico-Chimie des Materiaux Luminescents (LPCML), Domaine Scientifique de La Doua, Bat Kastler, 10 rue Andre Marie Ampere 69622 Villeurbanne Cedex (France); Ou, M G; Descamps, A; Perriat, P [Materiaux, Ingenierie et Sciences (MATEIS), CNRS UMR 5510, Universite de Lyon, INSA-Lyon, Domaine Scientifique de La Doua, 7 avenue Jean Capelle 69621 Villeurbanne Cedex (France); Vial, A; Bijeon, J-L [Institut Charles Delaunay, CNRS FRE 2848, Laboratoire de Nanotechnologie et d' Instrumentation Optique (LNIO), Universite de Technologie de Troyes, 12 rue Marie Curie BP 2060 10010 Troyes Cedex (France); Marquette, C A [Laboratoire de Genie Enzymatique et Biomoleculaire, UMR 5246 CNRS-ICBMS, Universite de Lyon, Universite Lyon 1, 69622 Villeurbanne Cedex (France); Jacquier, B [Universite de Lyon, Universite Lyon 1, CNRS UMR 5620, Laboratoire de Physico-Chimie des Materiaux Luminescents (LPCML), Domaine Scientifique de La Doua, Bat Kastler, 10 rue Andre Marie Ampere 69622 Villeurbanne Cedex (France)

    2008-01-23

    The paper shows how polysiloxane particles encapsulating fluorophores can be successfully used to detect biotin-streptavidin binding by two types of technique. After functionalization of the particles by streptavidin, the fixation of the biomolecule can indeed be detected by a shift of the localized surface plasmon resonance of the biotinylated gold dots used as substrate and by the luminescence of the fluorophores evidenced by scanning near-field optical microscopy. The development of particles allowing such a double detection opens a route for increasing the reliability of biological detection and for multi-labelling strategies crossing both detection principles.

  13. Chromatographic isolation and mass spectrometric identification of a base-induced mebendazole fluorophor.

    Science.gov (United States)

    Baeyens, W; Abdel Fattah, F; De Moerloose, P

    1986-10-01

    Sodium hydroxide treatment of the benzoylbenzimidazole anthelmintics mebendazole and flubendazole produces yellow solutions that possess practically no fluorescence characteristics under various conditions. However, when spotting the alkaline solutions on filter paper and examining the spots under U.V., strong bluish-white fluorescence is obtained. When pouring liquid nitrogen over the spots, a very intense bluish-white fluorescence followed by a long-lasting greenish phosphorescence is observed. These luminescence phenomena allow visualization of 1 ng of mebendazole and of 5 ng of flubendazole per spot. A preparative separation by means of column liquid chromatography was worked out for the isolation of the fluorophor in case of mebendazole. Combined spectroscopic methods indicated the formation of a primary amine function in position 2 of the imidazole nucleus by hydrolytic cleavage of the -NH-CO-bond. A discussion on the mechanism of fluorescence is given.

  14. Switchable 2,3-dithienylmaleimide bonded to different fluorophores:synthesis and photochromic properties

    Institute of Scientific and Technical Information of China (English)

    Lei XU; Zu-jin ZHAO; Ya-jun XING; Ping LU

    2008-01-01

    A series of photochromic diarylethene derivatives containing different fluorophores was synthesized by the Sonogashira coupling reaction and characterized by 1H nuclear magnetic resonance(NMR),13C NMR and mass spectroscopy.Photochromic conversion of Cz-MS was observed and its structural change was confirmed by proton NMR.Switchable absorption and emission phenomena were observed for these molecules.These phenomena were attributed to the reversible photochromic fluorescence resonance energy transfer(pcFRET)process.The design of this novel photochromic system of Py-4MS enables information to be processed in a non-destructive manner.This overcomes the problem of the destructive nature of tracking photochromism using ultraviolet(UV)-vis spectroscopy.

  15. Dependence of fluorescence intensity on the spectral overlap between fluorophores and plasmon resonant single silver nanoparticles.

    Science.gov (United States)

    Chen, Yeechi; Munechika, Keiko; Ginger, David S

    2007-03-01

    We investigate the fluorescence from dyes coupled to individual DNA-functionalized metal nanoparticles. We use single-particle darkfield scattering and fluorescence microscopy to correlate the fluorescence intensity of the dyes with the localized surface plasmon resonance (LSPR) spectra of the individual metal nanoparticles to which they are attached. For each of three different dyes, we observe a strong correlation between the fluorescence intensity of the dye and the degree of spectral overlap with the plasmon resonance of the nanoparticle. On average, we observe the brightest fluorescence from dyes attached to metal nanoparticles that have a LSPR scattering peak approximately 40-120 meV higher in energy than the emission peak of the fluorophore. These results should prove useful for understanding and optimizing metal-enhanced fluorescence.

  16. Laser induced fluorescence spectroscopy of chemo-drugs as biocompatible fluorophores: irinotecan, gemcitabine and navelbine

    Science.gov (United States)

    Motlagh, N. S. Hosseini; Parvin, P.; Ghasemi, F.; Atyabi, F.; Jelvani, S.; Abolhosseini, S.

    2016-07-01

    The fluorescence nature of chemo-drugs is useful for simultaneous cancer diagnosis and therapy. Here, the laser induced fluorescence (LIF) properties of irinotecan, gemcitabine and navelbine are extensively investigated. The UV photons provoke the desired transitions of the several chemo-drugs by virtue of the XeCl laser at 308 nm. It is shown that LIF spectra are strongly dependent on the fluorophore concentration, while no spectral shift is measured for irinotecan, gemcitabine and navelbine because of a large Stokes shift. On the other hand, doxorubicin is characterized by a large overlapping between absorption and emission spectra giving rise to a sensible red shift. The fluorescence extinction α and self-quenching k coefficients as well as the quantum yield η f of those chemo-drugs are determined accordingly. In fact, irinotecan shows the highest quantum efficiency among the chemo-drugs of interest.

  17. NLOphoric studies in thiazole containing symmetrical push-pull fluorophores - Combined experimental and DFT approach

    Science.gov (United States)

    Telore, Rahul D.; Satam, Manjaree A.; Sekar, Nagaiyan

    2015-10-01

    A series of donor-π-acceptor extended styryl chromophores having carbazole as electron donor with thiazole bridge and cyano group as the electron acceptors are investigated for nonlinear optical properties. The geometries of the extended styryls were optimized and electronic excitation properties were estimated using TD-DFT. The experimental solvatochromic shifts and density functional theory (DFT) computations are employed to understand the nonlinear optical properties of new generation push-pull fluorophores containing carbazole and thiazole cores. The solvatochromic polarizability values are calculated by two-level quantum mechanical model. Density Functional Theory is explored to determine the liner hyperpolarizability (α0), static first hyperpolarizability (β0) and second order hyperpolarizability (γ) for extended styryls. The calculation carried out by B3LYP method and 6-31G (d) basis set at ground state and excited state in different solvent polarity. The thiazole bridge in extended styryl enhances the nonlinear optical properties.

  18. Advancing with pyrrolopyrrole cyanines: a next generation class of near-infrared fluorophores

    Science.gov (United States)

    Wiktorowski, S.; Fischer, G. M.; Daltrozzo, E.; Zumbusch, A.

    2012-03-01

    Fluorescent dyes are the basis for a broad range of modern techniques in life and material sciences. Consequently, there is a pressing need for the development of new classes of NIR fluorophores in recent years. Pyrrolopyrrole Cyanines (PPCys) are a novel class of NIR chromophores that were first presented in 2007 by Fischer and coworkers.[1] Their optical properties are marked by strong and narrowband NIR absorptions, strong NIR fluorescence and hardly any absorption in the visible range. The absorption maxima can be tuned over a broad range while high fluorescence quantum yields are maintained. PPCys are attractive candidates for labelling applications or as selective NIR absorbers. Moreover, PPCys exhibit very high photostability. Due to these outstanding photophysical properties, PPCys are heading into a promising future as NIR dyes.

  19. Preliminary evaluation of endogenous milk fluorophores as tracer molecules for curd syneresis.

    Science.gov (United States)

    Fagan, C C; Ferreira, T G; Payne, F A; O'Donnell, C P; O'Callaghan, D J; Castillo, M

    2011-11-01

    A front-face fluorescence spectroscopy probe was installed in the wall of a laboratory-scale cheese vat. Excitation and emission filters were chosen for the selective detection of vitamin A, tryptophan, and riboflavin fluorescence. The evolution of the fluorescence of each fluorophore during milk coagulation and syneresis was monitored to determine if they had the potential to act as intrinsic tracers of syneresis and also coagulation. The fluorescence profiles for 2 of the fluorophores during coagulation could be divided into 3 sections relating to enzymatic hydrolysis of κ-casein, aggregation of casein micelles, and crosslinking. A parameter relating to coagulation kinetics was derived from the tryptophan and riboflavin profiles but this was not possible for the vitamin A response. The study also indicated that tryptophan and riboflavin may act as tracer molecules for syneresis, but this was not shown for vitamin A. The evolution of tryptophan and riboflavin fluorescence during syneresis followed a first-order reaction and had strong relationships with curd moisture and whey total solids content (r=0.86-0.96). Simple 1- and 2-parameter models were developed to predict curd moisture content, curd yield, and whey total solids using parameters derived from the sensor profiles (standard error of prediction=0.0005-0.394%; R(2)=0.963-0.999). The results of this study highlight the potential of tryptophan and riboflavin to act as intrinsic tracer molecules for noninvasive inline monitoring of milk coagulation and curd syneresis. Further work is required to validate these findings under a wider range of processing conditions.

  20. Semiconductor Nanocrystals for Biological Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Aihua; Gu, Weiwei; Larabell, Carolyn; Alivisatos, A. Paul

    2005-06-28

    Conventional organic fluorophores suffer from poor photo stability, narrow absorption spectra and broad emission feature. Semiconductor nanocrystals, on the other hand, are highly photo-stable with broad absorption spectra and narrow size-tunable emission spectra. Recent advances in the synthesis of these materials have resulted in bright, sensitive, extremely photo-stable and biocompatible semiconductor fluorophores. Commercial availability facilitates their application in a variety of unprecedented biological experiments, including multiplexed cellular imaging, long-term in vitro and in vivo labeling, deep tissue structure mapping and single particle investigation of dynamic cellular processes. Semiconductor nanocrystals are one of the first examples of nanotechnology enabling a new class of biomedical applications.

  1. Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

    DEFF Research Database (Denmark)

    Jensen, Pia Wrensted; Falconi, Mattia; Kristoffersen, Emil Laust

    2013-01-01

    such as biopsies. TDP1 removes covalent 3′DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5′fluorophore and a 3′quencher brought in close proximity by the secondary...

  2. Integration of organic fluorophores in the surface of polymer-coated colloidal nanoparticles for sensing the local polarity of the environment.

    Science.gov (United States)

    Amin, Faheem; Yushchenko, Dmytro A; Montenegro, Jose M; Parak, Wolfgang J

    2012-03-01

    Inorganic hydrophobically capped NPs such as quantum dots, superparamagnetic iron oxide, or gold nanoparticles can be modified to make them water-soluble by their embedding in an amphiphilic polymer shell. This polymer shell can be prefunctionalized by the integration of organic fluorophores, which allows the observation of the nanoparticles with fluorescence based techniques. The fluorophore could be either located more in the hydrophobic part of the inner polymer shell, or on the hydrophilic surface pointing towards solution. Herein we prepared gold nanoparticles decorated with the organic fluorophore FE, 4'-N,N-diethylamino-3-hydroxyflavone (FE), which possesses fluorescence sensitive to the polarity and hydrogen-bonding properties of the surrounding local environment. Based on the response of FE in the polymer shell to isopropanol, and CTAB compared to the response of free FE we conclude that the FE fluorophore is situated within the inner polymer shell. Nevertheless the fluorophore in the polymer shell can still sense polarity changes in solution.

  3. 3-dimensional imaging at nanometer resolutions

    Science.gov (United States)

    Werner, James H.; Goodwin, Peter M.; Shreve, Andrew P.

    2010-03-09

    An apparatus and method for enabling precise, 3-dimensional, photoactivation localization microscopy (PALM) using selective, two-photon activation of fluorophores in a single z-slice of a sample in cooperation with time-gated imaging for reducing the background radiation from other image planes to levels suitable for single-molecule detection and spatial location, are described.

  4. Photoacoustic imaging of a near-infrared fluorescent marker based on dual wavelength pump-probe excitation

    Science.gov (United States)

    Märk, Julia; Theiss, Christoph; Schmitt, Franz-Josef; Laufer, Jan

    2014-03-01

    Photoacoustic imaging has been used to determine the spatial distribution of fluorophores, such as exogenous dyes and genetically expressed proteins, from images acquired in phantoms and in vivo. Most methods involve the acquisition of multiwavelength images and rely on differences in the absorption spectra of the tissue chromophores to estimate the spatial distribution and abundance of the latter using spectral decomposition techniques, such as model based inversion schemes. However, the inversion of 3-D images can be computationally expensive. Experimental approaches to localising contrast agents may therefore be useful, especially if quantification is not essential. This work aims to develop a method for determining the spatial distribution of a near-infrared fluorescent cell marker from images acquired using dual wavelength excitation. The excitation wavelengths coincided with the absorption and emission spectrum of the fluorophore. The contrast mechanism relies on reducing the excited state lifetime of the fluorophore by inducing stimulated emission. This changes the amount of energy thermalized by the fluorophore, and hence the photoacoustic signal amplitude. Since this is not observed in endogenous chromophores, the background may be removed by subtracting two images acquired with and without pulse delay between the pump and probe pulses. To characterise the fluorophore, the signal amplitude is measured in a cuvette as a function of pulse delay, concentration, and fluence. The spatial distribution of the fluorophore is determined from images acquired in realistic tissue phantoms. This method may be suitable for in vivo applications, such as imaging of exogenous or genetically expressed fluorescent cell markers.

  5. Excitation energy transfer from a fluorophore to single-walled carbon nanotubes.

    Science.gov (United States)

    Swathi, R S; Sebastian, K L

    2010-03-14

    We study the process of electronic excitation energy transfer from a fluorophore to the electronic energy levels of a single-walled carbon nanotube. The matrix element for the energy transfer involves the Coulombic interaction between the transition densities on the donor and the acceptor. In the Forster approach, this is approximated as the interaction between the corresponding transition dipoles. For energy transfer from a dye to a nanotube, one can use the dipole approximation for the dye, but not for the nanotube. We have therefore calculated the rate using an approach that avoids the dipole approximation for the nanotube. We find that for the metallic nanotubes, the rate has an exponential dependence if the energy that is to be transferred, variant Planck's over 2piOmega is less than a threshold and a d(-5) dependence otherwise. The threshold is the minimum energy required for a transition other than the k(i, perpendicular)=0 and l=0 transition. Our numerical evaluation of the rate of energy transfer from the dye pyrene to a (5,5) carbon nanotube, which is metallic leads to a distance of approximately 165 A up to which energy transfer is appreciable. For the case of transfer to semiconducting carbon nanotubes, apart from the process of transfer to the electronic energy levels within the one electron picture, we also consider the possibility of energy transfer to the lowest possible excitonic state. Transfer to semiconducting carbon nanotubes is possible only if variant Planck's over 2piOmega > or = epsilon(g)-epsilon(b). The long range behavior of the rate of transfer has been found to have a d(-5) dependence if variant Planck's over 2piOmega > or = epsilon(g). But, when the emission energy of the fluorophore is in the range epsilon(g) > variant Planck's over 2piOmega > or = epsilon(g)-epsilon(b), the rate has an exponential dependence on the distance. For the case of transfer from pyrene to the semiconducting (6,4) carbon nanotube, energy transfer is found to

  6. Fluorescence Enhancement of Fluorophores Tethered to Different Sized Silver Colloids Deposited on Glass Substrate

    Science.gov (United States)

    Lukomska, Joanna; Malicka, Joanna; Gryczynski, Ignacy; Leonenko, Zoya; Lakowicz, Joseph R.

    2009-01-01

    We studied fluorescence enhancements of fluorescein tethered to silver colloids of different size. Thiolated 23-mer oligonucleotide (ss DNA-SH) was bound selectively to silver colloids deposited on 3-aminopropyltriethoxysilane (APS)-treated quartz slides. Fluoresceinlabeled complementary oligonucleotide (ss Fl-DNA) was added in an amount significantly lower than the amount of unlabeled DNA tethered to the colloids. The hybridization kinetics, observed as an increase in fluorescence emission, on small (30-40 nm) and large (> 120 nm) colloids were similar. However, the final fluorescence intensity of the sample with large colloids was about 50% higher than that observed for the sample with small colloids. The reference sample without ss DNA-SH was used to estimate the fluorescence enhancements of fluorescein tethered to the small colloids (E = 2.7) and to the large colloids (E = 4.1) due to its steady fluorescence signal. The proposed method, based on controlled hybridization with minimal amount of fluorophore labeled ss DNA, can be used to reliably estimate the fluorescence enhancements on any silver nanostructures. PMID:15578680

  7. Hyperfine interaction mechanism of magnetic field effects in sequential fluorophore and exciplex fluorescence

    Science.gov (United States)

    Dodin, Dmitry V.; Ivanov, Anatoly I.; Burshtein, Anatoly I.

    2013-03-01

    The magnetic field effect on the fluorescence of the photoexcited electron acceptor, 1A*, and the exciplex, 1[D+δA-δ] formed at contact of 1A* with an electron donor 1D, is theoretically explored in the framework of Integral Encounter Theory. It is assumed that the excited fluorophore is equilibrated with the exciplex that reversibly dissociates into the radical-ion pair. The magnetic field sensitive stage is the spin conversion in the resulting geminate radical-ion pair, 1, 3[D+…A-] that proceeds due to hyperfine interaction. We confirm our earlier conclusion (obtained with a rate description of spin conversion) that in the model with a single nucleus spin 1/2 the magnitude of the Magnetic Field Effect (MFE) also vanishes in the opposite limits of low and high dielectric permittivity of the solvent. Moreover, it is shown that MFE being positive at small hyperfine interaction A, first increases with A but approaching the maximum starts to decrease and even changes the sign.

  8. Incorporation of fluorophore-cholesterol conjugates into liposomal and mycobacterial membranes.

    Science.gov (United States)

    Wercholuk, Ashley N; Thuman, Jenna M; Stanley, Jordan L; Sargent, Andrew L; Anderson, Eric S; Allen, William E

    2016-03-01

    Fluorescently-labeled steroids that emit intense blue light in nonpolar solvent (λem (CH2Cl2)≈440nm, ΦF=0.70) were prepared by treating cholesteryl chloroformate with 4-amino-1,8-naphthalimides. The lipid portion of the conjugates embeds into liposomal membrane bilayers in minutes, leaving the fluorophore exposed to the external aqueous environment. This causes a 40-nm red-shift in λem and significant quenching. DFT optimizations predict the conjugates to be about 30Å long when fully extended, but rotation about the linker group can bring the compounds into an 'L'-shape. Such a conformation would allow the cholesteryl anchor to remain parallel to the acyl chains of a membrane while the fluorescent group resides in the interfacial region, instead of extending beyond it. When incubated with Mycobacterium smegmatis mc2 155, a bacterial species known to use natural cholesterol, the labeled steroids support growth and can be found localized in the membrane fraction of the cells using HPLC. These findings demonstrate stable integration of fluorescent cholesterols into bacterial membranes in vivo, indicating that these compounds may be useful for evaluating cholesterol uptake in prokaryotic organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Single-image measurements of monochromatic subdiffraction dimolecular separations

    CERN Document Server

    DeCenzo, Shawn H; Wang, Y M

    2010-01-01

    Measuring subdiffraction separations between single fluorescent particles is important for biological, nano-, and medical-technology studies. Major challenges include (i) measuring changing molecular separations with high temporal resolution while (ii) using identical fluorescent labels. Here we report a method that measures subdiffraction separations between two identical fluorophores by using a single image of milliseconds exposure time and a standard single-molecule fluorescent imaging setup. The fluorophores do not need to be bleached and the separations can be measured down to 40 nm with nanometer precision. The method is called single-molecule image deconvolution -- SMID, and in this article it measures the standard deviation (SD) of Gaussian-approximated combined fluorescent intensity profiles of the two subdiffraction-separated fluorophores. This study enables measurements of (i) subdiffraction dimolecular separations using a single image, lifting the temporal resolution of seconds to milliseconds, wh...

  10. Multiple analyte response and molecular logic operations by excited-state charge-transfer modulation in a bipyridine integrated fluorophore.

    Science.gov (United States)

    Sreejith, Sivaramapanicker; Divya, Kizhumuri P; Manojkumar, T K; Ajayaghosh, Ayyappanpillai

    2011-02-01

    The tunable excited-state properties of a new donor-π-acceptor-π-donor-type fluorophore 1 with a bipyridyl moiety and its ability to respond to different analytes in solution and on paper microchannels are described. Furthermore, the multiple analyte response of fluorophore 1 has been exploited to perform multiple logic operations. Molecule 1, by virtue of its excited-state charge transfer, exhibits solvatochromism and reversible modulation of its emission in response to multiple chemical inputs, thus resulting in different fluorescent signals. The intraligand charge-transfer (ILCT) emission of 1 at 574 nm has been modulated to three emission outputs by using different chemical inputs, such as Zn(2+), H(+), and ethylenediaminetetraacetic acid (EDTA). Thus, different logic operations such as AND, 2-input-INH, 3-input-INH, IMP, and a combination of these logic operations could be achieved.

  11. Images

    Data.gov (United States)

    National Aeronautics and Space Administration — Images for the website main pages and all configurations. The upload and access points for the other images are: Website Template RSW images BSCW Images HIRENASD...

  12. Sub-diffraction imaging on standard microscopes through photobleaching microscopy with non-linear processing.

    Science.gov (United States)

    Munck, Sebastian; Miskiewicz, Katarzyna; Sannerud, Ragna; Menchon, Silvia A; Jose, Liya; Heintzmann, Rainer; Verstreken, Patrik; Annaert, Wim

    2012-05-01

    Visualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.

  13. MagiProbe: a novel fluorescence quenching-based oligonucleotide probe carrying a fluorophore and an intercalator

    OpenAIRE

    Yamane, Akio

    2002-01-01

    Fluorescence is the favored signaling technology for molecular diagnoses. Fluorescence energy transfer-based methods are powerful homogeneous assay tools. A novel oligonucleotide probe, named MagiProbe, which is simple to use, is described, and information given about the duplex formed with a target. The probe internally has a fluorophore and an intercalator. Its fluorescence is quenched by the intercalator in the absence of a target sequence. On hybridization with a target sequence, the prob...

  14. Synthesis and spectroscopic characterization of Y-shaped fluorophores with an imidazole core containing crown ether moieties

    Energy Technology Data Exchange (ETDEWEB)

    Doğru, Ümit; Öztürk Ürüt, Gülsiye, E-mail: gulsiye.ozturk@deu.edu.tr; Bayramin, Dilek

    2015-07-15

    In this study three new Y-shaped fluorophores, 4,5-(2,2'-diphenyl)vinyl-{2-[(1,4,7,10-tetraoxa-13-azacyclopentadecyl) phenyl]}-1H-imidazole (1a), 4,5-{[2,2'-bis(4-methoxyphenyl)vinyl]-[2-(1,4,7,10-tetraoxa-13- azacyclopentadecyl)-phenyl]}-1H-imidazole (1b) and 4,5-(2,2'-diphenyl)vinyl-{2-(1,4,7,10,13-benzopentaoxacyclopentadecyl)} -1H-imidazole (1c) were synthesized. 1,6-Diphenylhexa-1,5-diene-3,4-dione (2a) and 1,6-bis(4-methoxyphenyl)hexa-1,5-diene-3,4-dione (2b) were synthesized as preliminary fluorophores and then reacted with 4-formylbenzo-aza-15-crown-5 (3a) and 4-formylbenzo-15-crown-5 (3b) to obtain the three Y-shaped fluorophores 1a, 1b and 1c. 4-formylbenzo-aza-15-crown-5 and 4-formylbenzo-15-crown-5 intermediates were synthesized with Vilsmeier–Haack reaction. The photophysical properties such as maximum absorption wavelengths, maximum emission wavelengths, Stokes' shifts, singlet energies, fluorescence quantum yields and photostabilities of the compounds were investigated by measuring absorption and emission spectra in a series of solvents of varying polarities of toluene (TOL), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), acetonitrile (ACN), and N,N-dimethylformamide (DMF). The three compounds 1a, 1b and 1c exhibited emission maxima in the 412–677 nm range. All the derivatives synthesized exhibited excellent photostability in all the solvents tested. - Highlights: • Three new Y-shaped fluorophores were synthesized for the first time. • Their absorption and emission properties were investigated. • All the derivatives synthesized exhibited excellent photostability.

  15. Energy transfer from silica core-surfactant shell nanoparticles to hosted molecular fluorophores.

    Science.gov (United States)

    Rampazzo, Enrico; Bonacchi, Sara; Juris, Riccardo; Montalti, Marco; Genovese, Damiano; Zaccheroni, Nelsi; Prodi, Luca; Rambaldi, Diana Cristina; Zattoni, Andrea; Reschiglian, Pierluigi

    2010-11-18

    Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with a rhodamine B derivative were prepared using micelles of F127 as nanoreactors for the hydrolysis and condensation of the silica precursor tetraethoxysilane (TEOS). The functionalization of the rhodamines with a triethoxysilane group allowed the covalent binding of the fluorophores to the silica core: no leaking of the dye was observed when the NPs were purified either by ultrafiltration (UF) or dialysis. The diameter of the core (d(c) = 10 ± 1 nm) was determined by TEM and subtracted from the hydrodynamic diameter, measured by DLS, (d(H) = 24 nm, PdI = 0.1) to calculate the shell thickness (∼7 nm). The presence of a single population of NPs with a radius compatible with the one measured by DLS after UF was confirmed by AF4-MALS-RI measurements. The concentration of the NPs was measured by MALS-RI. This allowed us to determine the average number of rhodamine molecules per NP (10). The ability of the NPs to host hydrophobic species as cyanines in the SS was confirmed by fluorescence anisotropy measurements. Steady-state and time-resolved fluorescence measurements allowed us to observe the occurrence of a very efficient Förster resonance energy transfer process from the covalently linked rhodamines to the hosted cyanines. In particular, the analysis of the TCSPC data and steady-state measurements revealed that the adsorption of a single cyanine molecule causes an almost complete quenching of the fluorescence of the NP. Thanks to these observations, it was possible to easily determine the concentration of the NPs by fluorescence titration experiments. Results are in good agreement with the concentration values obtained by MALS-RI. Finally, the hosted cyanine molecule could be extracted with (±)-2-octanol, demonstrating the reversibility of the adsorption process.

  16. Multiplane 3D superresolution optical fluctuation imaging

    CERN Document Server

    Geissbuehler, Stefan; Godinat, Aurélien; Bocchio, Noelia L; Dubikovskaya, Elena A; Lasser, Theo; Leutenegger, Marcel

    2013-01-01

    By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed ...

  17. Timing and Operating Mode Design for Time-Gated Fluorescence Lifetime Imaging Microscopy

    OpenAIRE

    Chao Liu; Xinwei Wang; Yan Zhou; Yuliang Liu

    2013-01-01

    Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM) can provide high resolution and high imaging frame during mature FLIM methods. An abstract ti...

  18. The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

    Science.gov (United States)

    Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

    2016-03-01

    Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

  19. Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores.

    Science.gov (United States)

    Nikiforov, Theo T; Beechem, Joseph M

    2006-10-01

    We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer. Then we prepared covalent conjugates of these quantum dots with biotin, fluorescein, and cortisol and established that the binding of these conjugates to suitable Alexa Fluor-labeled antibodies and streptavidin (in the case of biotin) can be efficiently detected by measuring the resonance energy transfer in homogeneous solutions. Finally, based on these observations, competitive binding assays for these three small analytes were developed. The performance of these assays as a function of the degree of labeling of the quantum dots was evaluated. It was found that decreasing the degree of loading of the quantum dots leads to decreases of the limits of detection. The results show the great potential of this FRET system for the development of new homogeneous binding assays.

  20. Self-assembled near-infrared dye nanoparticles as a selective protein sensor by activation of a dormant fluorophore.

    Science.gov (United States)

    Anees, Palapuravan; Sreejith, Sivaramapanicker; Ajayaghosh, Ayyappanpillai

    2014-09-24

    Design of selective sensors for a specific analyte in blood serum, which contains a large number of proteins, small molecules, and ions, is important in clinical diagnostics. While metal and polymeric nanoparticle conjugates have been used as sensors, small molecular assemblies have rarely been exploited for the selective sensing of a protein in blood serum. Herein we demonstrate how a nonspecific small molecular fluorescent dye can be empowered to form a selective protein sensor as illustrated with a thiol-sensitive near-IR squaraine (Sq) dye (λabs= 670 nm, λem= 700 nm). The dye self-assembles to form nonfluorescent nanoparticles (Dh = 200 nm) which selectively respond to human serum albumin (HSA) in the presence of other thiol-containing molecules and proteins by triggering a green fluorescence. This selective response of the dye nanoparticles allowed detection and quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably, the Sq dye in solution state is nonselective and responds to any thiol-containing proteins and small molecules. The sensing mechanism involves HSA specific controlled disassembly of the Sq nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum.

  1. Incorporating a piperidinyl group in the fluorophore extends the fluorescence lifetime of click-derived cyclam-naphthalimide conjugates.

    Science.gov (United States)

    Yu, Mingfeng; Ast, Sandra; Yu, Qun; Lo, Anthony T S; Flehr, Roman; Todd, Matthew H; Rutledge, Peter J

    2014-01-01

    Ligands incorporating a tetraazamacrocycle receptor, a 'click'-derived triazole and a 1,8-naphthalimide fluorophore have proven utility as probes for metal ions. Three new cyclam-based molecular probes are reported, in which a piperidinyl group has been introduced at the 4-position of the naphthalimide fluorophore. These compounds have been synthesized using the copper(I)-catalyzed azide-alkyne Huisgen cycloaddition and their photophysical properties studied in detail. The alkylamino group induces the expected red-shift in absorption and emission spectra relative to the simple naphthalimide derivatives and gives rise to extended fluorescence lifetimes in aqueous buffer. The photophysical properties of these systems are shown to be highly solvent-dependent. Screening the fluorescence responses of the new conjugates to a wide variety of metal ions reveals significant and selective fluorescence quenching in the presence of copper(II), yet no fluorescence enhancement with zinc(II) as observed previously for the simple naphthalimide derivatives. Reasons for this different behaviour are proposed. Cytotoxicity testing shows that these new cyclam-triazole-dye conjugates display little or no toxicity against either DLD-1 colon carcinoma cells or MDA-MB-231 breast carcinoma cells, suggesting a potential role for these and related systems in biological sensing applications.

  2. Incorporating a piperidinyl group in the fluorophore extends the fluorescence lifetime of click-derived cyclam-naphthalimide conjugates.

    Directory of Open Access Journals (Sweden)

    Mingfeng Yu

    Full Text Available Ligands incorporating a tetraazamacrocycle receptor, a 'click'-derived triazole and a 1,8-naphthalimide fluorophore have proven utility as probes for metal ions. Three new cyclam-based molecular probes are reported, in which a piperidinyl group has been introduced at the 4-position of the naphthalimide fluorophore. These compounds have been synthesized using the copper(I-catalyzed azide-alkyne Huisgen cycloaddition and their photophysical properties studied in detail. The alkylamino group induces the expected red-shift in absorption and emission spectra relative to the simple naphthalimide derivatives and gives rise to extended fluorescence lifetimes in aqueous buffer. The photophysical properties of these systems are shown to be highly solvent-dependent. Screening the fluorescence responses of the new conjugates to a wide variety of metal ions reveals significant and selective fluorescence quenching in the presence of copper(II, yet no fluorescence enhancement with zinc(II as observed previously for the simple naphthalimide derivatives. Reasons for this different behaviour are proposed. Cytotoxicity testing shows that these new cyclam-triazole-dye conjugates display little or no toxicity against either DLD-1 colon carcinoma cells or MDA-MB-231 breast carcinoma cells, suggesting a potential role for these and related systems in biological sensing applications.

  3. Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-dimethylamino-2,3-naphthalimide.

    Science.gov (United States)

    Vázquez, M Eugenio; Blanco, Juan B; Imperiali, B

    2005-02-02

    We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biological probe with emission in the 500-600 nm range and a marked response to changes in the environment polarity. The 6DMN fluorescence is red-shifted in polar protic environments, with the maximum emission intensity shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Additionally, the fluorescence quantum yield decreases more than 100-fold from chloroform (Phi = 0.225) to water (Phi = 0.002). The scope and applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid derivative (5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides, demonstrating that it can be manipulated under standard solid-phase peptide synthesis conditions. Peptides incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified in studies with Src homology 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm) upon binding to selected SH2 domains (e.g., Crk SH2, Abl SH2, and PI3K SH2). The peptides can be used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable throughout the titrations.

  4. Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer.

    Science.gov (United States)

    Murray, Sarah; McKenzie, Marian; Butler, Ruth; Baldwin, Samantha; Sutton, Kevin; Batey, Ian; Timmerman-Vaughan, Gail M

    2011-06-15

    Fluorophore-assisted carbohydrate electrophoresis (FACE) is an analytical method for characterizing carbohydrate chain length that has been applied to neutral, charged, and N-linked oligosaccharides and that has been implemented using diverse separation platforms, including polyacrylamide gel electrophoresis and capillary electrophoresis. In this article, we describe three substantial improvements to FACE: (i) reducing the amount of starch and APTS required in labeling reactions and systematically analyzing the effect of altering the starch and 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) concentrations on the reproducibility of the FACE peak area distributions; (ii) implementing FACE on a multiple capillary DNA sequencer (an ABI 3130xl), enabling higher throughput than is possible on other separation platforms; and (iii) developing a protocol for producing quantitative output of peak heights and areas using genetic marker analysis software. The results of a designed experiment to determine the effect of decreasing both the starch and fluorophore concentrations on the sensitivity and reproducibility of FACE electrophoregrams are presented. Analysis of the peak area distributions of the FACE electrophoregrams identified the labeling reaction conditions that resulted in the smallest variances in the peak area distributions while retaining strong fluorescence signals from the capillary-based DNA sequencer.

  5. IMAGING OF FLUOROPHORES IN CHROMATOGRAPHIC BEADS, RECONSTRUCTION OF RADIAL DENSITY DISTRIBUTIONS AND CHARACTERISATION OF PROTEIN UPTAKING PROCESSES

    Directory of Open Access Journals (Sweden)

    Bernd Stanislawski

    2010-11-01

    Full Text Available A new adjustment calculus is presented to determine the true intraparticle distribution of bound protein within chromatographic beads from confocal fluorescence slice series. The calculus does not require knowledge about optical properties of different chromatographic materials like refractive index and turbidity, but it depends on a parameter which can be adjusted interactively. The algorithm is of complexity O(n where n is the pixel number. From the reconstructed data we compute the parameters of the protein uptaking process using a model-based approach. It is demonstrated that the protein uptaking rates of the beads strongly dependent on the conditions of the fluid phase influencing the strength of protein surface interaction.

  6. Differential Laser-Induced Perturbation Spectroscopy for Analysis of Mixtures of the Fluorophores l-Phenylalanine, l-Tyrosine and l-Tryptophan Using a Fluorescence Probe.

    Science.gov (United States)

    Oztekin, Erman K; Hahn, David W

    2016-09-01

    Quantitative detection of common endogenous fluorophores is accomplished using differential laser-induced perturbation spectroscopy (DLIPS) with a 193-nm UV fluorescence probe and various UV perturbation wavelengths. In this study, DLIPS is explored as an alternative to traditional fluorescence spectroscopy alone, with a goal of exploring natural fluorophores pursuant to biological samples and tissue analysis. To this end, aromatic amino acids, namely, l-phenylalanine, l-tyrosine and l-tryptophan are mixed with differing mass ratios and then classified with various DLIPS schemes. Classification with a traditional fluorescence probe is used as a benchmark. The results show a 20% improvement in classification performance of the DLIPS method over the traditional fluorescence method using partial least squares (PLS) analysis. Additional multivariate analyses are explored, and the relevant photochemistry is elucidated in the context of perturbation wavelengths. We conclude that DLIPS is a promising biosensing approach with potential for in vivo analysis given the current findings with fluorophores relevant to biological tissues.

  7. Optimizing and Evaluating an Integrated SPECT-CmT System Dedicated to Improved 3-D Breast Cancer Imaging

    Science.gov (United States)

    2009-05-01

    the imaging system’s required clinica l performance. This evidence ranged from the ability of the CmT system to image close to the chest wall (see...year old woman undergoing dual-view screening mammograph y of her remaining int act breast seven years after a mastectomy) to completing a medica ...Telluride (CZT) gamma camera (model LumaGEM 3200S, Gamma Medica , Inc., Northridge, CA) with discretized crystals, each 2.3x2.3x5mm3 on a 2.5mm

  8. Near-Field Fluorescence and Topography Characterization of a Single Nanometre Fluorophore by Apertureless Tip-Enhanced Scanning Near-Field Microscopy

    Institute of Scientific and Technical Information of China (English)

    WU Xiao-Bin; WANG Jia; XU Ji-Ying; WANG Rui; TIAN Qian; YU Jian-Yuan

    2007-01-01

    Tip-enhanced near-field fluorescence and topography characterization of a single nanometre fluorophore is conducted by using an apertureless scanning near-field microscopy system. A fluorophore with size 80nm is mapped with a spatial resolution of 10nm. The corresponding near-field fluorescence data shows significant signal enhancement due to the apertureless tip-enhanced effect. With the nanometre spatial resolution capability and nanometre local tip-enhanced effect, the apertureless tip-enhanced scanning near-field microscopy may be further used to characterize a single molecule by realizing the local near-field spectrum assignment corresponding to topography at nanometre scale.

  9. Synthesis of fluorophore encapsulated silica nanoparticles for the evaluation of the biological fate and toxicity of food relevant nanoparticles

    Science.gov (United States)

    Zane, Andrew Paul

    We show that commercially available TiO2, SiO2, and ZnO nanoparticles are all internalized by C2BBe1 intestinal epithelial cells, but do not appear to be toxic, even after long term repeat-exposures. When particles were exposed to a simulated digestion protocol mimicking the stomach and intestinal environment, TiO2 particles did show mild toxicity by MTT assay, indicating a decrease in metabolic activity. IR spectra of these particles indicate presence of material from the digestion media, and these absorbed species may be responsible for the effects noted. Though the three particles were not significantly toxic, we note internalization by the intestinal epithelial cells, opening a possibility for absorption into circulation where they may localize in organs throughout the body. This will be observed by functionalizing the particles with fluorophores, after which they can be measured via fluorescence. To optimize the quantum yield efficiency, and thus the brightness, of one such fluorophore, we seek to improve a microwave synthesis of CdSe/CdS/ZnS quantum dots our lab has previously reported. By coupling the microwave reactor to a fluorescence spectrometer via fiber optic cables, we were able to monitor the development of the particles throughout the microwave heating. Time-dependent fluorescence shows the development of an early fluorescence peak at 502 nm attributed to CdSe cores. We then note two isosbestic points which we attribute to the development of CdS layer around CdSe cores, and eventually the formation of outer ZnS shell. We utilize this in situ monitoring along with a study of various nucleation temperatures ranging from 0 to 100°C, and pre-and-post microwave heating UV exposure treatments to obtain optimized CdSe/CdS/ZnS particles with a QY of 40%. This is an improvement over our previous particles' 13% QY, and the highest yet reported for an aqueous synthesis of CdSe/ZnS type particles. Finally, we incorporate these QDs as well as two organic

  10. Local viscosity and solvent relaxation experienced by rod-like fluorophores in AOT/4-chlorophenol/m-xylene organogels

    Science.gov (United States)

    Dandapat, Manika; Mandal, Debabrata

    2017-01-01

    Organogels prepared from AOT/4-chlorophenol/m-xylene are immobile in the macroscopic sense, with a well-characterized internal structure. However, the molecular level dynamics inside the gels is not too clear, although a very slow structural relaxation has been reported previously. Using a set of rod-like fluorophores, we find that the rotational mobility of a small guest molecule inside the gel can be extremely fast, indicating presence of sufficiently low-microviscosity domains. These domains consist of m-xylene solvent molecules trapped in the interstices of fiber bundles comprising columnar stacks of 4-chlorophenol surrounded by AOT molecules. However, interstitial trapping of m-xylene does retard its own dynamics, which explains the slow solvent relaxation inside the gels. Hence, the state of m-xylene in the organogel may be characterized as "bound", in contrast to the "free" state in neat m-xylene.

  11. And yet they glow: thiazole based push-pull fluorophores containing nitro groups and the influence of regioisomerism

    Science.gov (United States)

    Habenicht, Stefanie H.; Siegmann, Michael; Kupfer, Stephan; Kübel, Joachim; Weiß, Dieter; Cherek, Doreen; Möller, Uwe; Dietzek, Benjamin; Gräfe, Stefanie; Beckert, Rainer

    2015-06-01

    Reported is a study on the influence of regioisomerism on the photophysical properties in 4-hydroxy-1,3-thiazole-based push-pull-chromophores/fluorophores to evaluate the molecular structure-property relationship as a basic foundation for future design strategies concerning this class of dyes. Surprisingly, the nitro groups used as acceptors do not act as a fluorescence quencher, instead the derivatives synthesized exhibit quantum yields of 37-40%. Two 4-ethoxy-1,3-thiazole derivatives which differ only in the positioning of their electron donating (methoxy) and electron withdrawing (nitro) groups have been synthesized and examined in terms of their photophysical properties, i.e. UV/Vis absorption and fluorescence emission spectra. Additionally, quantum chemical calculations have been performed to unravel the underlying fundamental transitions and to explain the experimental results.

  12. Methods for labeling skeletal muscle ion channels site-specifically with fluorophores suitable for FRET-based structural analysis.

    Science.gov (United States)

    Mahalingam, Mohana; Fessenden, James D

    2015-01-01

    Skeletal muscle excitation-contraction coupling is triggered by the concerted action of two enormous Ca(2+) channel complexes, the dihydropyridine receptor and the type 1 ryanodine receptor. Recent advances in our understanding of the structure of these large Ca(2+) channels have been driven by fluorescence resonance energy transfer (FRET)-based analysis. A methodological challenge in conducting these FRET measurements is the ability to site-specifically label these huge ion channels with donor and acceptor fluorophores capable of undergoing energy transfer. In this chapter, we detail specific protocols for tagging large membrane proteins with these fluorescent probes using three orthogonal labeling methods: fluorescent protein fusions, biarsenical reagents directed to engineered tetracysteine tags, and Cy3/5 nitrilotriacetic acid conjugates that bind to poly-histidine tags. © 2015 Elsevier Inc. All rights reserved.

  13. A state space based approach to localizing single molecules from multi-emitter images.

    Science.gov (United States)

    Vahid, Milad R; Chao, Jerry; Ward, E Sally; Ober, Raimund J

    2017-01-28

    Single molecule super-resolution microscopy is a powerful tool that enables imaging at sub-diffraction-limit resolution. In this technique, subsets of stochastically photoactivated fluorophores are imaged over a sequence of frames and accurately localized, and the estimated locations are used to construct a high-resolution image of the cellular structures labeled by the fluorophores. Available localization methods typically first determine the regions of the image that contain emitting fluorophores through a process referred to as detection. Then, the locations of the fluorophores are estimated accurately in an estimation step. We propose a novel localization method which combines the detection and estimation steps. The method models the given image as the frequency response of a multi-order system obtained with a balanced state space realization algorithm based on the singular value decomposition of a Hankel matrix, and determines the locations of intensity peaks in the image as the pole locations of the resulting system. The locations of the most significant peaks correspond to the locations of single molecules in the original image. Although the accuracy of the location estimates is reasonably good, we demonstrate that, by using the estimates as the initial conditions for a maximum likelihood estimator, refined estimates can be obtained that have a standard deviation close to the Cramér-Rao lower bound-based limit of accuracy. We validate our method using both simulated and experimental multi-emitter images.

  14. Intraoperative optical biopsy for brain tumors using spectro-lifetime properties of intrinsic fluorophores

    Science.gov (United States)

    Vasefi, Fartash; Kittle, David S.; Nie, Zhaojun; Falcone, Christina; Patil, Chirag G.; Chu, Ray M.; Mamelak, Adam N.; Black, Keith L.; Butte, Pramod V.

    2016-04-01

    We have developed and tested a system for real-time intra-operative optical identification and classification of brain tissues using time-resolved fluorescence spectroscopy (TRFS). A supervised learning algorithm using linear discriminant analysis (LDA) employing selected intrinsic fluorescence decay temporal points in 6 spectral bands was employed to maximize statistical significance difference between training groups. The linear discriminant analysis on in vivo human tissues obtained by TRFS measurements (N = 35) were validated by histopathologic analysis and neuronavigation correlation to pre-operative MRI images. These results demonstrate that TRFS can differentiate between normal cortex, white matter and glioma.

  15. Stroboscopic fluorescence lifetime imaging.

    Science.gov (United States)

    Holton, Mark D; Silvestre, Oscar R; Errington, Rachel J; Smith, Paul J; Matthews, Daniel R; Rees, Paul; Summers, Huw D

    2009-03-30

    We report a fluorescence lifetime imaging technique that uses the time integrated response to a periodic optical excitation, eliminating the need for time resolution in detection. A Dirac pulse train of variable period is used to probe the frequency response of the total fluorescence per pulse leading to a frequency roll-off that is dependent on the relaxation rate of the fluorophores. The technique is validated by demonstrating wide-field, realtime, lifetime imaging of the endocytosis of inorganic quantum dots by a cancer cell line. Surface charging of the dots in the intra-cellular environment produces a switch in the fluorescence lifetime from approximately 40 ns to technique offers lifetime based imaging at video rates with standard CCD cameras and has application in probing millisecond cell dynamics and in high throughput imaging assays.

  16. Effect of probe diffusion on the SOFI imaging accuracy

    Science.gov (United States)

    Vandenberg, Wim; Dedecker, Peter

    2017-01-01

    Live-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density. PMID:28333166

  17. [Imaging].

    Science.gov (United States)

    Chevrot, A; Drapé, J L; Godefroy, D; Dupont, A M; Pessis, E; Sarazin, L; Minoui, A

    1997-01-01

    The panoply of imaging techniques useful in podology is essentially limited to X-rays. Standard "standing" and "lying" X-rays furnish most of the required information. Arthrography is sometimes performed, in particular for trauma or tumour of the ankle. CT scan and MRI make a decisive contribution in difficult cases, notably in fractures and in small fractures without displacement. The two latter techniques are useful in tendon, ligament and muscular disorders, where echography is also informative. Rigorous analysis of radiographies and a good knowledge of foot disorders make these imaging techniques efficacious.

  18. Enhanced emission of fluorophores on shrink-induced wrinkled composite structures.

    Science.gov (United States)

    Sharma, Himanshu; Digman, Michelle A; Felsinger, Natasha; Gratton, Enrico; Khine, Michelle

    2014-01-01

    We introduce a manufacturable and scalable method for creating tunable wrinkled ferromagnetic-metallic structures to enhance fluorescence signals. Thin layers of nickel (Ni) and gold (Au) were deposited onto a pre-stressed thermoplastic (shrink wrap film) polymer. Heating briefly forced the metal films to buckle when the thermoplastic retracted, resulting in multi-scale composite 'wrinkles'. This is the first demonstration of leveraging the plasmons in such hybrid nanostructures by metal enhanced fluorescence (MEF) in the near-infrared wavelengths. We observed more than three orders of magnitude enhancement in the fluorescence signal of a single molecule of goat anti-mouse immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate, FITC, (FITC-IgG) by two-photon excitation with these structures. These large enhancements in the fluorescence signal at the nanoscale gaps between the composite wrinkles corresponded to shortened lifetimes due to localized surface plasmons. To characterize these structures, we combined fluctuation correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), and two-photon microscopy to spatially and temporally map the hot spots with high resolution.

  19. Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin.

    Science.gov (United States)

    Zalewski, P D; Jian, X; Soon, L L; Breed, W G; Seamark, R F; Lincoln, S F; Ward, A D; Sun, F Z

    1996-01-01

    The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.

  20. Synthesis of highly fluorescent silica nanoparticles in a reverse microemulsion through double-layered doping of organic fluorophores

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hyojong, E-mail: hyojong@hallym.ac.kr; Pak, Joonsung [Hallym University, Department of Chemistry (Korea, Republic of)

    2013-05-15

    Water-soluble, highly fluorescent double-layered silica nanoparticles (FL-DLSN) have been successfully synthesized through a reverse (water-in-oil) microemulsion method. The microemulsion was prepared by mixing a surfactant (Brij35), co-surfactant, organic solvent, water, and fluorescein as an organic fluorophore. The sizes of the silica nanoparticles were successfully controlled in the reverse microemulsion using Brij35 by changing the water-to-Brij35 ratio and by adding HCl. Initially, tetraethylorthosilicate was hydrolyzed by adding NH{sub 4}OH as a catalyst and then polymerized to generate core fluorescent silica nanoparticles with fluorescein. 3-(Aminopropyl)triethoxysilane (APTS) was sequentially added into the reaction mixture, and reacted on the surface of pre-generated core silica nanoparticles to form the second layer in the form of a shell. The second silica layer that was derived from the condensation of APTS effectively protected the fluorescein dye within the silica matrix. This is a novel and simple synthetic approach to generate highly fluorescent, monodispersed silica nanoparticles by doping organic molecules into a silica matrix.Graphical Abstract.

  1. Gray-Tone Lithography Implementation of Drexhage's Method for Calibrating Radiative and Nonradiative Decay Constants of Fluorophores

    CERN Document Server

    Kwadrin, Andrej; 10.1021/jp3048423

    2013-01-01

    We present a straightforward method to realize non-planar dielectric structures with a controlled height profile for use in calibration of fluorophores. Calibration of fluorescence quantum efficiency and intrinsic radiative and nonradiative decay rates of emitters is possible by using changes in the local density of optical states, provided one can control the emitter-surface distance with nanometer accuracy. We realize a method that is accurate yet fast to implement. We fabricate PMMA wedges (4 mm x 4 mm x 2 \\mu m) by gray-tone UV-lithography of Shipley S1813G2. Its applicability as dielectric spacer is demonstrated in Drexhage experiments for three different emitters in the visible and near-infrared wavelength regime. The decay-rate dependence of the fluorescent state of emitters on the distance to a silver mirror is observed and compared to calculations based on the local density of states. Quantitative values for (non)radiative decay rates and quantum efficiencies are extracted. Furthermore, we discuss ho...

  2. Measuring number of fluorophores labelling cDNA strands, in solution, with Fluorescence Correlation Spectroscopy and photobleaching

    CERN Document Server

    Delon, Antoine; Lambert, Emeline; Lerbs-Mache, Silva; Mache, Régis; Derouard, Jacques; Motto-Ros, Vincent; Galland, Rémi

    2009-01-01

    We present different approaches that aim at determining, in solution, the brightness and the number of Alexa Fluor 647 molecules labelling the C bases of two sequences of cDNA, corresponding to two transcripts of different sizes, a short and a long transcript (123 and 306 base long, with 45 and 74 dCTP residues, respectively). In each case, the Alexa labeled bases have been incorporated during reverse-transcription. Two kinds of experiments have been performed and combined: photobleaching and Fluorescence Correlation Spectroscopy (together with the factorial cumulant analysis method). As a result, we show that the photobleaching cross-section of labelled cDNA strands is about half that of free Alexa in aqueous solution, while their brightness is about twice. The factorial cumulant analysis put into evidence the fact that the brightness of cDNA strands varies from molecule to molecule, due to the statistical distribution of the number of Alexa fluorophores labelling cDNA. Our measurements are consistent with a...

  3. DNA visualization in single molecule studies carried out with optical tweezers: Covalent versus non-covalent attachment of fluorophores.

    Science.gov (United States)

    Suei, Sandy; Raudsepp, Allan; Kent, Lisa M; Keen, Stephen A J; Filichev, Vyacheslav V; Williams, Martin A K

    2015-10-16

    In this study, we investigated the use of the covalent attachment of fluorescent dyes to double-stranded DNA (dsDNA) stretched between particles using optical tweezers (OT) and compared the mechanical properties of the covalently-functionalized chain to that of unmodified DNA and to DNA bound to a previously uncharacterized groove-binder, SYBR-gold. Modified DNA species were obtained by covalently linking azide-functionalized organic fluorophores onto the backbone of DNA chains via the alkyne moieties of modified bases that were incorporated during PCR. These DNA molecules were then constructed into dumbbells by attaching polystyrene particles to the respective chain ends via biotin or digoxigenin handles that had been pre-attached to the PCR primers which formed the ends of the synthesized molecule. Using the optical tweezers, the DNA was stretched by separating the two optically trapped polystyrene particles. Displacements of the particles were measured in 3D using an interpolation-based normalized cross-correlation method and force-extension curves were calculated and fitted to the worm-like chain model to parameterize the mechanical properties of the DNA. Results showed that both the contour and persistence length of the covalently-modified dsDNAs were indistinguishable from that of the unmodified dsDNA, whereas SYBR-gold binding perturbed the contour length of the chain in a force-dependent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Luminescence imaging using radionuclides: a potential application in molecular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong Chan [Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Il An, Gwang [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Park, Se-Il [Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Oh, Jungmin [Korea Basic Science Institute Chuncheon Center, Gangwon-do 200-701 (Korea, Republic of); Kim, Hong Joo [Department of Physics and Energy Science, Kyungpook National University, Daegu 702-710 (Korea, Republic of); Su Ha, Yeong; Wang, Eun Kyung [Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Min Kim, Kyeong; Kim, Jung Young [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Lee, Jaetae [Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Welch, Michael J. [Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Yoo, Jeongsoo, E-mail: yooj@knu.ac.k [Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2011-04-15

    Introduction: Nuclear and optical imaging are complementary in many aspects and there would be many advantages when optical imaging probes are prepared using radionuclides rather than classic fluorophores, and when nuclear and optical dual images are obtained using single imaging probe. Methods: The luminescence intensities of various radionuclides having different decay modes have been assayed using luminescence imaging and in vitro luminometer. Radioiodinated Herceptin was injected into a tumor-bearing mouse, and luminescence and microPET images were obtained. The plant dipped in [{sup 32}P]phosphate solution was scanned in luminescence mode. Radio-TLC plate was also imaged in the same imaging mode. Results: Radionuclides emitting high energy {beta}{sup +}/{beta}{sup -} particles showed higher luminescence signals. NIH3T6.7 tumors were detected in both optical and nuclear imaging. The uptake of [{sup 32}P]phosphate in plant was easily followed by luminescence imaging. Radio-TLC plate was visualized and radiochemical purity was quantified using luminescence imaging. Conclusion: Many radionuclides with high energetic {beta}{sup +} or {beta}{sup -} particles during decay were found to be imaged in luminescence mode due mainly to Cerenkov radiation. 'Cerenkov imaging' provides a new optical imaging platform and an invaluable bridge between optical and nuclear imaging. New optical imaging probes could be easily prepared using well-established radioiodination methods. Cerenkov imaging will have more applications in the research field of plant science and autoradiography.

  5. IRDye78 Conjugates for Near-Infrared Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Atif Zaheer

    2002-10-01

    Full Text Available The detection of human malignancies by near-infrared (NIR fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic, we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.

  6. Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

    Energy Technology Data Exchange (ETDEWEB)

    Gartia, Manas Ranjan [Department of Nuclear, Plasma and Radiological Engineering, University of Illinois, Urbana, IL 61801 (United States); Hsiao, Austin; Logan Liu, G [Department of Bioengineering, University of Illinois, Urbana, IL 61801 (United States); Sivaguru, Mayandi [Institute for Genomic Biology, University of Illinois, Urbana, IL 61801 (United States); Chen Yi, E-mail: loganliu@illinois.edu [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)

    2011-09-07

    We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

  7. Dual view on sliding phases in U (1 ) symmetric systems

    Science.gov (United States)

    Vayl, S.; Kuklov, A. B.; Oganesyan, V.

    2017-03-01

    The proposal of sliding phases (SP) is revisited from the perspective of duality. A generic argument is formulated as essentially a no-go theorem for SP in translationally invariant nonfrustrated systems with short-range interactions—classical or quantum. Its validity is demonstrated on an asymmetric bilayer and its multilayer variation models where the duality allows obtaining asymptotically exact analytical solution. This solution is in drastic contrast with the perturbative renormalization group prediction and is strongly supported by Monte Carlo simulations. An alternative path toward finding SP is suggested. Its key ingredient is a long-range gauge-type interaction suppressing the interlayer Josephson coupling.

  8. Multiphoton fluorescence lifetime imaging of human hair.

    Science.gov (United States)

    Ehlers, Alexander; Riemann, Iris; Stark, Martin; König, Karsten

    2007-02-01

    In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.

  9. Excited-state intramolecular proton transfer (ESIPT) inspired azole-quinoline based fluorophores: Synthesis and photophysical properties study

    Energy Technology Data Exchange (ETDEWEB)

    Padalkar, Vikas S.; Sekar, Nagaiyan, E-mail: n.sekar@ictmumbai.edu.in

    2014-11-15

    7-Hydroxy-3-(4-nitrophenyl)quinoline-6-carboxylic acid was obtained by the condensation reaction of p-amino salicylic acid and 4-nitrophenylmalonadialdehyde which was obtained from phenylacetonitrile through nitration, hydrolysis and Vilsmeier reaction. 7-Hydroxy-3-(4-nitrophenyl) quinoline-6-carboxylic acid was condensed with different o-aminophenols or o-aminothiophenol in ethanol in the presence of phosphorustrichloride. Synthesized quinoline contained benzimidazole and benzothiazole moieties. Photophysical behaviors of these compounds in solvents of different polarities were studied using UV–vis and fluorescence spectroscopy. The compounds showed single absorption in all the studied solvents. The dual emissions (normal emission and ESIPT emission) as well as large Stokes' shift emission pattern were observed for the synthesized fluorophores. The photophysical study shows that the emission properties of the compounds depend on the solvent polarity. The photophysical properties of the compounds were compared with structurally analogous ESIPT quinoline. Thermal stability of the compounds was studied using thermogravimetric analysis and results show that compounds are thermally stable up to 300 °C. The synthesized quinoline derivatives were characterized using elemental analysis, FT-IR and {sup 1}H –NMR, {sup 13}C –NMR spectroscopy and mass spectral analysis. - Highlights: • First and unique study of quinoline derivatives contain ESIPT azole unit at 6-position and hydroxyl group at 7-position. • Compounds are fluorescent with considerable quantum yields. • All compounds showed absorption in ultraviolet region and emission in visible region with large Stokes' shift. • The photophysical properties of new compounds were compared with reported ESIPT quinoline analogous.

  10. Near-Infrared Fluorescence Enhanced (NIR-FE) Molecular Imaging of Live Cells on Gold Substrates

    CERN Document Server

    Hong, Guosong; Welsher, Kevin; Chen, Zhuo; Robinson, Joshua T; Wang, Hailiang; Zhang, Bo; Dai, Hongjie

    2011-01-01

    Low quantum yields of near infrared (NIR) fluorophores have limited their capabilities as imaging probes in a transparent, low background imaging window. Here for the first time we reported near-infrared fluorescence enhance (NIR-FE) cell imaging using nanostructured Au substrate, which was employed as a general platform for both single-walled carbon nanotubes (SWNTs) and organic fluorescent labels in the NIR region. Fluorescence intensity, as well as cell targeting specificity, was greatly improved by this novel imaging technique. With NIR-FE imaging, we were able to image SWNT-stained cells at short exposure time of 300ms, and push the detectable limit of SWNT staining of cells down to an ultralow concentration of ~50 pM. Further, different degrees of fluorescence enhancement for endocytosed, intracellular SWNTs vs. nanotubes on the cell membrane at the cell/gold interface were observed, suggesting the possibility of using this technique to track the transmembrane behavior of NIR fluorophores.

  11. Monitoring the metabolic state of fungal hyphae and the presence of melanin by nonlinear spectral imaging

    NARCIS (Netherlands)

    Knaus, H.; Blab, G.; Agronskaia, A.V.; van den Heuvel, D.J.; Gerritsen, H.C.; Wösten, H.A.B.

    2013-01-01

    Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluore

  12. Quasi-real-time fluorescence imaging with lifetime dependent contrast

    Science.gov (United States)

    Jiang, Pei-Chi; Grundfest, Warren S.; Stafsudd, Oscar M.

    2011-08-01

    Conventional fluorescence lifetime imaging requires complicated algorithms to extract lifetimes of fluorophores and acquisition of multiple data points at progressively longer delay times to characterize tissues. To address diminishing signal-to-noise ratios at these progressively longer time delays, we report a time-resolved fluorescence imaging method, normalized fluorescence yield imaging that does not require the extraction of lifetimes. The concept is to extract the ``contrast'' instead of the lifetime value of the fluorophores by using simple mathematical algorithms. This process converts differences in decay times directly to different intensities. The technique was verified experimentally using a gated iCCD camera and an ultraviolet light-emitting diode light source. It was shown that this method can distinguish between chemical dyes (Fluorescein and Rhodamine-B) and biomedical samples, such as powders of elastin and collagen. Good contrast was obtained between fluorophores that varied by less than 6% in lifetime. Additionally, it was shown that long gate times up to 16 ns achieve good contrast depending upon the samples to be studied. These results support the feasibility of time-resolved fluorescence imaging without lifetime extraction, which has a potential clinical role in noninvasive real-time imaging.

  13. Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

    Directory of Open Access Journals (Sweden)

    James D Fessenden

    Full Text Available The type 1 ryanodine receptor (RyR1 is an intracellular Ca(2+ release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET method using a fused green fluorescent protein (GFP donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33% was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10 tag. A high degree of energy transfer (∼66% indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

  14. Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

    Science.gov (United States)

    Fessenden, James D; Mahalingam, Mohana

    2013-01-01

    The type 1 ryanodine receptor (RyR1) is an intracellular Ca(2+) release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

  15. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    Science.gov (United States)

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T; Herbert, Alex; Lando, David; Etheridge, Thomas J; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M; Klenerman, David; Lee, Steven F

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

  16. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    Directory of Open Access Journals (Sweden)

    Matthieu Palayret

    Full Text Available Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

  17. Full-direct method for imaging pharmacokinetic parameters in dynamic fluorescence molecular tomography

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guanglei, E-mail: guangleizhang@bjtu.edu.cn [Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084 (China); Department of Biomedical Engineering, School of Computer and Information Technology, Beijing Jiaotong University, Beijing 100044 (China); Pu, Huangsheng; Liu, Fei; Bai, Jing [Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084 (China); He, Wei [China Institute of Sport Science, Beijing 100061 (China); Luo, Jianwen, E-mail: luo-jianwen@tsinghua.edu.cn [Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084 (China); Center for Biomedical Imaging Research, School of Medicine, Tsinghua University, Beijing 100084 (China)

    2015-02-23

    Images of pharmacokinetic parameters (also known as parametric images) in dynamic fluorescence molecular tomography (FMT) can provide three-dimensional metabolic information for biological studies and drug development. However, the ill-posed nature of FMT and the high temporal variation of fluorophore concentration together make it difficult to obtain accurate parametric images in small animals in vivo. In this letter, we present a method to directly reconstruct the parametric images from the boundary measurements based on hybrid FMT/X-ray computed tomography (XCT) system. This method can not only utilize structural priors obtained from the XCT system to mitigate the ill-posedness of FMT but also make full use of the temporal correlations of boundary measurements to model the high temporal variation of fluorophore concentration. The results of numerical simulation and mouse experiment demonstrate that the proposed method leads to significant improvements in the reconstruction quality of parametric images.

  18. Full-direct method for imaging pharmacokinetic parameters in dynamic fluorescence molecular tomography

    Science.gov (United States)

    Zhang, Guanglei; Pu, Huangsheng; He, Wei; Liu, Fei; Luo, Jianwen; Bai, Jing

    2015-02-01

    Images of pharmacokinetic parameters (also known as parametric images) in dynamic fluorescence molecular tomography (FMT) can provide three-dimensional metabolic information for biological studies and drug development. However, the ill-posed nature of FMT and the high temporal variation of fluorophore concentration together make it difficult to obtain accurate parametric images in small animals in vivo. In this letter, we present a method to directly reconstruct the parametric images from the boundary measurements based on hybrid FMT/X-ray computed tomography (XCT) system. This method can not only utilize structural priors obtained from the XCT system to mitigate the ill-posedness of FMT but also make full use of the temporal correlations of boundary measurements to model the high temporal variation of fluorophore concentration. The results of numerical simulation and mouse experiment demonstrate that the proposed method leads to significant improvements in the reconstruction quality of parametric images.

  19. Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate.

    Science.gov (United States)

    Stump, Matthew R; Gloss, Lisa M

    2008-02-01

    Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.

  20. Folded or nonfolded fluorophores incorporating naphthalene:Their fluorescent quenching driven by charge transfer or co-aggregation in the aqueous-organic binary solvent

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Folded or nonfolded fluorophores incorporating naphthalene were synthesized and characterized by steady state fluorescence technique.Paraquat as an excellent quenching reagent quenched the fluorescence of Nel6 or nDs(n = 1-4) driven by charge transfer.Under aggregation of nDs,α-CD did not quench the fluorescence of 1D.At lower concentration,the quenching tendency ofα-CD against nDs is 2D>3D>4D,while at higher concentration,the tendency is 2D<3D<4D.α-CD showed the selective recognition on its fluorescent ...

  1. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    Science.gov (United States)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  2. Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP.

    Directory of Open Access Journals (Sweden)

    Shlomo S Dellal

    Full Text Available Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C(5-maleimide (AM546. Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM or acidic external solution (pH 6.5 elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.

  3. In-vivo optical detection of cancer using chlorin e6 – polyvinylpyrrolidone induced fluorescence imaging and spectroscopy

    OpenAIRE

    Soo Khee; Bhuvaneswari Ramaswamy; Thong Patricia SP; Chin William WL; Heng Paul WS; Olivo Malini

    2009-01-01

    Abstract Background Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. Methods Fluorescence imaging was performed on MGH human...

  4. A small-molecule dye for NIR-II imaging.

    Science.gov (United States)

    Antaris, Alexander L; Chen, Hao; Cheng, Kai; Sun, Yao; Hong, Guosong; Qu, Chunrong; Diao, Shuo; Deng, Zixin; Hu, Xianming; Zhang, Bo; Zhang, Xiaodong; Yaghi, Omar K; Alamparambil, Zita R; Hong, Xuechuan; Cheng, Zhen; Dai, Hongjie

    2016-02-01

    Fluorescent imaging of biological systems in the second near-infrared window (NIR-II) can probe tissue at centimetre depths and achieve micrometre-scale resolution at depths of millimetres. Unfortunately, all current NIR-II fluorophores are excreted slowly and are largely retained within the reticuloendothelial system, making clinical translation nearly impossible. Here, we report a rapidly excreted NIR-II fluorophore (∼90% excreted through the kidneys within 24 h) based on a synthetic 970-Da organic molecule (CH1055). The fluorophore outperformed indocyanine green (ICG)-a clinically approved NIR-I dye-in resolving mouse lymphatic vasculature and sentinel lymphatic mapping near a tumour. High levels of uptake of PEGylated-CH1055 dye were observed in brain tumours in mice, suggesting that the dye was detected at a depth of ∼4 mm. The CH1055 dye also allowed targeted molecular imaging of tumours in vivo when conjugated with anti-EGFR Affibody. Moreover, a superior tumour-to-background signal ratio allowed precise image-guided tumour-removal surgery.

  5. A small-molecule dye for NIR-II imaging

    Science.gov (United States)

    Antaris, Alexander L.; Chen, Hao; Cheng, Kai; Sun, Yao; Hong, Guosong; Qu, Chunrong; Diao, Shuo; Deng, Zixin; Hu, Xianming; Zhang, Bo; Zhang, Xiaodong; Yaghi, Omar K.; Alamparambil, Zita R.; Hong, Xuechuan; Cheng, Zhen; Dai, Hongjie

    2016-02-01

    Fluorescent imaging of biological systems in the second near-infrared window (NIR-II) can probe tissue at centimetre depths and achieve micrometre-scale resolution at depths of millimetres. Unfortunately, all current NIR-II fluorophores are excreted slowly and are largely retained within the reticuloendothelial system, making clinical translation nearly impossible. Here, we report a rapidly excreted NIR-II fluorophore (~90% excreted through the kidneys within 24 h) based on a synthetic 970-Da organic molecule (CH1055). The fluorophore outperformed indocyanine green (ICG)--a clinically approved NIR-I dye--in resolving mouse lymphatic vasculature and sentinel lymphatic mapping near a tumour. High levels of uptake of PEGylated-CH1055 dye were observed in brain tumours in mice, suggesting that the dye was detected at a depth of ~4 mm. The CH1055 dye also allowed targeted molecular imaging of tumours in vivo when conjugated with anti-EGFR Affibody. Moreover, a superior tumour-to-background signal ratio allowed precise image-guided tumour-removal surgery.

  6. Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta.

    Directory of Open Access Journals (Sweden)

    Yutaka Kodama

    Full Text Available Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence. As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.

  7. A fluorescence turn-on method for real-time monitoring of protease activity based on the electron transfer between a fluorophore labeled oligonucleotide and cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Dongli [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022 (China); University of Chinese Academy of Sciences, Beijing, 100049 (China); Li, Yongxin; Chen, Jian [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022 (China); Yu, Cong, E-mail: congyu@ciac.jl.cn [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022 (China)

    2013-06-19

    Graphical abstract: -- Highlights: •Rapid detection of protease activity with fairly good sensitivity and selectivity. •A turn-on fluorescence assay reduces the likelihood of false positive signals. •Cytochrome c efficiently quench the fluorescence of the FAM single stranded DNA. •The enzymatic reaction could be monitored in real-time. •The assay could be used for the screening of potential protease inhibitors. -- Abstract: A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC{sub 50} values are 3.23 μg mL{sup −1} and 0.303 μg mL{sup −1} for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.

  8. Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120.

    Science.gov (United States)

    Videau, Patrick; Oshiro, Reid T; Cozy, Loralyn M; Callahan, Sean M

    2014-09-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of 'new' (recently differentiated) and 'old' (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.

  9. Fluorescence Lifetime Imaging System for in Vivo Studies

    Directory of Open Access Journals (Sweden)

    Moinuddin Hassan

    2007-07-01

    Full Text Available In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA. Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH, making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]. Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.

  10. Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

    Science.gov (United States)

    Lien, Chi-Hsiang; Abrigo, Gerald; Chen, Pei-Hsuan; Chien, Fan-Ching

    2017-02-01

    Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore-labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0±0.7 μm and the shifting distance of the temporal focal plane was less than 0.95 μm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

  11. Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing.

    Science.gov (United States)

    Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G; Menon, Rajesh; Gerton, Jordan M; Jorgensen, Erik M; Zalevsky, Zeev

    2013-12-16

    Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

  12. Two-photon excited fluorescence of intrinsic fluorophores enables label-free assessment of adipose tissue function

    Science.gov (United States)

    Alonzo, Carlo Amadeo; Karaliota, Sevasti; Pouli, Dimitra; Liu, Zhiyi; Karalis, Katia P.; Georgakoudi, Irene

    2016-08-01

    Current methods for evaluating adipose tissue function are destructive or have low spatial resolution. These limit our ability to assess dynamic changes and heterogeneous responses that occur in healthy or diseased subjects, or during treatment. Here, we demonstrate that intrinsic two-photon excited fluorescence enables functional imaging of adipocyte metabolism with subcellular resolution. Steady-state and time-resolved fluorescence from intracellular metabolic co-factors and lipid droplets can distinguish the functional states of excised white, brown, and cold-induced beige fat. Similar optical changes are identified when white and brown fat are assessed in vivo. Therefore, these studies establish the potential of non-invasive, high resolution, endogenous contrast, two-photon imaging to identify distinct adipose tissue types, monitor their functional state, and characterize heterogeneity of induced responses.

  13. Mueller-matrix mapping of optically anisotropic fluorophores of molecular biological tissues in the diagnosis of death causes

    Science.gov (United States)

    Ushenko, A. G.; Dubolazov, A. V.; Ushenko, V. A.; Ushenko, Yu. A.; Pidkamin, L. Y.; Soltys, I. V.; Zhytaryuk, V. G.; Pavlyukovich, N.

    2016-09-01

    A model of generalized optical anisotropy of polycrystalline networks of albumin and globulin of human brain liquor has been suggested. The polarization-phase method of spatial and frequency differentiation of linear and circular birefringence coordinate distributions have been analytically substantiated. A set of criteria of the dynamics of necrotic changes of polarization-phase images of liquor polycrystalline films for determination of death coming prescription has been detected and substantiated.

  14. 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.

    Directory of Open Access Journals (Sweden)

    David Baddeley

    Full Text Available BACKGROUND: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. METHODOLOGY/PRINCIPAL FINDINGS: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev. while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev. was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk. CONCLUSIONS/SIGNIFICANCE: Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

  15. In vivo neuronal calcium imaging in C. elegans.

    Science.gov (United States)

    Chung, Samuel H; Sun, Lin; Gabel, Christopher V

    2013-04-10

    The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon and GCaMP allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

  16. Fluorescence Imaging In Vivo up to 1700 nm

    CERN Document Server

    Diao, Shuo; Hong, Guosong; Antaris, Alexander L; Chang, Junlei; Wu, Justin Z; Zhang, Bo; Kuo, Calvin J; Dai, Hongjie

    2015-01-01

    Compared to visible and near-infrared regions below ~ 900 nm, imaging in the second near-infrared window beyond 1000 nm (NIR-II, 1000-1700 nm) is promising for deep-tissue high-resolution optical imaging in vivo owing to reduced scattering of photons traversing through tissues. Here, we succeeded fluorescence imaging in vivo in the long 1500-1700 nm (NIR-IIb) region using a novel, chemical separation enriched large-diameter semiconducting single-walled carbon nanotube material. Imaging in the 1500-1700 nm window resolved 3-4 um wide capillary blood vessels at ~ 3 millimeters depth through the intact body and brain of mice with the ability of blood-flow speed mapping in individual capillary vessels. Further, non-invasive single fluorophore imaging inside the tumor of a live mouse was achieved in the 1500-1700 nm window. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting up to 1700 nm for a new paradigm of high performance in vivo optical imaging.

  17. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

    2011-07-01

    Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

  18. Tomographic imaging of flourescence resonance energy transfer in highly light scattering media

    Science.gov (United States)

    Soloviev, Vadim Y.; McGinty, James; Tahir, Khadija B.; Laine, Romain; Stuckey, Daniel W.; Mohan, P. Surya; Hajnal, Joseph V.; Sardini, Alessandro; French, Paul M. W.; Arridge, Simon R.

    2010-02-01

    Three-dimensional localization of protein conformation changes in turbid media using Förster Resonance Energy Transfer (FRET) was investigated by tomographic fluorescence lifetime imaging (FLIM). FRET occurs when a donor fluorophore, initially in its electronic excited state, transfers energy to an acceptor fluorophore in close proximity through non-radiative dipole-dipole coupling. An acceptor effectively behaves as a quencher of the donor's fluorescence. The quenching process is accompanied by a reduction in the quantum yield and lifetime of the donor fluorophore. Therefore, FRET can be localized by imaging changes in the quantum yield and the fluorescence lifetime of the donor fluorophore. Extending FRET to diffuse optical tomography has potentially important applications such as in vivo studies in small animal. We show that FRET can be localized by reconstructing the quantum yield and lifetime distribution from time-resolved non-invasive boundary measurements of fluorescence and transmitted excitation radiation. Image reconstruction was obtained by an inverse scattering algorithm. Thus we report, to the best of our knowledge, the first tomographic FLIM-FRET imaging in turbid media. The approach is demonstrated by imaging a highly scattering cylindrical phantom concealing two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically-encoded calcium FRET sensor. A 10mM calcium chloride solution was added to one of the wells to induce a protein conformation change upon binding to TN-L15, resulting in FRET and a corresponding decrease in the donor fluorescence lifetime. The resulting fluorescence lifetime distribution, the quantum efficiency, absorption and scattering coefficients were reconstructed.

  19. Structure-Guided Design and Synthesis of a Mitochondria-Targeting Near-Infrared Fluorophore with Multimodal Therapeutic Activities.

    Science.gov (United States)

    Tan, Xu; Luo, Shenglin; Long, Lei; Wang, Yu; Wang, Dechun; Fang, Shengtao; Ouyang, Qin; Su, Yongping; Cheng, Tianmin; Shi, Chunmeng

    2017-10-05

    An urgent challenge for imaging-guided disease-targeted multimodal therapy is to develop the appropriate multifunctional agents to meet the requirements for potential applications. Here, a rigid cyclohexenyl substitution in the middle of a polymethine linker and two asymmetrical amphipathic N-alkyl side chains to indocyanine green (ICG) (the only FDA-approved NIR contrast agent) are introduced, and a new analog, IR-DBI, is developed with simultaneous cancer-cell mitochondrial targeting, NIR imaging, and chemo-/PDT/PTT/multimodal therapeutic activities. The asymmetrical and amphipathic structural modification renders IR-DBI a close binding to albumin protein site II to form a drug-protein complex and primarily facilitates its preferential accumulation at tumor sites via the enhanced permeability and retention (EPR) effect. The released IR-DBI dye is further actively taken up by cancer cells through organic-anion-transporting polypeptide transporters, and the lipophilic cationic property leads to its selective accumulation in the mitochondria of cancer cells. Finally, based on the high albumin-binding affinity, IR-DBI is modified into human serum albumin (HSA) via self-assembly to produce a nanosized complex, which exhibits significant improvement in the cancer targeting and multimodal cancer treatment with better biocompatibility. This finding may present a practicable strategy to develop small-molecule-based cancer theranostic agents for simultaneous cancer diagnostics and therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Fluorescence and Bioluminescence Imaging of Orthotopic Brain Tumors in Mice.

    Science.gov (United States)

    McKinnon, Emilie; Moore, Alfred; Dixit, Suraj; Zhu, Yun; Broome, Ann-Marie

    2017-01-01

    Optical imaging strategies, such as fluorescence and bioluminescence imaging, are non-invasive, in vivo whole body imaging techniques utilized to study cancer. Optical imaging is widely used in preclinical work because of its ease of use and cost-friendliness. It also provides the opportunity to study animals and biological responses longitudinally over time. Important considerations include depth of tissue penetration, photon scattering, absorption and the choice of light emitting probe, all of which affect the resolution (image quality and data information) and the signal to noise ratio of the image. We describe how to use bioluminescence and fluorescence imaging to track a chemotherapeutic delivery nanocarrier conjugated with a fluorophore to determine its localization in vivo.

  1. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    CERN Document Server

    Sivankutty, Siddharth; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2013-01-01

    We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modi?cation on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve high resolution surface imaging, especially for membrane imaging in biological samples

  2. Fluorescence polarization imaging for delineating nonmelanoma skin cancers

    Science.gov (United States)

    Yaroslavsky, A. N.; Neel, V.; Anderson, R. R.

    2004-09-01

    We present a method for detecting nonmelanoma skin cancers using exogenous fluorescence polarization. We built an automated system that permits exogenous fluorescence polarization imaging. It includes a tunable linearly polarized monochromatic light source and a CCD camera equipped with a rotating linear polarizer and a filter to reject excitation light. Two fluorophores that are retained in tumors, toluidine blue and methylene blue, are employed. We demonstrate that fluorescence polarization imaging can be used for accurate delineation of nonmelanoma cancers. The results suggest that this optical technique may be suitable for real-time noninvasive demarcation of epithelial cancers.

  3. Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing.

    Science.gov (United States)

    Tonniges, J; Hansen, M; Duncan, J; Bassett, M J; Fritzsch, B; Gray, B D; Easwaran, A; Nichols, M G

    2010-08-01

    Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue.

  4. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

  5. Single-wavelength-controlled in situ dynamic super-resolution fluorescence imaging for block copolymer nanostructures via blue-light-switchable FRAP.

    Science.gov (United States)

    Gong, Wen-Liang; Yan, Jie; Zhao, Ling-Xi; Li, Chong; Huang, Zhen-Li; Tang, Ben Zhong; Zhu, Ming-Qiang

    2016-11-02

    Photoswitchable fluorophores are promising in single-molecule optical devices and super-resolution fluorescence imaging, especially in single-molecule photo-activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). However, the scarcity of current photoswitchable fluorophores stimulates researchers to develop complicated optical systems and processing software, in accordance with the limited photoswitchable fluorescent proteins and organic fluorophores. Previous efforts to develop synthetic photoswitchable fluorophores have exhibited their promising potential in super-resolution fluorescence imaging. Here, we have designed and synthesized a fluorescence molecular switch with reversible green emission, a napthalimide-hexaarylbiimidazole conjugate (NI-N-HABI), which exhibits strong fluorescence in the emissive state, with fast thermal fading of the photochromism and spontaneous fluorescence recovery after photobleaching (FRAP) induced by blue-light. The photoswitchable fluorophore enables the red-edge wavelength of the optical response to red-shift from the initial near-UV region at less than 400 nm, to 500 nm. The relatively fast fading speed of NI-N-HABI and its sensitivity to longer blue-light irradiation (400-500 nm) have allowed simplification of the optical microscopic system from a two-wavelength laser source to a single-wavelength laser. We applied NI-N-HABI in single-wavelength-controlled in situ dynamic super-resolution fluorescence imaging for the self-assembly and solvent annealing of amphiphilic block polymers, with 50 nm of optical resolution. Single-wavelength-controlled dynamic super-resolution fluorescence imaging facilitates nanoscale optical visualization for the dynamic physical and chemical fluctuation processes of stimuli-responsive nanostructures.

  6. Errors in confocal fluorescence ratiometric imaging microscopy due to chromatic aberration.

    Science.gov (United States)

    Lin, Yuxiang; Gmitro, Arthur F

    2011-01-01

    Confocal fluorescence ratiometric imaging is an optical technique used to measure a variety of important biological parameters. A small amount of chromatic aberration in the microscope system can introduce a variation in the signal ratio dependent on the fluorophore concentration gradient along the optical axis and lead to bias in the measurement. We present a theoretical model of this effect. Experimental results and simulations clearly demonstrate that this error can be significant and should not be ignored.

  7. In vivo imaging of cancer cells with electroporation of quantum dots and multispectral imaging

    Science.gov (United States)

    Yoo, Jung Sun; Won, Nayoun; Kim, Hong Bae; Bang, Jiwon; Kim, Sungjee; Ahn, Saeyoung; Soh, Kwang-Sup

    2010-06-01

    Our understanding of dissemination and growth of cancer cells is limited by our inability for long-term followup of this process in vivo. Fluorescence molecular imaging has the potential to track cancer cells with high contrast and sensitivity in living animals. For this purpose, intracellular delivery of near-infrared fluorescence quantum dots (QDs) by electroporation offers considerable advantages over organic fluorophores and other cell tagging methods. In this research we developed a multispectral imaging system that could eliminate two major parameters compromising in vivo fluorescence imaging performance, i.e., variations in the tissue optical properties and tissue autofluorescence. We demonstrated that electroporation of QDs and multispectral imaging allowed in vivo assessment of cancer development and progression in the xenograft mouse tumor model for more than 1 month, providing a powerful means to learn more about the biology of cancer and metastasis.

  8. Optical molecular imaging for detection of Barrett's-associated neoplasia

    Institute of Scientific and Technical Information of China (English)

    Nadhi Thekkek; Sharmila Anandasabapathy; Rebecca Richards-Kortum

    2011-01-01

    Recent advancements in the endoscopic imaging of Barrett's esophagus can be used to probe a wide range of optical properties that are altered with neoplastic progression.This review summarizes relevant changes in optical properties as well as imaging approaches that measures those changes.Wide-field imaging approaches include narrow-band imaging that measures changes in light scattering and absorption,and autofluorescence imaging that measure changes in endogenous fluorophores.High-resolution imaging approaches include optical coherence tomography,endocytoscopy,confocal microendoscopy,and high-resolution microendoscopy.These technologies,some coupled with an appropriate contrast agent,can measure differences in glandular morphology,nuclear morphology,or vascular alterations associated with neoplasia.Advances in targeted contrast agents are further discussed.Studies that have explored these technologies are highlighted;as are the advantages and limitations of each.

  9. PolyA-tailed and fluorophore-labeled aptamer-gold nanoparticle conjugate for fluorescence turn-on bioassay using iodide-induced ligand displacement.

    Science.gov (United States)

    Li, Wei; Dong, Yifan; Wang, Xi; Li, Hui; Xu, Danke

    2015-04-15

    Depending on the strong affinity of polyA sequence to gold (or silver) surface, applicability of polyA-tailed DNA-gold (or silver) nanoparticle conjugates in homogeneous and heterogeneous protein assays was first demonstrated. Interestingly, when using polyA-tailed, fluophore-labeled DNA-AuNP conjugate, it was found that iodide and thiosulfate anions could act as the ligand displacing reagent to detach polyA-tailed DNA strands from AuNP surface and simultaneously activate the AuNP-quenched fluorophores by destroying the polyA-AuNP interaction via a divide-and-conquer strategy. Based on this new discovery, we have developed a novel, cost-effective and sandwich-type fluorescence turn-on aptasensor for highly sensitive and specific thrombin detection, what took advantage of aptamer-conjugated magnetic beads (apt-MBs) for protein capture and separation, and iodide-induced fluorescence recovery of activatable polyA-based AuNP probes through ligand displacement for fluorescence turn-on detection. This proposed aptasensor could detect thrombin specifically with a detection limit as low as 89pM, which was better than or comparable to many existing fluorescent thrombin assays. Importantly, employment of such polyA-based AuNP conjugate not only avoids the use of thiolated oligonucleotides and thiol-containing displacing reagents, but also offers new possibilities for fabricating convenient and cost-effective bioanalytical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. IMAGES, IMAGES, IMAGES

    Energy Technology Data Exchange (ETDEWEB)

    Marcus, A.

    1980-07-01

    The role of images of information (charts, diagrams, maps, and symbols) for effective presentation of facts and concepts is expanding dramatically because of advances in computer graphics technology, increasingly hetero-lingual, hetero-cultural world target populations of information providers, the urgent need to convey more efficiently vast amounts of information, the broadening population of (non-expert) computer users, the decrease of available time for reading texts and for decision making, and the general level of literacy. A coalition of visual performance experts, human engineering specialists, computer scientists, and graphic designers/artists is required to resolve human factors aspects of images of information. The need for, nature of, and benefits of interdisciplinary effort are discussed. The results of an interdisciplinary collaboration are demonstrated in a product for visualizing complex information about global energy interdependence. An invited panel will respond to the presentation.

  11. In vitro and in vivo on-chip biofluorescence imaging using a CMOS image sensor

    Science.gov (United States)

    Ng, David C.; Matsuo, Masamichi; Tokuda, Takashi; Kagawa, Keiichiro; Nunoshita, Masahiro; Ohta, Jun

    2006-02-01

    We have designed and fabricated a 176×144-pixels (QCIF) CMOS image sensor for on-chip bio-fluorescence imaging of the mouse brain. In our approach, a single CMOS image sensor chip without additional optics is used. This enables imaging at arbitrary depths into the brain; a clear advantage compared to existing optical microscopy methods. Packaging of the chip represents a challenge for in vivo imaging. We developed a novel packaging process whereby an excitation filter is applied onto the sensor. This eliminates the use of a filter cube found in conventional fluorescence microscopes. The fully packaged chip is about 350 μm thick. Using the device, we demonstrated in vitro on-chip fluorescence imaging of a 400 μm thick mouse brain slice detailing the hippocampus. The image obtained compares favorably to the image captured by conventional microscopes in terms of image resolution. In order to study imaging in vivo, we also developed a phantom media. In situ fluorophore measurement shows that detection through the turbid medium of up to 1 mm thickness is possible. We have successfully demonstrated imaging deep into the hippocampal region of the mouse brain where quantitative fluorometric measurements was made. This work is expected to lead to a promising new tool for imaging the brain in vivo.

  12. Nanoparticle imaging probes for molecular imaging with computed tomography and application to cancer imaging

    Science.gov (United States)

    Roeder, Ryan K.; Curtis, Tyler E.; Nallathamby, Prakash D.; Irimata, Lisa E.; McGinnity, Tracie L.; Cole, Lisa E.; Vargo-Gogola, Tracy; Cowden Dahl, Karen D.

    2017-03-01

    Precision imaging is needed to realize precision medicine in cancer detection and treatment. Molecular imaging offers the ability to target and identify tumors, associated abnormalities, and specific cell populations with overexpressed receptors. Nuclear imaging and radionuclide probes provide high sensitivity but subject the patient to a high radiation dose and provide limited spatiotemporal information, requiring combined computed tomography (CT) for anatomic imaging. Therefore, nanoparticle contrast agents have been designed to enable molecular imaging and improve detection in CT alone. Core-shell nanoparticles provide a powerful platform for designing tailored imaging probes. The composition of the core is chosen for enabling strong X-ray contrast, multi-agent imaging with photon-counting spectral CT, and multimodal imaging. A silica shell is used for protective, biocompatible encapsulation of the core composition, volume-loading fluorophores or radionuclides for multimodal imaging, and facile surface functionalization with antibodies or small molecules for targeted delivery. Multi-agent (k-edge) imaging and quantitative molecular imaging with spectral CT was demonstrated using current clinical agents (iodine and BaSO4) and a proposed spectral library of contrast agents (Gd2O3, HfO2, and Au). Bisphosphonate-functionalized Au nanoparticles were demonstrated to enhance sensitivity and specificity for the detection of breast microcalcifications by conventional radiography and CT in both normal and dense mammary tissue using murine models. Moreover, photon-counting spectral CT enabled quantitative material decomposition of the Au and calcium signals. Immunoconjugated Au@SiO2 nanoparticles enabled highly-specific targeting of CD133+ ovarian cancer stem cells for contrast-enhanced detection in model tumors.

  13. Sunlight-induced changes in chromophores and fluorophores of wastewater-derived organic matter in receiving waters--the role of salinity.

    Science.gov (United States)

    Yang, Xiaofang; Meng, Fangang; Huang, Guocheng; Sun, Li; Lin, Zheng

    2014-10-01

    Wastewater-derived organic matter (WOM) is an important constituent of discharge to urban rivers and is suspected of altering the naturally occurring dissolved organic matter (DOM) in water systems. This study investigated sunlight-induced changes in chromophores and fluorophores of WOM with different salinities (S = 0, 10, 20 and 30) that were collected from two wastewater treatment plants (WWTP-A and WWTP-B). The results showed that exposure to sunlight for 5.3 × 10(5) J/m(2) caused significant decreases in UV254-absorbing WOM (45-59% loss) compared to gross dissolved organic carbon (<15% loss). An increase in salinity accelerated the overall photo-degradation rates of the UV254-absorbing chromophores from both WOM and natural DOM. In addition, irradiated WOM at a higher salinity had a larger molecular size than that at a lower salinity. However, natural DOM did not display such behavior. Parallel factor analysis of the excitation-emission matrix determined the presence of two humic-like components (C1 and C2) and two protein-like components (C3 and C4). All the components in WOM followed second-order kinetics, except for the C4 component in WWTP-A, which fit zero-order photoreaction kinetics. The photo-degradation of the C1 component in both WWTPs appeared to be independent of salinity; however, the photo-degradation rates of the C2 and C3 components in both WWTPs and C4 in WWTP-B increased significantly with increasing salinity. In comparison, the photo-degradation of the C1 component was significantly facilitated by increased salinity in natural DOM, fitting first-order photoreaction kinetics. As such, the current knowledge concerning the photo-degradation of naturally occurring DOM cannot be extrapolated for the understanding of WOM photo-degradation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

    Science.gov (United States)

    Wong, Wai-Ting; Chan, Kwok-Chu; So, Pui-Kin; Yap, Hong-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Wong, Kwok-Yin; Zhao, Yanxiang

    2011-09-09

    The Ω-loop at the active site of β-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of β-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered β-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the Ω-loop and to detect the binding of diverse substrates. Our results show that this engineered β-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the Ω-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the Ω-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum β-lactam antibiotics. The oxyimino-cephalosporin-specific fluorescence profile of our engineered β-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.

  15. A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium.

    Science.gov (United States)

    Chernomordik, Victor; Gandjbakhche, Amir H; Hassan, Moinuddin; Pajevic, Sinisa; Weiss, George H

    2010-12-01

    We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the object being to estimate the position and lifetime of the fluorophore. Such information can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.

  16. Bright fluorescent chemosensor platforms for imaging endogenous pools of neuronal zinc.

    Science.gov (United States)

    Chang, Christopher J; Nolan, Elizabeth M; Jaworski, Jacek; Burdette, Shawn C; Sheng, Morgan; Lippard, Stephen J

    2004-02-01

    A series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn(2+). A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of "off-on" photoinduced electron transfer (PET) probes by controlling fluorophore pK(a) values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn(2+); the novel ZP3 fluorophore images endogenous stores of Zn(2+) in live hippocampal neurons and slices, including the first fluorescence detection of Zn(2+) in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pK(a) can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn(2+) in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications.

  17. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

    Science.gov (United States)

    Ha, Taekjip; Tinnefeld, Philip

    2012-05-01

    Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own “personalities” regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

  18. Statistical image segmentation for the detection of skin lesion borders in UV fluorescence excitation

    Science.gov (United States)

    Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Franco, Walfre

    2016-04-01

    The skin contains several fluorescent molecules or fluorophores that serve as markers of structure, function and composition. UV fluorescence excitation photography is a simple and effective way to image specific intrinsic fluorophores, such as the one ascribed to tryptophan which emits at a wavelength of 345 nm upon excitation at 295 nm, and is a marker of cellular proliferation. Earlier, we built a clinical UV photography system to image cellular proliferation. In some samples, the naturally low intensity of the fluorescence can make it difficult to separate the fluorescence of cells in higher proliferation states from background fluorescence and other imaging artifacts -- like electronic noise. In this work, we describe a statistical image segmentation method to separate the fluorescence of interest. Statistical image segmentation is based on image averaging, background subtraction and pixel statistics. This method allows to better quantify the intensity and surface distributions of fluorescence, which in turn simplify the detection of borders. Using this method we delineated the borders of highly-proliferative skin conditions and diseases, in particular, allergic contact dermatitis, psoriatic lesions and basal cell carcinoma. Segmented images clearly define lesion borders. UV fluorescence excitation photography along with statistical image segmentation may serve as a quick and simple diagnostic tool for clinicians.

  19. Vibrational imaging based on stimulated Raman scattering microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nandakumar, P; Kovalev, A; Volkmer, A [3. Physikalisches Institut, Universitaet Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart (Germany)], E-mail: a.volkmer@physik.uni-stuttgart.de

    2009-03-15

    A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.

  20. Vibrational imaging based on stimulated Raman scattering microscopy

    Science.gov (United States)

    Nandakumar, P.; Kovalev, A.; Volkmer, A.

    2009-03-01

    A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.

  1. Phi optics: from image to knowledge (Conference Presentation)

    Science.gov (United States)

    Chiritescu, Catalin

    2016-03-01

    Optical microscopy of live cells and tissues provides the main insight for life science researchers in academia and bio-pharma. The cells have very small features, are transparent, and require long term observations (hours to days) to measure the effects of drugs and diseases. New technologies - under the umbrella term of Quantitative Phase Imaging (QPI) - have come to light in the past decade to challenge and complement the current state of the art solutions that use fluorophores. Phi Optics talk will outline their lessons learned in the process of bringing an academic idea to the commercial space.

  2. Nanobiodevices for Biomolecule Analysis and Imaging

    Science.gov (United States)

    Yasui, Takao; Kaji, Noritada; Baba, Yoshinobu

    2013-06-01

    Nanobiodevices have been developed to analyze biomolecules and cells for biomedical applications. In this review, we discuss several nanobiodevices used for disease-diagnostic devices, molecular imaging devices, regenerative medicine, and drug-delivery systems and describe the numerous advantages of nanobiodevices, especially in biological, medical, and clinical applications. This review also outlines the fabrication technologies for nanostructures and nanomaterials, including top-down nanofabrication and bottom-up molecular self-assembly approaches. We describe nanopillar arrays and nanowall arrays for the ultrafast separation of DNA or protein molecules and nanoball materials for the fast separation of a wide range of DNA molecules, and we present examples of applications of functionalized carbon nanotubes to obtain information about subcellular localization on the basis of mobility differences between free fluorophores and fluorophore-labeled carbon nanotubes. Finally, we discuss applications of newly synthesized quantum dots to the screening of small interfering RNA, highly sensitive detection of disease-related proteins, and development of cancer therapeutics and diagnostics.

  3. Bioorthogonal Chemical Imaging for Biomedicine

    Science.gov (United States)

    Min, Wei

    2017-06-01

    Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because relatively bulky fluorescent labels could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, we have developed a bioorthogonal chemical imaging platform. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, multiplicity and biocompatibility for imaging small biomolecules in live systems including tissues and organisms. Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. This bioorthogonal chemical imaging platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, further chemical and spectroscopic strategies allow for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species, bringing small molecules under the illumination of modern light microscopy.

  4. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging.

    Science.gov (United States)

    Geissbuehler, Stefan; Sharipov, Azat; Godinat, Aurélien; Bocchio, Noelia L; Sandoz, Patrick A; Huss, Anja; Jensen, Nickels A; Jakobs, Stefan; Enderlein, Jörg; Gisou van der Goot, F; Dubikovskaya, Elena A; Lasser, Theo; Leutenegger, Marcel

    2014-12-18

    Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm(3) without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.

  5. Label-free nonlinear optical imaging of mouse retina.

    Science.gov (United States)

    He, Sicong; Ye, Cong; Sun, Qiqi; Leung, Christopher K S; Qu, Jianan Y

    2015-03-01

    A nonlinear optical (NLO) microscopy system integrating stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was developed to image fresh mouse retinas. The morphological and functional details of various retinal layers were revealed by the endogenous NLO signals. Particularly, high resolution label-free imaging of retinal neurons and nerve fibers in the ganglion cell and nerve fiber layers was achieved by capturing endogenous SRS and TPEF signals. In addition, the spectral and temporal analysis of TPEF images allowed visualization of different fluorescent components in the retinal pigment epithelium (RPE). Fluorophores with short TPEF lifetime, such as A2E, can be differentiated from other long-lifetime components in the RPE. The NLO imaging method would provide important information for investigation of retinal ganglion cell degeneration and holds the potential to study the biochemical processes of visual cycle in the RPE.

  6. Binding of heavy metal ions in aggregates of microbial cells, EPS and biogenic iron minerals measured in-situ using metal- and glycoconjugates-specific fluorophores

    Science.gov (United States)

    Hao, Likai; Guo, Yuan; Byrne, James M.; Zeitvogel, Fabian; Schmid, Gregor; Ingino, Pablo; Li, Jianli; Neu, Thomas R.; Swanner, Elizabeth D.; Kappler, Andreas; Obst, Martin

    2016-05-01

    Aggregates consisting of bacterial cells, extracellular polymeric substances (EPS) and Fe(III) minerals formed by Fe(II)-oxidizing bacteria are common at bulk or microscale chemical interfaces where Fe cycling occurs. The high sorption capacity and binding capacity of cells, EPS, and minerals controls the mobility and fate of heavy metals. However, it remains unclear to which of these component(s) the metals will bind in complex aggregates. To clarify this question, the present study focuses on 3D mapping of heavy metals sorbed to cells, glycoconjugates that comprise the majority of EPS constituents, and Fe(III) mineral aggregates formed by the phototrophic Fe(II)-oxidizing bacteria Rhodobacter ferrooxidans SW2 using confocal laser scanning microscopy (CLSM) in combination with metal- and glycoconjugates-specific fluorophores. The present study evaluated the influence of glycoconjugates, microbial cell surfaces, and (biogenic) Fe(III) minerals, and the availability of ferrous and ferric iron on heavy metal sorption. Analyses in this study provide detailed knowledge on the spatial distribution of metal ions in the aggregates at the sub-μm scale, which is essential to understand the underlying mechanisms of microbe-mineral-metal interactions. The heavy metals (Au3+, Cd2+, Cr3+, CrO42-, Cu2+, Hg2+, Ni2+, Pd2+, tributyltin (TBT) and Zn2+) were found mainly sorbed to cell surfaces, present within the glycoconjugates matrix, and bound to the mineral surfaces, but not incorporated into the biogenic Fe(III) minerals. Statistical analysis revealed that all ten heavy metals tested showed relatively similar sorption behavior that was affected by the presence of sorbed ferrous and ferric iron. Results in this study showed that in addition to the mineral surfaces, both bacterial cell surfaces and the glycoconjugates provided most of sorption sites for heavy metals. Simultaneously, ferrous and ferric iron ions competed with the heavy metals for sorption sites on the organic

  7. Increasing the imaging depth through computational scattering correction (Conference Presentation)

    Science.gov (United States)

    Koberstein-Schwarz, Benno; Omlor, Lars; Schmitt-Manderbach, Tobias; Mappes, Timo; Ntziachristos, Vasilis

    2016-03-01

    Imaging depth is one of the most prominent limitations in light microscopy. The depth in which we are still able to resolve biological structures is limited by the scattering of light within the sample. We have developed an algorithm to compensate for the influence of scattering. The potential of algorithm is demonstrated on a 3D image stack of a zebrafish embryo captured with a selective plane illumination microscope (SPIM). With our algorithm we were able shift the point in depth, where scattering starts to blur the imaging and effect the image quality by around 30 µm. For the reconstruction the algorithm only uses information from within the image stack. Therefore the algorithm can be applied on the image data from every SPIM system without further hardware adaption. Also there is no need for multiple scans from different views to perform the reconstruction. The underlying model estimates the recorded image as a convolution between the distribution of fluorophores and a point spread function, which describes the blur due to scattering. Our algorithm performs a space-variant blind deconvolution on the image. To account for the increasing amount of scattering in deeper tissue, we introduce a new regularizer which models the increasing width of the point spread function in order to improve the image quality in the depth of the sample. Since the assumptions the algorithm is based on are not limited to SPIM images the algorithm should also be able to work on other imaging techniques which provide a 3D image volume.

  8. In vivo optical imaging to visualize photodynamic therapy-induced immune responses

    Science.gov (United States)

    Mitra, Soumya; Foster, Thomas H.

    2009-02-01

    Motivated by recent successes in growing intradermal tumors in the ears of mice and establishing the feasibility of in vivo confocal imaging of anatomic vessels in these tumors using fluorophore-conjugated antibodies to CD31, we are exploring a number of applications of optical fluorescence imaging in superficial murine tumor models in vivo. Immune responses induced by photodynamic therapy (PDT) are dynamic processes that occur in a spatially and temporally specific manner. To visualize these processes noninvasively, we have made progress in developing optical molecular imaging strategies that take advantage of intradermal injection of fluorophore-conjugated-antibodies against surface antigens on immune cells. This enables confocal imaging of the fluorescently labeled host cells to depths of at least 100 microns, and using this technique we have achieved in vivo imaging of granulocyte (GR-1)- and major histocompatibility complex class II (MHC-II)-positive cell trafficking in tumors in response to PDT. The latter include macrophages and dendritic cells. Data from tumors that were subjected to PDT with the photosensitizer, HPPH, reveals a significantly enhanced level of GR-1+ cell infiltration compared to untreated control tumor. The temporal kinetics of GR-1+ and MHC-II+ cells at different time intervals post-PDT are being examined. The ability to image host responses in vivo without excising or perturbing the tissue has opened up opportunities to explore means of optimizing them to therapeutic advantage.

  9. Optimal parameters for near infrared fluorescence imaging of amyloid plaques in Alzheimer's disease mouse models

    Energy Technology Data Exchange (ETDEWEB)

    Raymond, S B [Harvard-MIT Division of Health Sciences and Technology, 77 Mass Ave., E25-519, Cambridge, MA 02139 (United States); Kumar, A T N; Boas, D A [Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, 149 13th St Charlestown, MA 02129 (United States); Bacskai, B J [Alzheimer' s Research Unit, Department of Neurology, Massachusetts General Hospital, 114 16th St, Charlestown, MA 02129 (United States)], E-mail: bbacskai@partners.org

    2009-10-21

    Amyloid-{beta} plaques are an Alzheimer's disease biomarker which present unique challenges for near-infrared fluorescence tomography because of size (<50 {mu}m diameter) and distribution. We used high-resolution simulations of fluorescence in a digital Alzheimer's disease mouse model to investigate the optimal fluorophore and imaging parameters for near-infrared fluorescence tomography of amyloid plaques. Fluorescence was simulated for amyloid-targeted probes with emission at 630 and 800 nm, plaque-to-background ratios from 1-1000, amyloid burden from 0-10%, and for transmission and reflection measurement geometries. Fluorophores with high plaque-to-background contrast ratios and 800 nm emission performed significantly better than current amyloid imaging probes. We tested idealized fluorophores in transmission and full-angle tomographic measurement schemes (900 source-detector pairs), with and without anatomical priors. Transmission reconstructions demonstrated strong linear correlation with increasing amyloid burden, but underestimated fluorescence yield and suffered from localization artifacts. Full-angle measurements did not improve upon the transmission reconstruction qualitatively or in semi-quantitative measures of accuracy; anatomical and initial-value priors did improve reconstruction localization and accuracy for both transmission and full-angle schemes. Region-based reconstructions, in which the unknowns were reduced to a few distinct anatomical regions, produced highly accurate yield estimates for cortex, hippocampus and brain regions, even with a reduced number of measurements (144 source-detector pairs)

  10. Super-resolved imaging with ultimate time resolution

    CERN Document Server

    Ashida, Yuto

    2015-01-01

    Precisely and accurately locating point objects is a long-standing common thread in science. Super-resolved imaging of single molecules has revolutionized our view of quasi-static nanostructures $\\it{in-vivo}$. A wide-field approach based on localizing individual fluorophores has emerged as a versatile method to surpass the standard resolution limit. In those techniques, the super-resolution is realized by sparse photoactivation and localization together with the statistical analysis based on point spread functions. Nevertheless, the slow temporal resolution of super-resolved imaging severely restricts the utility to the study of live-cell phenomena. Clearly, a major breakthrough to observe fast, nanoscale dynamics needs to be made. Here we present a super-resolved imaging method that achieves the theoretical-limit time resolution. By invoking information theory, we can achieve the robust localization of overlapped light emitters at an order of magnitude faster speed than the conventional super-resolution mic...

  11. Model Convolution: A Computational Approach to Digital Image Interpretation

    Science.gov (United States)

    Gardner, Melissa K.; Sprague, Brian L.; Pearson, Chad G.; Cosgrove, Benjamin D.; Bicek, Andrew D.; Bloom, Kerry; Salmon, E. D.

    2010-01-01

    Digital fluorescence microscopy is commonly used to track individual proteins and their dynamics in living cells. However, extracting molecule-specific information from fluorescence images is often limited by the noise and blur intrinsic to the cell and the imaging system. Here we discuss a method called “model-convolution,” which uses experimentally measured noise and blur to simulate the process of imaging fluorescent proteins whose spatial distribution cannot be resolved. We then compare model-convolution to the more standard approach of experimental deconvolution. In some circumstances, standard experimental deconvolution approaches fail to yield the correct underlying fluorophore distribution. In these situations, model-convolution removes the uncertainty associated with deconvolution and therefore allows direct statistical comparison of experimental and theoretical data. Thus, if there are structural constraints on molecular organization, the model-convolution method better utilizes information gathered via fluorescence microscopy, and naturally integrates experiment and theory. PMID:20461132

  12. A general approach to break the concentration barrier in single-molecule imaging

    KAUST Repository

    Loveland, Anna B.

    2012-09-09

    Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule\\'s signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations. © 2012 Nature America, Inc. All rights reserved.

  13. Application and further development of diffusion based 2D chemical imaging techniques in the rhizosphere

    Science.gov (United States)

    Hoefer, Christoph; Santner, Jakob; Borisov, Sergey; Kreuzeder, Andreas; Wenzel, Walter; Puschenreiter, Markus

    2015-04-01

    Two dimensional chemical imaging of root processes refers to novel in situ methods to investigate and map solutes at a high spatial resolution (sub-mm). The visualization of these solutes reveals new insights in soil biogeochemistry and root processes. We derive chemical images by using data from DGT-LA-ICP-MS (Diffusive Gradients in Thin Films and Laser Ablation Inductively Coupled Plasma Mass Spectrometry) and POS (Planar Optode Sensors). Both technologies have shown promising results when applied in aqueous environment but need to be refined and improved for imaging at the soil-plant interface. Co-localized mapping using combined DGT and POS technologies and the development of new gel combinations are in our focus. DGTs are smart and thin (images of the target area are plotted using imaging software. POS are, similar to DGT, thin sensor foils containing a fluorophore coating depending on the target analyte. The measurement principle is based on excitation of the flourophore by a specific wavelength and emission of the fluorophore depending on the presence of the analyte. The emitted signal is captured using optical filters and a DSLR camera. While DGT analysis is destructive, POS measurements can be performed continuously during the application. Both semi-quantitative techniques allow an in situ application to visualize chemical processes directly at the soil-plant interface. Here, we present a summary of results from rhizotron experiments with different plants in metal contaminated and agricultural soils.

  14. Precise diagnosis in different scenarios using photoacoustic and fluorescence imaging with dual-modality nanoparticles

    Science.gov (United States)

    Peng, Dong; Du, Yang; Shi, Yiwen; Mao, Duo; Jia, Xiaohua; Li, Hui; Zhu, Yukun; Wang, Kun; Tian, Jie

    2016-07-01

    Photoacoustic imaging and fluorescence molecular imaging are emerging as important research tools for biomedical studies. Photoacoustic imaging offers both strong optical absorption contrast and high ultrasonic resolution, and fluorescence molecular imaging provides excellent superficial resolution, high sensitivity, high throughput, and the ability for real-time imaging. Therefore, combining the imaging information of both modalities can provide comprehensive in vivo physiological and pathological information. However, currently there are limited probes available that can realize both fluorescence and photoacoustic imaging, and advanced biomedical applications for applying this dual-modality imaging approach remain underexplored. In this study, we developed a dual-modality photoacoustic-fluorescence imaging nanoprobe, ICG-loaded Au@SiO2, which was uniquely designed, consisting of gold nanorod cores and indocyanine green with silica shell spacer layers to overcome fluorophore quenching. This nanoprobe was examined by both PAI and FMI for in vivo imaging on tumor and ischemia mouse models. Our results demonstrated that the nanoparticles can specifically accumulate at the tumor and ischemic areas and be detected by both imaging modalities. Moreover, this dual-modality imaging strategy exhibited superior advantages for a precise diagnosis in different scenarios. The new nanoprobe with the dual-modality imaging approach holds great potential for diagnosis and stage classification of tumor and ischemia related diseases.Photoacoustic imaging and fluorescence molecular imaging are emerging as important research tools for biomedical studies. Photoacoustic imaging offers both strong optical absorption contrast and high ultrasonic resolution, and fluorescence molecular imaging provides excellent superficial resolution, high sensitivity, high throughput, and the ability for real-time imaging. Therefore, combining the imaging information of both modalities can provide

  15. MMP-2/9-Specific Activatable Lifetime Imaging Agent

    Directory of Open Access Journals (Sweden)

    Marcus T.M. Rood

    2015-05-01

    Full Text Available Optical (molecular imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy3 and Cy5 luminescence and a restoration of Ir(ppy3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents.

  16. Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

    Science.gov (United States)

    Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J. Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

    2016-02-01

    Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery

  17. Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

    2016-12-01

    Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

  18. A detection instrument for enhanced-fluorescence and label-free imaging on photonic crystal surfaces.

    Science.gov (United States)

    Block, Ian D; Mathias, Patrick C; Ganesh, Nikhil; Jones, Sarah I; Dorvel, Brian R; Chaudhery, Vikram; Vodkin, Lila O; Bashir, Rashid; Cunningham, Brian T

    2009-07-20

    We report on the design and demonstration of an optical imaging system capable of exciting surface-bound fluorophores within the resonant evanescent electric field of a photonic crystal surface and gathering fluorescence emission that is directed toward the imaging objective by the photonic crystal. The system also has the ability to quantify shifts in the local resonance angle induced by the adsorption of biomolecules on the photonic crystal surface for label-free biomolecular imaging. With these two capabilities combined within a single detection system, we demonstrate label-free images self-registered to enhanced fluorescence images with 328x more sensitive fluorescence detection relative to a glass surface. This technique is applied to a DNA microarray where label-free quantification of immobilized capture DNA enables improved quality control and subsequent enhanced fluorescence detection of dye-tagged hybridized DNA yields 3x more genes to be detected versus commercially available microarray substrates.

  19. Biosynthetic labeling and two-color imaging of phospholipids in cells.

    Science.gov (United States)

    Jao, Cindy Y; Roth, Mary; Welti, Ruth; Salic, Adrian

    2015-02-09

    Phospholipids with a choline head group are abundant components of all biological membranes, performing critical functions in cellular structure, metabolism, and signaling. In spite of their importance, our ability to visualize choline phospholipids in vivo remains very limited. We present a simple and robust chemical strategy to image choline phospholipids, based on the metabolic incorporation of azidocholine analogues, that accurately reflects the normal biosynthetic incorporation of choline into cellular phospholipids. Azidocholine-labeled phospholipids can be imaged in cells with high sensitivity and resolution, following derivatization with fluorophores, by bio-orthogonal chemical reactions compatible with live-cell imaging. We used this method to visualize the subcellular localization of choline phospholipids. We also demonstrate that double metabolic labeling with azidocholine and propargylcholine allows sensitive two-color imaging of choline phospholipids. Our method represents a powerful approach to directly image phospholipids, and to study their dynamics in cells and tissues.

  20. Semiconducting Polymer Nanoparticles with Persistent Near-Infrared Luminescence for In Vivo Optical Imaging.

    Science.gov (United States)

    Palner, Mikael; Pu, Kanyi; Shao, Shirley; Rao, Jianghong

    2015-09-21

    Materials with persistent luminescence are attractive for in vivo optical imaging since they have a long lifetime that allows the separation of excitation of fluorophores and image acquisition for time-delay imaging, thus eliminating tissue autofluorescence associated with fluorescence imaging. Persistently luminescent nanoparticles have previously been fabricated from toxic rare-earth metals. This work reports that nanoparticles made of the conjugated polymer MEH-PPV can generate luminescence persisting for an hour upon single excitation. A near-infrared dye was encapsulated in the conjugated polymer nanoparticle to successfully generate persistent near-infrared luminescence through resonance energy transfer. This new persistent luminescence nanoparticles have been demonstrated for optical imaging applications in living mice.

  1. Optical fiber based imaging of bioengineered tissue construct

    Science.gov (United States)

    Sapoznik, Etai; Niu, Guoguang; Lu, Peng; Zhou, Yu; Xu, Yong; Soker, Shay

    2016-04-01

    Imaging cells and tissues through opaque and turbid media is challenging and presents a major barrier for monitoring maturation and remodeling of bioengineered tissues. The fiber optics based imaging system described here offers a new approach for fluorescent cell imaging. A micro imaging channel is embedded in a Polycaprolactone (PCL) electrospun scaffold designed for cell seeding, which allows us to use an optical fiber to locally deliver excitation laser close to the fluorescent cells. The emission is detected by an Electron Multiplying Charge Coupled Device (EMCCD) detector and image reconstruction of multiple excitation points is achieved with a working distance of several centimeters. The objective of this study is to assess the effects of system parameters on image reconstruction outcomes. Initial studies using fluorescent beads indicated that scaffold thickness had a small effect on image quality, whereas scaffold composition (collagen content), fluorophore spectra, and the reconstruction window size had a large effect. The results also suggest that a far-red fluorescent emission is preferential when using collagenous scaffolds with a thickness of up to 500 μm. Using these optimized parameters, we were able to image fluorescently labeled cells on a scaffold with a resolution of 15-20 μm, and have also measured muscle progenitor cell differentiation and scaffold surface coverage with endothelial cells. In the future, this imaging platform can be applied to other bioengineered tissues for non-invasive monitoring both in vitro and in vivo.

  2. Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

    2017-02-01

    Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

  3. Single-Molecule Spectroscopy, Imaging, and Photocontrol: Foundations for Super-Resolution Microscopy (Nobel Lecture).

    Science.gov (United States)

    Moerner, W E William E

    2015-07-06

    The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super-resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.

  4. Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy*

    Science.gov (United States)

    Moerner, W. E. William E.

    2015-10-01

    The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super-resolution microscopy with single molecules. In the room-temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts as a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and selected current developments are summarized.

  5. Real-time dynamic imaging of virus distribution in vivo.

    Directory of Open Access Journals (Sweden)

    Sean E Hofherr

    Full Text Available The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.

  6. Use of fluorescence lifetime imaging (FLIM) for latent fingerprints detection

    Science.gov (United States)

    Wang, Peng; Chao, Zhi Xia; Seah, Leong K.; Murukeshan, Vadakke M.

    2005-04-01

    Fluorescence lifetime imaging (FLIM) in frequency domain enables the mapping of the spatial distribution of fluorescence lifetimes of a specimen. FLIM can provide unique information about fluorophores and hence is widely used in biology and for medical diagnostics. In this paper, a theoretical analysis for the fluorescence lifetime determination of latent fingerprint samples is described, which is followed by the feasibility study of using FLIM in frequency domain for latent fingerprints detection. Experiments are carried out with fingerprint on green paper substrate and postcard substrate treated with certain fluorescent powder. The total phase lag and demodulation factor are calculated to determine the lifetimes pixel by pixel. The resulting fluorescence lifetime image of fingerprint revealed an improvement in the contrast, and was able to detect the latent fingerprint clearly.

  7. Fluorescence imaging of chromosomal DNA using click chemistry

    Science.gov (United States)

    Ishizuka, Takumi; Liu, Hong Shan; Ito, Kenichiro; Xu, Yan

    2016-09-01

    Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics.

  8. Fluorescence imaging of chromosomal DNA using click chemistry

    Science.gov (United States)

    Ishizuka, Takumi; Liu, Hong Shan; Ito, Kenichiro; Xu, Yan

    2016-01-01

    Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics. PMID:27620982

  9. Tri-modality small animal imaging system

    Energy Technology Data Exchange (ETDEWEB)

    Kundu, B.K.; Stolin, A.V.; Pole, J.; Baumgart, L.; Fontaine, M.; Wojcik, R.; Kross, B.; Zorn, C.; Majewski, S.; Williams, M.B.

    2006-02-01

    Our group is developing a scanner that combines x-ray, single gamma, and optical imaging on the same rotating gantry. Two functional modalities (SPECT and optical) are included because they have different strengths and weaknesses in terms of spatial and temporal decay lengths in the context of in vivo imaging, and because of the recent advent of multiple reporter gene constructs. The effect of attenuation by biological tissue on the detected intensity of the emitted signal was measured for both gamma and optical imaging. Attenuation by biological tissue was quantified for both the bioluminescent emission of luciferace and for the emission light of the near infrared fluorophore cyanine 5.5, using a fixed excitation light intensity. Experiments were performed to test the feasibility of using either single gamma or x-ray imaging to make depth-dependent corrections to the measured optical signal. Our results suggest that significant improvements in quantitation of optical emission are possible using straightforward correction techniques based on information from other modalities. Development of an integrated scanner in which data from each modality are obtained with the animal in a common configuration will greatly simplify this process.

  10. FRET and colocalization analyzer--a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug-in.

    Science.gov (United States)

    Hachet-Haas, Muriel; Converset, Noël; Marchal, Olivier; Matthes, Hans; Gioria, Sophie; Galzi, Jean-Luc; Lecat, Sandra

    2006-12-01

    Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.

  11. Novel Strategy for Preparing Dual-Modality Optical/PET Imaging Probes via Photo-Click Chemistry.

    Science.gov (United States)

    Sun, Lingyi; Ding, Jiule; Xing, Wei; Gai, Yongkang; Sheng, Jing; Zeng, Dexing

    2016-05-18

    Preparation of small molecule based dual-modality probes remains a challenging task due to the complicated synthetic procedure. In this study, a novel concise and generic strategy for preparing dual-modality optical/PET imaging probes via photo-click chemistry was developed, in which the diazole photo-click linker functioned not only as a bridge between the targeting-ligand and the PET imaging moiety, but also as the fluorophore for optical imaging. A dual-modality AE105 peptidic probe was successfully generated via this strategy and subsequently applied in the fluorescent staining of U87MG cells and the (68)Ga based PET imaging of mice bearing U87MG xenograft. In addition, dual-modality monoclonal antibody cetuximab has also been generated via this strategy and labeled with (64)Cu for PET imaging studies, broadening the application of this strategy to include the preparation of macromolecule based imaging probes.

  12. Pulsed photoacoustic flow imaging with a handheld system.

    Science.gov (United States)

    van den Berg, Pim J; Daoudi, Khalid; Steenbergen, Wiendelt

    2016-02-01

    Flow imaging is an important technique in a range of disease areas, but estimating low flow speeds, especially near the walls of blood vessels, remains challenging. Pulsed photoacoustic flow imaging can be an alternative since there is little signal contamination from background tissue with photoacoustic imaging. We propose flow imaging using a clinical photoacoustic system that is both handheld and portable. The system integrates a linear array with 7.5 MHz central frequency in combination with a high-repetition-rate diode laser to allow high-speed photoacoustic imaging--ideal for this application. This work shows the flow imaging performance of the system in vitro using microparticles. Both two-dimensional (2-D) flow images and quantitative flow velocities from 12 to 75  mm/s were obtained. In a transparent bulk medium, flow estimation showed standard errors of ∼7% the estimated speed; in the presence of tissue-realistic optical scattering, the error increased to 40% due to limited signal-to-noise ratio. In the future, photoacoustic flow imaging can potentially be performed in vivo using fluorophore-filled vesicles or with an improved setup on whole blood.

  13. Precise diagnosis in different scenarios using photoacoustic and fluorescence imaging with dual-modality nanoparticles.

    Science.gov (United States)

    Peng, Dong; Du, Yang; Shi, Yiwen; Mao, Duo; Jia, Xiaohua; Li, Hui; Zhu, Yukun; Wang, Kun; Tian, Jie

    2016-08-14

    Photoacoustic imaging and fluorescence molecular imaging are emerging as important research tools for biomedical studies. Photoacoustic imaging offers both strong optical absorption contrast and high ultrasonic resolution, and fluorescence molecular imaging provides excellent superficial resolution, high sensitivity, high throughput, and the ability for real-time imaging. Therefore, combining the imaging information of both modalities can provide comprehensive in vivo physiological and pathological information. However, currently there are limited probes available that can realize both fluorescence and photoacoustic imaging, and advanced biomedical applications for applying this dual-modality imaging approach remain underexplored. In this study, we developed a dual-modality photoacoustic-fluorescence imaging nanoprobe, ICG-loaded Au@SiO2, which was uniquely designed, consisting of gold nanorod cores and indocyanine green with silica shell spacer layers to overcome fluorophore quenching. This nanoprobe was examined by both PAI and FMI for in vivo imaging on tumor and ischemia mouse models. Our results demonstrated that the nanoparticles can specifically accumulate at the tumor and ischemic areas and be detected by both imaging modalities. Moreover, this dual-modality imaging strategy exhibited superior advantages for a precise diagnosis in different scenarios. The new nanoprobe with the dual-modality imaging approach holds great potential for diagnosis and stage classification of tumor and ischemia related diseases.

  14. Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues

    Science.gov (United States)

    Yu, Yingqiu; Lee, Anthony M. D.; Wang, Hequn; Tang, Shuo; Zhao, Jianhua; Lui, Harvey; Zeng, Haishan

    2012-07-01

    There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.

  15. Multicolor excitation two-photon microscopy: in vivo imaging of cells and tissues

    Science.gov (United States)

    Li, Dong; Zheng, Wei; Qu, Jianan Y.

    2010-02-01

    Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system. Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular, with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early development of cancer in epithelial tissue.

  16. Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

    Science.gov (United States)

    Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

    2009-02-01

    Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

  17. In-vivo optical detection of cancer using chlorin e6 – polyvinylpyrrolidone induced fluorescence imaging and spectroscopy

    Directory of Open Access Journals (Sweden)

    Soo Khee

    2009-01-01

    Full Text Available Abstract Background Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6 formulated in polyvinylpyrrolidone (PVP as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. Methods Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. Results Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. Conclusion Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer.

  18. Functional imaging of small tissue volumes with diffuse optical tomography

    Science.gov (United States)

    Klose, Alexander D.; Hielscher, Andreas H.

    2006-03-01

    Imaging of dynamic changes in blood parameters, functional brain imaging, and tumor imaging are the most advanced application areas of diffuse optical tomography (DOT). When dealing with the image reconstruction problem one is faced with the fact that near-infrared photons, unlike X-rays, are highly scattered when they traverse biological tissue. Image reconstruction schemes are required that model the light propagation inside biological tissue and predict measurements on the tissue surface. By iteratively changing the tissue-parameters until the predictions agree with the real measurements, a spatial distribution of optical properties inside the tissue is found. The optical properties can be related to the tissue oxygenation, inflammation, or to the fluorophore concentration of a biochemical marker. If the model of light propagation is inaccurate, the reconstruction process will lead to an inaccurate result as well. Here, we focus on difficulties that are encountered when DOT is employed for functional imaging of small tissue volumes, for example, in cancer studies involving small animals, or human finger joints for early diagnosis of rheumatoid arthritis. Most of the currently employed image reconstruction methods rely on the diffusion theory that is an approximation to the equation of radiative transfer. But, in the cases of small tissue volumes and tissues that contain low scattering regions diffusion theory has been shown to be of limited applicability Therefore, we employ a light propagation model that is based on the equation of radiative transfer, which promises to overcome the limitations.

  19. Design and synthesis of an on-off "click" fluorophore that executes a logic operation and detects heavy and transition metal ions in water and living cells.

    Science.gov (United States)

    Midya, Ganesh Chandra; Paladhi, Sushovan; Bhowmik, Sudipta; Saha, Subhadeep; Dash, Jyotirmayee

    2013-05-14

    We have designed and synthesized a novel fluorescent molecular probe using the Cu(i)-catalyzed Huisgen cycloaddition of 1,3-diethynyl-6-fluoroisoquinoline with 1-(2-azidoethyl)pyrrolidine. This water soluble "click" fluorescent chemosensor displays good sensitivity towards heavy and transition metal ions. It shows pronounced fluorescence enhancement and high selectivity for Zn(2+) over other biologically relevant metal ions in water at pH 7.0. The fluorescence response of the bis-triazole derivative in the presence of Zn(2+) is switchable and reversible as a function of pH. The chemosensor also exhibits fluorescence quenching with Fe(2+) and Cu(2+) in water at pH 7.0. A modified YES logic gate property has been proposed using the "turn-on" and "turn-off" behavior of the bis-triazole with Zn(2+) and Fe(2+). The sensor is cell membrane permeable and applicable for intracellular Zn(2+) imaging.

  20. Evaluation of luminol chemiluminescence based on simultaneous introducing of coumarin derivatives as green fluorophores and chitosan-induced Au/Ag alloy nanoparticle as catalyst for the sensitive determination of glucose.

    Science.gov (United States)

    Chaichi, M J; Alijanpour, S O; Asghari, S; Shadlou, S

    2015-03-01

    We report herein the development of a novel chemiluminescence system based on simultaneous introducing of synthetic coumarin derivatives and chitosan-induced Au/Ag alloy NPs on the luminol CL system and suggest how it may be useful for determination of glucose. Chitosan-induced Au/Ag nanoalloys in the coumarin derivatives intensified-luminol CL system, in addition to catalyze CL reaction can make a change in the process of coumarin derivatives effect as fluorophore on the luminol CL system. This phenomenon is caused by interaction between active functional groups of coumarin derivatives and chitosan. The interaction strength depends on the coumarin derivatives' structure and their substituents. Considering the inevitable trend luminol radical and superoxide anion radical to absorption on the surface of the embedded Au/Ag nanoalloy in the chitosan matrix, it can be concluded that chitosan acts as a platform for all reagents involved in the CL reaction including coumarin derivatives, Au/Ag nanoalloy and luminol, and electron-transfer taking place on it; Placing all chemiluminescent reagents together on the chitosan network can lead to a powerful CL due to increasing rigidity of CL system. The most efficient coumarin derivative on the Au/Ag nanoalloy-fluorophore-luminol-H2O2 CL system, in relation to interaction capability with chitosan' functional groups, was selected and the CL condition in presence of it was optimized. Whereas the glucose oxidase-mediated oxidation of glucose yields gluconic acid and H2O2, under optimum condition the most efficient CL system was applied to detection of glucose due to enzymatically production of hydrogen peroxide. The linear response range of 1.5 × 10(-6)-5.0 × 10(-3) M and the detection limit (defined as the concentration that could be detected at the signal-to-noise ratio of 3) of 7.5 × 10(-7) M was found for the glucose standards. Also, the developed method was successfully applied to determination of glucose in

  1. Molecular Imaging Probes for Positron Emission Tomography and Optical Imaging of Sentinel Lymph Node and Tumor

    Science.gov (United States)

    Qin, Zhengtao

    Molecular imaging is visualizations and measurements of in vivo biological processes at the molecular or cellular level using specific imaging probes. As an emerging technology, biocompatible macromolecular or nanoparticle based targeted imaging probes have gained increasing popularities. Those complexes consist of a carrier, an imaging reporter, and a targeting ligand. The active targeting ability dramatically increases the specificity. And the multivalency effect may further reduce the dose while providing a decent signal. In this thesis, sentinel lymph node (SLN) mapping and cancer imaging are two research topics. The focus is to develop molecular imaging probes with high specificity and sensitivity, for Positron Emission Tomography (PET) and optical imaging. The objective of this thesis is to explore dextran radiopharmaceuticals and porous silicon nanoparticles based molecular imaging agents. Dextran polymers are excellent carriers to deliver imaging reporters or therapeutic agents due to its well established safety profile and oligosaccharide conjugation chemistry. There is also a wide selection of dextran polymers with different lengths. On the other hand, Silicon nanoparticles represent another class of biodegradable materials for imaging and drug delivery. The success in fluorescence lifetime imaging and enhancements of the immune activation potency was briefly discussed. Chapter 1 begins with an overview on current molecular imaging techniques and imaging probes. Chapter 2 presents a near-IR dye conjugated probe, IRDye 800CW-tilmanocept. Fluorophore density was optimized to generate the maximum brightness. It was labeled with 68Ga and 99mTc and in vivo SLN mapping was successfully performed in different animals, such as mice, rabbits, dogs and pigs. With 99mTc labeled IRDye 800CW-tilmanocept, chapter 3 introduces a two-day imaging protocol with a hand-held imager. Chapter 4 proposed a method to dual radiolabel the IRDye 800CW-tilmanocept with both 68Ga and

  2. Development of a Hybrid Nanoprobe for Triple-Modality MR/SPECT/Optical Fluorescence Imaging

    Science.gov (United States)

    Madru, Renata; Svenmarker, Pontus; Ingvar, Christian; Ståhlberg, Freddy; Engels, Stefan-Andersson; Knutsson, Linda; Strand, Sven-Erik

    2014-01-01

    Hybrid clinical imaging is an emerging technology, which improves disease diagnosis by combining already existing technologies. With the combination of high-resolution morphological imaging, i.e., MRI/CT, and high-sensitive molecular detection offered by SPECT/PET/Optical, physicians can detect disease progression at an early stage and design patient-specific treatments. To fully exploit the possibilities of hybrid imaging a hybrid probe compatible with each imaging technology is required. Here, we present a hybrid nanoprobe for triple modality MR/SPECT/Fluorescence imaging. Our imaging agent is comprised of superparamagnetic iron oxide nanoparticles (SPIONs), labeled with 99mTc and an Alexa fluorophore (AF), together forming 99mTc-AF-SPIONs. The agent was stable in human serum, and, after subcutaneous injection in the hind paw of Wistar rats, showed to be highly specific by accumulating in the sentinel lymph node. All three modalities clearly visualized the imaging agent. Our results show that a single imaging agent can be used for hybrid imaging. The use of a single hybrid contrast agent permits simultaneous hybrid imaging and, more conventionally, allow for single modality imaging at different time points. For example, a hybrid contrast agent enables pre-operative planning, intra-operative guidance, and post-operative evaluation with the same contrast agent. PMID:26852675

  3. Single-photon sensitive fast ebCMOS camera system for multiple-target tracking of single fluorophores: application to nano-biophotonics

    Science.gov (United States)

    Cajgfinger, Thomas; Chabanat, Eric; Dominjon, Agnes; Doan, Quang T.; Guerin, Cyrille; Houles, Julien; Barbier, Remi

    2011-03-01

    Nano-biophotonics applications will benefit from new fluorescent microscopy methods based essentially on super-resolution techniques (beyond the diffraction limit) on large biological structures (membranes) with fast frame rate (1000 Hz). This trend tends to push the photon detectors to the single-photon counting regime and the camera acquisition system to real time dynamic multiple-target tracing. The LUSIPHER prototype presented in this paper aims to give a different approach than those of Electron Multiplied CCD (EMCCD) technology and try to answer to the stringent demands of the new nano-biophotonics imaging techniques. The electron bombarded CMOS (ebCMOS) device has the potential to respond to this challenge, thanks to the linear gain of the accelerating high voltage of the photo-cathode, to the possible ultra fast frame rate of CMOS sensors and to the single-photon sensitivity. We produced a camera system based on a 640 kPixels ebCMOS with its acquisition system. The proof of concept for single-photon based tracking for multiple single-emitters is the main result of this paper.

  4. High-sensitivity chemical imaging for biomedicine by SRS microscopy (Conference Presentation)

    Science.gov (United States)

    Min, Wei

    2017-02-01

    Innovations in spectroscopy principles and microscopy technology have significantly impacted modern biology and medicine. While most of the contemporary bio-imaging modalities harness electronic transition, nuclear spin or radioactivity, vibrational spectroscopy has not been widely used yet. Here we will discuss an emerging chemical imaging platform, stimulated Raman scattering (SRS) microscopy, which can enhance the otherwise feeble spontaneous Raman eight orders of magnitude by virtue of stimulated emission. When coupled with stable isotopes (e.g., deuterium and 13C) or bioorthogonal chemical moieties (e.g., alkynes), SRS microscopy is well suited for probing in vivo metabolic dynamics of small bio-molecules which cannot be labeled by bulky fluorophores. Physical principle of the underlying optical spectroscopy and exciting biomedical applications such as imaging lipid metabolism, protein synthesis, DNA replication, protein degradation, RNA synthesis, glucose uptake, drug trafficking and tumor metabolism will be presented.

  5. Ratiometric and near-infrared molecular probes for the detection and imaging of zinc ions.

    Science.gov (United States)

    Carol, Priya; Sreejith, Sivaramapanicker; Ajayaghosh, Ayyappanpillai

    2007-03-05

    The detection and imaging of Zn2+ in biological samples are of paramount interest owing to the role of this cation in physiological functions. This is possible only with molecular probes that specifically bind to Zn2+ and result in changes in emission properties. A "turn-on" emission or shift in the emission color upon binding to Zn2+ should be ideal for in vivo imaging. In this context, ratiometric and near-IR probes are of particular interest. Therefore, in the area of chemosensors or molecular probes, the design of fluorophores that allow ratiometric sensing or imaging in the near-IR region is attracting the attention of chemists. The purpose of this Focus Review is to highlight recent developments in this area and stress the importance of further research for future applications.

  6. Design, Synthesis and Evaluation of Dual-Modality Glyco-Nanoparticles for Tumor Imaging

    Directory of Open Access Journals (Sweden)

    Zhi Yang

    2013-05-01

    Full Text Available d-Glucosamine (DG was conjugated to a core-cross linked polymeric micelle (CCPM system equipped with both a near-infrared fluorophore (NIRF and a gamma emitter (111In. The resultant nano-scale tumor-targeting imaging tracer, 111In-DG-NIRF-CCPM, selectively accumulated in a human epithelial carcinoma A-431 xenograft model in mice. At 24 hrs post injection, the tumor uptake was 2.62 ± 0.80 % of the injected dose per gram of tissue (%ID/g. Tumors were clearly delineated in both single-photon emission computed tomography (SPECT and optical imaging. The results suggest that the prepared imaging tracer is a promising agent for tumor diagnosis.

  7. Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase.

    Science.gov (United States)

    Okkelman, Irina A; Dmitriev, Ruslan I; Foley, Tara; Papkovsky, Dmitri B

    2016-01-01

    Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

  8. Multi-pulse pumping for far-field super-resolution imaging

    Science.gov (United States)

    Requena, Sebastian; Raut, Sangram; Doan, Hung; Kimball, Joe; Fudala, Rafal; Borejdo, Julian; Gryczynski, Ignacy; Strzhemechny, Yuri; Gryczynski, Zygmunt

    2016-02-01

    Recently, far-field optical imaging with a resolution significantly beyond diffraction limit has attracted tremendous attention allowing for high resolution imaging in living objects. Various methods have been proposed that are divided in to two basic approaches; deterministic super-resolution like STED or RESOLFT and stochastic super-resolution like PALM or STORM. We propose to achieve super-resolution in far-field fluorescence imaging by the use of controllable (on-demand) bursts of pulses that can change the fluorescence signal of long-lived component over one order of magnitude. We demonstrate that two beads, one labeled with a long-lived dye and another with a short-lived dye, separated by a distance lower than 100 nm can be easily resolved in a single experiment. The proposed method can be used to separate two biological structures in a cell by targeting them with two antibodies labeled with long-lived and short-lived fluorophores.

  9. Body Image

    Science.gov (United States)

    ... About Us Contact Us Text size | Print | Body Image Developing a positive body image and a healthy mental attitude is crucial to ... on for tips to have a healthy body image. Topics About body image When you look in ...

  10. An excited-state intramolecular photon transfer fluorescence probe for localizable live cell imaging of cysteine

    Science.gov (United States)

    Liu, Wei; Chen, Wen; Liu, Si-Jia; Jiang, Jian-Hui

    2017-03-01

    Small molecule probes suitable for selective and specific fluorescence imaging of some important but low-concentration intracellular reactive sulfur species such as cysteine (Cys) pose a challenge in chemical biology. We present a readily available, fast-response fluorescence probe CHCQ-Ac, with 2-(5‧-chloro-2-hydroxyl-phenyl)-6-chloro-4(3 H)-quinazolinone (CHCQ) as the fluorophore and acrylate group as the functional moiety, that enables high-selectivity and high-sensitivity for detecting Cys in both solution and biological system. After specifically reacted with Cys, the probe undergoes a seven-membered intramolecular cyclization and released the fluorophore CHCQ with excited-state intramolecular photon transfer effect. A highly fluorescent, insoluble aggregate was then formed to facilitate high-sensitivity and high-resolution imaging. The results showed that probe CHCQ-Ac affords a remarkably large Stokes shift and can detect Cys under physiological pH condition with no interference from other analytes. Moreover, this probe was proved to have excellent chemical stability, low cytotoxicity and good cell permeability. Our design of this probe provides a novel potential tool to visualize and localize cysteine in bioimaging of live cells that would greatly help to explore various Cys-related physiological and pathological cellular processes in cell biology and diagnostics.

  11. Evaluation of PpIX formation in Cervical Intraepithelial Neoplasia I (CIN) using widefield fluorescence images

    Science.gov (United States)

    Carbinatto, Fernanda M.; Inada, Natalia M.; Fortunato, Thereza C.; Lombardi, Welington; da Silva, Eduardo V.; Vollet Filho, José D.; Kurachi, Cristina; Pratavieira, Sebastião.; Bagnato, Vanderlei S.

    2016-03-01

    Optical techniques has been described as auxiliary technology for screening of neoplasia because shows the potential for tissues differentiation in real-time and it is a noninvasive detection and safe. However, only endogenous fluorophores presents the lesion may be insufficient and needed of the administration of the fluorophores synthesized, such as, precursor molecule of protoporphyrin IX (PpIX) induced by 5- aminolevulinic acid and your derivatives. Topical application of methylaminolevulinate (MAL), induces formation of the endogenous photosensitizer, PpIX in tissues where carcinogenesis has begun. The PpIX tend to accumulate in premalignant and malignant tissues and the illumination with light with appropriate wavelength beginning to excitation of PpIX fluorescence, which helps to localize PpIX-rich areas and identify potentially malignant tissues. The aim of the study is to evaluate the production of PpIX in the cervix with CIN I through of the fluorescence images captured after 1 hour of cream application. It was possible to visualize PpIX fluorescence in cervix and it was possible to observe the selectivity in fluorescence in squamous-columnar junction, which a pre-cancerous condition (CIN) and usually is localized. Through the image processing it was possible to quantify the increase of red fluorescence. For the CIN I the increase of red fluorescence was approximately of 4 times indicating a good PpIX formation.

  12. Enhancing the output current of a CdTe solar cell via a CN-free hydrocarbon luminescent down-shifting fluorophore with intramolecular energy transfer and restricted internal rotation characteristics.

    Science.gov (United States)

    Li, Yilin; Olsen, Joseph; Dong, Wen-Ji

    2015-04-01

    A CN-free hydrocarbon fluorophore (Perylene-TPE) was synthesized as a new luminescent down-shifting (LDS) material. Its photophysical properties in both the solution state and the solid state were studied. The unity fluorescence quantum yield of Perylene-TPE observed in its solid state is considered to be from the characteristics of intramolecular energy transfer (IET) and restricted internal rotation (RIR). This is supported by the results from theoretical calculations and spectroscopic measurements. For the photovoltaic application of Perylene-TPE, a theoretical modeling study suggests that using the LDS film of Perylene-TPE may increase the output short circuit current density (Jsc) of a CdTe solar cell by 2.95%, enhance the spectral response of a CdTe solar cell at 400 nm by 41%, and shift the incident solar photon distribution from short-wavelength (500 nm). Experimentally, placing a LDS film of Perylene-TPE on a CdTe solar cell can enhance its output Jsc by as high as 3.30 ± 0.31%, which is comparable to the current commercially available LDS material – Y083 (3.28% ± 0.37%).

  13. Near-Infrared Fluorescent NanoGUMBOS for Biomedical Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bwambok, David [Louisiana State University; El-Zahab, Bilal [Lousianna State University; Challa, Santhosh [Louisiana State University; Li, Min [Lousianna State University; Chandler, Lin [Horiba Jobin Yvon Inc.; Baker, Gary A [ORNL; Warner, Isiah M [ORNL

    2009-01-01

    Herein, we report on near-infrared (NIR) fluorescent nanoparticles generated from an emergent class of materials we refer to as a Group of Uniform Materials Based on Organic Salts (GUMBOS). GUMBOS are largely frozen ionic liquids, although the concept is more general and is also easily applied to solid ionic materials with melting points in excess of 100 C. Nanoparticles based on GUMBOS (nanoGUMBOS) derived from a NIR fluorophore are prepared using a reprecipitation method and evaluated for in vivo fluorescence imaging. Due to their uniformity, single-step preparation, and composite nature, nanoGUMBOS help to resolve issues with dye leakage problems innate to alternate cellular stains and unlock a myriad of applications for these materials, highlighting exciting possibilities for multifunctional nanoGUMBOS.

  14. Fluorescence Lifetime Imaging of Free and Protein-Bound NADH

    Science.gov (United States)

    Lakowicz, Joseph R.; Szmacinski, Henryk; Nowaczyk, Kazimierz; Johnson, Michael L.

    1992-02-01

    We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challenging case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images, each obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe an imaging procedure that allows specific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.

  15. Trans-Stent B-Mode Ultrasound and Passive Cavitation Imaging.

    Science.gov (United States)

    Haworth, Kevin J; Raymond, Jason L; Radhakrishnan, Kirthi; Moody, Melanie R; Huang, Shao-Ling; Peng, Tao; Shekhar, Himanshu; Klegerman, Melvin E; Kim, Hyunggun; McPherson, David D; Holland, Christy K

    2016-02-01

    Angioplasty and stenting of a stenosed artery enable acute restoration of blood flow. However, restenosis or a lack of re-endothelization can subsequently occur depending on the stent type. Cavitation-mediated drug delivery is a potential therapy for these conditions, but requires that particular types of cavitation be induced by ultrasound insonation. Because of the heterogeneity of tissue and stochastic nature of cavitation, feedback mechanisms are needed to determine whether the sustained bubble activity is induced. The objective of this study was to determine the feasibility of passive cavitation imaging through a metal stent in a flow phantom and an animal model. In this study, an endovascular stent was deployed in a flow phantom and in porcine femoral arteries. Fluorophore-labeled echogenic liposomes, a theragnostic ultrasound contrast agent, were injected proximal to the stent. Cavitation images were obtained by passively recording and beamforming the acoustic emissions from echogenic liposomes insonified with a low-frequency (500 kHz) transducer. In vitro experiments revealed that the signal-to-noise ratio for detecting stable cavitation activity through the stent was greater than 8 dB. The stent did not significantly reduce the signal-to-noise ratio. Trans-stent cavitation activity was also detected in vivo via passive cavitation imaging when echogenic liposomes were insonified by the 500-kHz transducer. When stable cavitation was detected, delivery of the fluorophore into the arterial wall was observed. Increased echogenicity within the stent was also observed when echogenic liposomes were administered. Thus, both B-mode ultrasound imaging and cavitation imaging are feasible in the presence of an endovascular stent in vivo. Demonstration of this capability supports future studies to monitor restenosis with contrast-enhanced ultrasound and pursue image-guided ultrasound-mediated drug delivery to inhibit restenosis.

  16. The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging.

    Science.gov (United States)

    Backlund, Mikael P; Lew, Matthew D; Backer, Adam S; Sahl, Steffen J; Moerner, W E

    2014-03-17

    Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

  17. Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

    Science.gov (United States)

    Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

    2016-05-24

    Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

  18. Cytosolic NADH-NAD+ Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

    Science.gov (United States)

    Mongeon, Rebecca; Venkatachalam, Veena

    2016-01-01

    Abstract Aim: Cytosolic NADH-NAD+ redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD+ redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. Results: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD+ ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Innovation: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Conclusion: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD+ ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553–563. PMID:26857245

  19. Wide field-of-view fluorescence imaging of coral reefs.

    Science.gov (United States)

    Treibitz, Tali; Neal, Benjamin P; Kline, David I; Beijbom, Oscar; Roberts, Paul L D; Mitchell, B Greg; Kriegman, David

    2015-01-13

    Coral reefs globally are declining rapidly because of both local and global stressors. Improved monitoring tools are urgently needed to understand the changes that are occurring at appropriate temporal and spatial scales. Coral fluorescence imaging tools have the potential to improve both ecological and physiological assessments. Although fluorescence imaging is regularly used for laboratory studies of corals, it has not yet been used for large-scale in situ assessments. Current obstacles to effective underwater fluorescence surveying include limited field-of-view due to low camera sensitivity, the need for nighttime deployment because of ambient light contamination, and the need for custom multispectral narrow band imaging systems to separate the signal into meaningful fluorescence bands. Here we describe the Fluorescence Imaging System (FluorIS), based on a consumer camera modified for greatly increased sensitivity to chlorophyll-a fluorescence, and we show high spectral correlation between acquired images and in situ spectrometer measurements. This system greatly facilitates underwater wide field-of-view fluorophore surveying during both night and day, and potentially enables improvements in semi-automated segmentation of live corals in coral reef photographs and juvenile coral surveys.

  20. Fluorescent Dendrimer Nanoconjugates as Advanced Probes for Biological Imaging

    Science.gov (United States)

    Reilly, Daniel; Kim, Sung Hoon; Katzenellenbogen, John A.; Schroeder, Charles M.

    2014-03-01

    Recent advances in fluorescence microscopy have enabled improvements in spatial resolution for biological imaging. However, there is a strong need for development of advanced fluorescent probes to enable a molecular-scale understanding of biological events. In this work, we report the development of a new class of probes for fluorescence imaging based on dye-conjugated dendrimer nanoconjugates. We utilize molecular-scale dendritic scaffolds as fluorescent probes, thereby enabling conjugation of multiple dyes and linkers to the scaffold periphery. In particular, we use polyamidoamine dendrimers as molecular scaffolds, wherein dye conjugation can be varied over a wide range. Single molecule fluorescence imaging shows that dendrimer nanoconjugates are far brighter than single fluorophores, resulting in increased localization precision. In addition, we further developed a new set of remarkably photostable probes by conjugating photoprotective triplet state quenchers directly onto the dendritic scaffold. We observe large increases in the photobleaching times compared to single dyes and reduced transient dark states (blinking). Overall, we believe that these new probes will allow for single molecule imaging over long time scales, enabling new vistas in biological imaging.

  1. Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

    Science.gov (United States)

    Sakhalkar, H. S.; Dewhirst, M.; Oliver, T.; Cao, Y.; Oldham, M.

    2007-04-01

    Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB

  2. Indexing Images.

    Science.gov (United States)

    Rasmussen, Edie M.

    1997-01-01

    Focuses on access to digital image collections by means of manual and automatic indexing. Contains six sections: (1) Studies of Image Systems and their Use; (2) Approaches to Indexing Images; (3) Image Attributes; (4) Concept-Based Indexing; (5) Content-Based Indexing; and (6) Browsing in Image Retrieval. Contains 105 references. (AEF)

  3. Advances in frequency-domain fluorometry, gigahertz instrumentation, time-dependent photomigration, and fluorescence lifetime imaging

    Science.gov (United States)

    Lakowicz, Joseph R.; Gryczynski, Ignacy; Szmacinski, Henryk; Nowaczyk, Kazimierz; Johnson, Michael L.

    1992-02-01

    During the past seven years, there have been remarkable advances in the frequency-domain method for measurement of time-resolved emission or light scattering. In this presentation we describe the recent extension of the frequency range to 10 GHz using a specially designed microchannel plate PMT. Experimental data will be shown for measurement of picosecond rotational diffusion and for sub-picosecond resolution of time delays. The resolution of ps to ns timescale processes is not obtained at the expense of sensitivity or is it shown by measurements on the intrinsic tryptophan emission from hemoglobin. We also describe a time- resolved reflectance imaging experiment on a scattering medium containing an absorbing object. Time-resolved imaging of the back-scattered light is realized by means of a RF-phase- sensitive camera, synchronized to the laser pulses. By processing the stored images, a final image can be created, the contrast of which is based only on time differences of the back- scattered photons. This image reveals the presence and position of the absorber within the scattering medium. And finally, we describe a new methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image, and not the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. FLIM has numerous potential applications in cell biology and imaging.

  4. Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

    Directory of Open Access Journals (Sweden)

    Aqib H Zehri

    2014-01-01

    Full Text Available Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM, two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery.

  5. Image Gallery

    Science.gov (United States)

    ... Accredited Practices EER Endowment for Education & Research Journal Image Gallery Click each image to enlarge. Fetal nose/lips Fetal nose, upper ... in the third trimester of pregnancy; the ultrasound image shows the chest of each fetus with the ...

  6. Image Statistics

    Energy Technology Data Exchange (ETDEWEB)

    Wendelberger, Laura Jean [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-08-08

    In large datasets, it is time consuming or even impossible to pick out interesting images. Our proposed solution is to find statistics to quantify the information in each image and use those to identify and pick out images of interest.

  7. From multispectral imaging of autofluorescence to chemical and sensory images of lipid oxidation in cod caviar paste.

    Science.gov (United States)

    Airado-Rodríguez, Diego; Høy, Martin; Skaret, Josefine; Wold, Jens Petter

    2014-05-01

    The potential of multispectral imaging of autofluorescence to map sensory flavour properties and fluorophore concentrations in cod caviar paste has been investigated. Cod caviar paste was used as a case product and it was stored over time, under different headspace gas composition and light exposure conditions, to obtain a relevant span in lipid oxidation and sensory properties. Samples were divided in two sets, calibration and test sets, with 16 and 7 samples, respectively. A third set of samples was prepared with induced gradients in lipid oxidation and sensory properties by light exposure of certain parts of the sample surface. Front-face fluorescence emission images were obtained for excitation wavelength 382 nm at 11 different channels ranging from 400 to 700 nm. The analysis of the obtained sets of images was divided in two parts: First, in an effort to compress and extract relevant information, multivariate curve resolution was applied on the calibration set and three spectral components and their relative concentrations in each sample were obtained. The obtained profiles were employed to estimate the concentrations of each component in the images of the heterogeneous samples, giving chemical images of the distribution of fluorescent oxidation products, protoporphyrin IX and photoprotoporphyrin. Second, regression models for sensory attributes related to lipid oxidation were constructed based on the spectra of homogeneous samples from the calibration set. These models were successfully validated with the test set. The models were then applied for pixel-wise estimation of sensory flavours in the heterogeneous images, giving rise to sensory images. As far as we know this is the first time that sensory images of odour and flavour are obtained based on multispectral imaging.

  8. Super-Resolution Molecular and Functional imaging of Nanoscale Architectures in Life and Materials Science

    Directory of Open Access Journals (Sweden)

    Satoshi eHabuchi

    2014-06-01

    Full Text Available Super-resolution fluorescence microscopy has been revolutionizing the way in which we investigate the structures, dynamics, and functions of a wide range of nanoscale systems. In this review, I describe the current state of various super-resolution fluorescence microscopy techniques along with the latest developments of fluorophores and labeling for the super-resolution microscopy. I discuss the applications of super-resolution microscopy in the fields of life science and materials science with a special emphasis on quantitative molecular imaging and nanoscale functional imaging. These studies open new opportunities for unraveling the physical, chemical, and optical properties of a wide range of nanoscale architectures together with their nanostructures and will enable the development of new (bio-nanotechnology.

  9. Super-Resolution Molecular and Functional Imaging of Nanoscale Architectures in Life and Materials Science

    KAUST Repository

    Habuchi, Satoshi

    2014-06-12

    Super-resolution (SR) fluorescence microscopy has been revolutionizing the way in which we investigate the structures, dynamics, and functions of a wide range of nanoscale systems. In this review, I describe the current state of various SR fluorescence microscopy techniques along with the latest developments of fluorophores and labeling for the SR microscopy. I discuss the applications of SR microscopy in the fields of life science and materials science with a special emphasis on quantitative molecular imaging and nanoscale functional imaging. These studies open new opportunities for unraveling the physical, chemical, and optical properties of a wide range of nanoscale architectures together with their nanostructures and will enable the development of new (bio-)nanotechnology.

  10. Enhanced two-photon excited fluorescence from imaging agents using true thermal light

    Science.gov (United States)

    Jechow, Andreas; Seefeldt, Michael; Kurzke, Henning; Heuer, Axel; Menzel, Ralf

    2013-12-01

    Two-photon excited fluorescence (TPEF) is a standard technique in modern microscopy, but is still affected by photodamage to the probe. It has been proposed that TPEF can be enhanced using entangled photons, but this has proven challenging. Recently, it was shown that some features of entangled photons can be mimicked with thermal light, which finds application in ghost imaging, subwavelength lithography and metrology. Here, we use true thermal light from a superluminescent diode to demonstrate TPEF that is enhanced compared to coherent light, using two common fluorophores and luminescent quantum dots, which suit applications in imaging and microscopy. We find that the TPEF rate is directly proportional to the measured degree of second-order coherence, as predicted by theory. Our results show that photon bunching in thermal light can be exploited in two-photon microscopy, with the photon statistic providing a new degree of freedom.

  11. The Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images

    Energy Technology Data Exchange (ETDEWEB)

    Syed, Aleem [Ames Laboratory; Lesoine, Michael D [Ames Laboratory; Bhattacharjee, Ujjal [Ames Laboratory; Petrich, Jacob W [Ames Laboratory; Smith, Emily A [Ames Laboratory

    2014-03-03

    Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion (STED) fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F-actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a tenfold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel-by-pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal-to-noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40- to 400-nm spatial regime.

  12. Facile Synthesis of Biocompatible Fluorescent Nanoparticles for Cellular Imaging and Targeted Detection of Cancer Cells.

    Science.gov (United States)

    Tang, Fu; Wang, Chun; Wang, Xiaoyu; Li, Lidong

    2015-11-18

    In this work, we report the facile synthesis of functional core-shell structured nanoparticles with fluorescence enhancement, which show specific targeting of cancer cells. Biopolymer poly-l-lysine was used to coat the silver core with various shell thicknesses. Then, the nanoparticles were functionalized with folic acid as a targeting agent for folic acid receptor. The metal-enhanced fluorescence effect was observed when the fluorophore (5-(and-6)-carboxyfluorescein-succinimidyl ester) was conjugated to the modified nanoparticle surface. Cellular imaging assay of the nanoparticles in folic acid receptor-positive cancer cells showed their excellent biocompatibility and selectivity. The as-prepared functional nanoparticles demonstrate the efficiency of the metal-enhanced fluorescence effect and provide an alternative approach for the cellular imaging and targeting of cancer cells.

  13. Molecular probes for nonlinear optical imaging of biological membranes

    Science.gov (United States)

    Blanchard-Desce, Mireille H.; Ventelon, Lionel; Charier, Sandrine; Moreaux, Laurent; Mertz, Jerome

    2001-12-01

    Second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) are nonlinear optical (NLO) phenomena that scale with excitation intensity squared, and hence give rise to an intrinsic 3-dimensional resolution when used in microscopic imaging. TPEF microscopy has gained widespread popularity in the biology community whereas SHG microscopy promises to be a powerful tool because of its sensitivity to local asymmetry. We have implemented an approach toward the design of NLO-probes specifically adapted for SHG and/or TPEF imaging of biological membranes. Our strategy is based on the design of nanoscale amphiphilic NLO-phores. We have prepared symmetrical bolaamphiphilic fluorophores combining very high two-photon absorption (TPA) cross-sections in the visible red region and affinity for cellular membranes. Their incorporation and orientation in lipid membranes can be monitored via TPEF anisotropy. We have also prepared amphiphilic push-pull chromophores exhibiting both large TPA cross-sections and very large first hyperpolarizabilities in the near-IR region. These NLO-probes have proved to be particularly useful for imaging of biological membranes by simultaneous SHG and TPEF microscopy and offer attractive prospects for real-time imaging of fundamental biological processes such as adhesion, fusion or reporting of membrane potentials.

  14. Tumor-stem cells interactions by fluorescence imaging

    Science.gov (United States)

    Meleshina, Aleksandra V.; Cherkasova, Elena I.; Sergeeva, Ekaterina; Turchin, Ilya V.; Kiseleva, Ekaterina V.; Dashinimaev, Erdem B.; Shirmanova, Marina V.; Zagaynova, Elena V.

    2013-02-01

    Recently, great deal of interest is investigation the function of the stem cells (SC) in tumors. In this study, we studied «recipient-tumor- fluorescent stem cells » system using the methods of in vivo imaging and laser scanning microscopy (LSM). We used adipose-derived adult stem (ADAS) cells of human lentiviral transfected with the gene of fluorescent protein Turbo FP635. ADAS cells were administrated into nude mice with transplanted tumor HeLa Kyoto (human cervical carcinoma) at different stages of tumor growth (0-8 days) intravenously or into tumor. In vivo imaging was performed on the experimental setup for epi - luminescence bioimaging (IAP RAS, Nizhny Novgorod). The results of the imaging showed localization of fluorophore tagged stem cells in the spleen on day 5-9 after injection. The sensitivity of the technique may be improved by spectral separation autofluorescence and fluorescence of stem cells. We compared the results of in vivo imaging and confocal laser scanning microscopy (LSM 510 META, Carl Zeiss, Germany). Internal organs of the animals and tumor tissue were investigated. It was shown that with i.v. injection of ADAS, bright fluorescent structures with spectral characteristics corresponding to TurboFP635 protein are locally accumulated in the marrow, lungs and tumors of animals. These findings indicate that ADAS cells integrate in the animal body with transplanted tumor and can be identified by fluorescence bioimaging techniques in vivo and ex vivo.

  15. A Dual-Modality System for Both Multi-Color Ultrasound-Switchable Fluorescence and Ultrasound Imaging

    Science.gov (United States)

    Kandukuri, Jayanth; Yu, Shuai; Cheng, Bingbing; Bandi, Venugopal; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Yuan, Baohong

    2017-01-01

    Simultaneous imaging of multiple targets (SIMT) in opaque biological tissues is an important goal for molecular imaging in the future. Multi-color fluorescence imaging in deep tissues is a promising technology to reach this goal. In this work, we developed a dual-modality imaging system by combining our recently developed ultrasound-switchable fluorescence (USF) imaging technology with the conventional ultrasound (US) B-mode imaging. This dual-modality system can simultaneously image tissue acoustic structure information and multi-color fluorophores in centimeter-deep tissue with comparable spatial resolutions. To conduct USF imaging on the same plane (i.e., x-z plane) as US imaging, we adopted two 90°-crossed ultrasound transducers with an overlapped focal region, while the US transducer (the third one) was positioned at the center of these two USF transducers. Thus, the axial resolution of USF is close to the lateral resolution, which allows a point-by-point USF scanning on the same plane as the US imaging. Both multi-color USF and ultrasound imaging of a tissue phantom were demonstrated. PMID:28165390

  16. Selective detection of luminescence from semiconductor quantum dots by nanosecond time-gated imaging with a colour-masked CCD detector.

    Science.gov (United States)

    Mitchell, A C; Dad, S; Morgan, C G

    2008-05-01

    Quantum dots are of considerable interest as highly detectable labels with broad absorption, narrow spectral emission and good quantum yields. The luminescence emission has a longer decay time than that of the most common fluorophores, leading to facile rejection of much background emission (such as autofluorescence from biological samples) by means of gated detection. Here, it is shown that a new technique, true-colour nanosecond time-gated luminescence imaging, can be used for selective detection of quantum dot luminescence and should prove valuable for multiplexed detection on the basis of both spectral emission profile and luminescence decay time.

  17. Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins.

    Science.gov (United States)

    Kobayashi, Tomonori; Komatsu, Toru; Kamiya, Mako; Campos, Cláudia; González-Gaitán, Marcos; Terai, Takuya; Hanaoka, Kenjiro; Nagano, Tetsuo; Urano, Yasuteru

    2012-07-11

    Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O(6)-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag.

  18. Mapping Sentinel Lymph Node Metastasis by Dual-probe Optical Imaging

    Science.gov (United States)

    Yang, Xiangyu; Wang, Zhe; Zhang, Fuwu; Zhu, Guizhi; Song, Jibin; Teng, Gao-Jun; Niu, Gang; Chen, Xiaoyuan

    2017-01-01

    Purpose: Sentinel lymph node biopsy (SLNB) has emerged as the preferred standard procedure in patients with breast cancer, melanoma and other types of cancer. Herein, we developed a method to intra-operatively map SLNs and differentiate tumor metastases within SLNs at the same time, with the aim to provide more accurate and real-time intraoperative guidance. Experimental Design: Hyaluronic acid (HA), a ligand of lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, is employed as a SLN mapping agent after being conjugated with a near-infrared fluorophore (Cy5.5). Different sized HAs (5, 10 and 20K) were tested in normal mice and mice with localized inflammation to optimize LN retention time and signal to background ratio. Cetuximab, an antibody against epidermal growth factor receptor (EGFR), and trastuzumab, an antibody against human epidermal growth factor receptor 2 (HER2), were labeled with near-infrared fluorophore (IRDye800) for detecting metastatic tumors. LN metastasis model was developed by hock injection of firefly luciferase engineered human head neck squamous carcinoma cancer UM-SCC-22B cells or human ovarian cancer SKOV-3 cells. The metastases within LNs were confirmed by bioluminescence imaging (BLI). IRDye800-Antibodies were intravenously administered 24 h before local administration of Cy5.5-HA. Optical imaging was then performed to identify nodal metastases. Results: Binding of HA with LYVE-1 was confirmed by ELISA and fluorescence staining. HA with a size of 10K was chosen based on the favorable migration and retention profile. After sequential administration of IRDye800-antibodies intravenously and Cy5.5-HA locally to a mouse model with LN metastases and fluorescence optical imaging, partially metastasized LNs were successfully distinguished from un-metastasized LNs and fully tumor occupied LNs, based on the different signal patterns. Conclusions: Fluorophore conjugated HA is a potential lymphatic mapping agent for SLNB. Dual-tracer imaging

  19. Diffusion of flexible random-coil dextran polymers measured in anisotropic brain extracellular space by integrative optical imaging.

    Science.gov (United States)

    Xiao, Fanrong; Nicholson, Charles; Hrabe, Jan; Hrabetová, Sabina

    2008-08-01

    There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D*(major) and D*(minor), decreased with molecular size. The diffusion anisotropy ratio (DAR = D*(major)/D*(minor)) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at approximately 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

  20. 3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.

    Energy Technology Data Exchange (ETDEWEB)

    Nieman, Linda T.; Sinclair, Michael B.; Davidson, George S.; Van Benthem, Mark Hilary; Haaland, David Michael; Timlin, Jerilyn Ann; Sasaki, Darryl Yoshio; Bachand, George David; Jones, Howland D. T.

    2007-02-01

    A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

  1. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    Science.gov (United States)

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

  2. Target-to-background enhancement in multispectral endoscopy with background autofluorescence mitigation for quantitative molecular imaging

    Science.gov (United States)

    Yang, Chenying; Hou, Vivian W.; Girard, Emily J.; Nelson, Leonard Y.; Seibel, Eric J.

    2014-07-01

    Fluorescence molecular imaging with exogenous probes improves specificity for the detection of diseased tissues by targeting unambiguous molecular signatures. Additionally, increased diagnostic sensitivity is expected with the application of multiple molecular probes. We developed a real-time multispectral fluorescence-reflectance scanning fiber endoscope (SFE) for wide-field molecular imaging of fluorescent dye-labeled molecular probes at nanomolar detection levels. Concurrent multichannel imaging with the wide-field SFE also allows for real-time mitigation of the background autofluorescence (AF) signal, especially when fluorescein, a U.S. Food and Drug Administration approved dye, is used as the target fluorophore. Quantitative tissue AF was measured for the ex vivo porcine esophagus and murine brain tissues across the visible and near-infrared spectra. AF signals were then transferred to the unit of targeted fluorophore concentration to evaluate the SFE detection sensitivity for sodium fluorescein and cyanine. Next, we demonstrated a real-time AF mitigation algorithm on a tissue phantom, which featured molecular probe targeted cells of high-grade dysplasia on a substrate containing AF species. The target-to-background ratio was enhanced by more than one order of magnitude when applying the real-time AF mitigation algorithm. Furthermore, a quantitative estimate of the fluorescein photodegradation (photobleaching) rate was evaluated and shown to be insignificant under the illumination conditions of SFE. In summary, the multichannel laser-based flexible SFE has demonstrated the capability to provide sufficient detection sensitivity, image contrast, and quantitative target intensity information for detecting small precancerous lesions in vivo.

  3. Image processing

    NARCIS (Netherlands)

    Heijden, van der F.; Spreeuwers, L.J.; Blanken, H.M.; Vries de, A.P.; Blok, H.E.; Feng, L

    2007-01-01

    The field of image processing addresses handling and analysis of images for many purposes using a large number of techniques and methods. The applications of image processing range from enhancement of the visibility of cer- tain organs in medical images to object recognition for handling by industri

  4. Image city

    DEFF Research Database (Denmark)

    2003-01-01

    Image city exhibition explores a condition of mediation, through a focus on image and sound narratives with a point of departure on a number of Asian cities.......Image city exhibition explores a condition of mediation, through a focus on image and sound narratives with a point of departure on a number of Asian cities....

  5. Photothermal imaging

    Science.gov (United States)

    Lapotko, Dmitry; Antonishina, Elena

    1995-02-01

    An automated image analysis system with two imaging regimes is described. Photothermal (PT) effect is used for imaging of a temperature field or absorption structure of the sample (the cell) with high sensitivity and spatial resolution. In a cell study PT-technique enables imaging of live non-stained cells, and the monitoring of the cell shape/structure. The system includes a dual laser illumination unit coupled to a conventional optical microscope. A sample chamber provides automated or manual loading of up to 3 samples and cell positioning. For image detection a 256 X 256 10-bit CCD-camera is used. The lasers, scanning stage, and camera are controlled by PC. The system provides optical (transmitted light) image, probe laser optical image, and PT-image acquisition. Operation rate is 1 - 1.5 sec per cell for a cycle: cell positioning -- 3 images acquisition -- image parameters calculation. A special database provides image/parameters storage, presentation, and cell diagnostic according to quantitative image parameters. The described system has been tested during live and stained blood cell studies. PT-images of the cells have been used for cell differentiation. In experiments with the red blood cells (RBC) that originate from normal and anaemia blood parameters for disease differentiation have been found. For white blood cells in PT-images the details of cell structure have found that absent in their optical images.

  6. Rapid Visualization of Human Tumor Xenografts through Optical Imaging with a Near-Infrared Fluorescent Anti–Epidermal Growth Factor Receptor Nanobody

    Directory of Open Access Journals (Sweden)

    Sabrina Oliveira

    2012-01-01

    Full Text Available Given that overexpression of the epidermal growth factor receptor (EGFR is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

  7. Diffuse reflectance and fluorescence multispectral imaging system for assessment of skin

    Science.gov (United States)

    Saknite, Inga; Jakovels, Dainis; Spigulis, Janis

    2014-05-01

    The diffuse reflectance multispectral imaging technique has been used for distant mapping of in vivo skin chromophores (hemoglobin and melanin). The fluorescence multispectral imaging is not so common for skin applications due to complicity of data acquisition and processing, but could provide additional information about skin fluorophores. Both techniques are compatible, and could be combined into a multimodal solution. The multispectral imaging system Nuance based on liquid crystal tunable filters was adapted for diffuse reflectance and fluorescence spectral imaging of in vivo skin. Uniform illumination was achieved by LED ring light. Combination of four LEDs (warm white, 770 nm, 830 nm and 890 nm) was used to support diffuse reflectance mode in spectral range 450-950 nm. 405 nm LEDs were used for excitation of skin autofluorescence. Multispectral imaging system was adapted for spectral working range of 450-950 nm with scanning step of 10 nm and spectral resolution of 15 nm. An average field of view was 50x35 mm in size with spatial resolution 0,05 mm (the pixel size). Due to spectrally different illumination intensity and system sensitivity, various exposure times (from 7…500 ms) were used for each image acquisition. The proposed approach was tested for different skin lesions: benign nevus, hemangioma, basalioma and halo nevus. Spectral image cubes of different skin lesions were acquired and analyzed to test its diagnostic potential.

  8. Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

    Science.gov (United States)

    Cheglakov, Zoya

    Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide

  9. Enhanced speed in fluorescence imaging using beat frequency multiplexing

    Science.gov (United States)

    Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

    2016-03-01

    Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

  10. Multivalent viral capsids with internal cargo for fibrin imaging.

    Directory of Open Access Journals (Sweden)

    Allie C Obermeyer

    Full Text Available Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.

  11. Cerenkov radiation energy transfer (CRET imaging: a novel method for optical imaging of PET isotopes in biological systems.

    Directory of Open Access Journals (Sweden)

    Robin S Dothager

    Full Text Available BACKGROUND: Positron emission tomography (PET allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β(+-emitting radionuclides in small animals and humans. Upon β(+ decay, the initial velocity of high-energy β(+ particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms. PRINCIPAL FINDINGS: To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes (64Cu and (18F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705 to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with (99mTc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET and fluorescence resonance energy transfer (FRET, herein we define the Cerenkov radiation energy transfer (CRET ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [(18F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [(18F]FDG showed CRET ratios in vivo as high as 3.5±0.3. CONCLUSIONS: Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals.

  12. Quantitative imaging of intracellular signaling for personalized pancreatic cancer therapy in an in vivo avatar (Conference Presentation)

    Science.gov (United States)

    Samkoe, Kimberley S.; Schultz, Emily; Park, Yeonjae; Fischer, Dawn; Pogue, Brian W.; Smith, Kerrington; Tichauer, Kenneth M.; Gibbs, Summer L.

    2017-02-01

    Pancreatic ductal adenocarcinomas (PDAC) are notoriously difficult to treat and in general, molecular targeted therapies have failed even when the targeted protein is overexpressed in the tumor tissue. Genetic mutations in extracellular receptors and downstream signaling proteins (i.e., RAS signaling pathway) and convoluted intracellular cross-talk between cell signaling pathways are likely reasons that these promising therapies fail. Monitoring the complex relationship between intracellular protein signaling is difficult and to-date, standard techniques that are used (Western blot, flow cytometry, immunohistochemistry, etc.) are invasive, static and do not accurately represent in vivo structure-function relationships. Here, we describe the development of an in ovo avatar using patient derived tumors grown on the chicken chorioallantoic membrane (CAM) and the novel fluorescence-based Quantitative Protein Expression Tracking (QUIET) methodology to bridge the gap between oncology, genomics and patient outcomes. Previously developed paired-agent imaging, was extended to a three-compartment model system in QUIET, which utilizes three types of imaging agents: novel fluorophore conjugated cell permeable targeted and untargeted small molecule paired-agents, in addition to a tumor perfusion agent that is not cell membrane permeable. We have demonstrated the ability to quantify the intracellular binding domain of a trans-membrane protein in vitro using cell permeable fluorescent agents (erlotinib-TRITC and control isotype-BODIPY FL). In addition, we have demonstrated imaging protocols to simultaneously image up to 6 spectrally distinct organic fluorophores in in ovo avatars using the Nuance EX (Perkin Elmer) and established proof-of-principle intracellular and extracellular protein concentrations of epidermal growth factor receptor using QUIET and traditional paired-agent imaging.

  13. Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

    Science.gov (United States)

    Biteen, Julie

    2013-03-01

    Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

  14. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Yankelevich, Diego R. [Department of Electrical and Computer Engineering, University of California, 3101 Kemper Hall, Davis, California 95616 (United States); Department of Biomedical Engineering, University of California, 451 Health Sciences Drive, Davis, California 95616 (United States); Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Marcu, Laura, E-mail: lmarcu@ucdavis.edu [Department of Biomedical Engineering, University of California, 451 Health Sciences Drive, Davis, California 95616 (United States); Elson, Daniel S. [Hamlyn Centre for Robotic Surgery, Department of Surgery and Cancer, Imperial College London, Exhibition Road, London SW7 2AZ (United Kingdom)

    2014-03-15

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  15. Spinal imaging and image analysis

    CERN Document Server

    Yao, Jianhua

    2015-01-01

    This book is instrumental to building a bridge between scientists and clinicians in the field of spine imaging by introducing state-of-the-art computational methods in the context of clinical applications.  Spine imaging via computed tomography, magnetic resonance imaging, and other radiologic imaging modalities, is essential for noninvasively visualizing and assessing spinal pathology. Computational methods support and enhance the physician’s ability to utilize these imaging techniques for diagnosis, non-invasive treatment, and intervention in clinical practice. Chapters cover a broad range of topics encompassing radiological imaging modalities, clinical imaging applications for common spine diseases, image processing, computer-aided diagnosis, quantitative analysis, data reconstruction and visualization, statistical modeling, image-guided spine intervention, and robotic surgery. This volume serves a broad audience as  contributions were written by both clinicians and researchers, which reflects the inte...

  16. A new wide field-of-view confocal imaging system and its applications in drug discovery and pathology

    Science.gov (United States)

    Li, Gang; Damaskinos, Savvas; Dixon, Arthur E.; Lee, Lucy E. J.

    2005-11-01

    Conventional widefield light microscopy and confocal scanning microscopy have been indispensable for pathology and drug discovery research. Clinical specimens from diseased tissues are examined, new drug candidates are tested on drug targets, and the morphological and molecular biological changes of cells and tissues are observed. High throughput screening of drug candidates requires highly efficient screening instruments. A standard biomedical slide is 1 by 3 inches (25.4 by 76.2 mm) in size. A typical tissue specimen is 10 mm in diameter. To form a high resolution image of the entire specimen, a conventional widefield light microscope must acquire a large number of small images of the specimen, and then tile them together, which is tedious, inefficient and error-prone. A patented new wide field-of-view confocal scanning laser imaging system has been developed for tissue imaging, which is capable of imaging an entire microscope slide without tiling. It is capable of operating in brightfield, reflection and epi-fluorescence imaging modes. Three (red, green and blue (RGB)) lasers are used to produce brightfield and reflection images, and to excite various fluorophores. This new confocal system makes examination of large biomedical specimens more efficient, and makes fluorescence examination of large specimens possible for the first time without tiling. Description of the new confocal technology and applications of the imaging system in pathology and drug discovery research, for example, imaging large tissue specimens, tissue microarrays, and zebrafish sections, are reported in this paper.

  17. Signal peptide peptidase (SPP dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM in intact cells

    Directory of Open Access Journals (Sweden)

    Nyborg Andrew C

    2006-11-01

    Full Text Available Abstract Background Signal peptide peptidase (SPP is an intramembrane cleaving protease identified by its cleavage of several type II membrane signal peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo. Results Fluorescence lifetime imaging microscopy (FLIM can be is used to determine intra- or intermolecular interactions by fluorescently labeling epitopes on one or two different molecules. If the donor and acceptor fluorophores are less than 10 nm apart, the donor fluorophore lifetime shortens proportionally to the distance between the fluorophores. In this study, we used two types of fluorescence energy transfer (FRET pairs; cyan fluorescent protein (CFP with yellow fluorescent protein (YFP or Alexa 488 with Cy3 to differentially label the NH2- or COOH-termini of SPP molecules. A cell based SPP activity assay was used to show that all tagged SPP proteins are proteolytically active. Using FLIM we were able to show that the donor fluorophore lifetime of the CFP tagged SPP construct in living cells significantly decreases when either a NH2- or COOH-terminally YFP tagged SPP construct is co-transfected, indicating close proximity between two different SPP molecules. These data were then confirmed in cell lines stably co-expressing V5- and FLAG-tagged SPP constructs. Conclusion Our FLIM data strongly suggest dimer formation between two separate SPP proteins

  18. Medical Imaging.

    Science.gov (United States)

    Barker, M. C. J.

    1996-01-01

    Discusses four main types of medical imaging (x-ray, radionuclide, ultrasound, and magnetic resonance) and considers their relative merits. Describes important recent and possible future developments in image processing. (Author/MKR)

  19. Musculoskeletal imaging

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, M.; Baur-Melnyk, A.; Glaser, C. [Univ. of Munich-Grosshadern Campus (Germany). Dept. of Clinical Radiology

    2008-07-01

    The book covers on musculoskeletal imaging epidemiology and imaging signs for the following topics: tumours, inflammatory diseases, degenerative diseases, metabolic disorders, developmental disorders, osteonecrosis, intra-articular lesions, ligament and tendon injuries, fractures and dislocations.

  20. Microwave imaging

    CERN Document Server

    Pastorino, Matteo

    2010-01-01

    An introduction to the most relevant theoretical and algorithmic aspects of modern microwave imaging approaches Microwave imaging-a technique used in sensing a given scene by means of interrogating microwaves-has recently proven its usefulness in providing excellent diagnostic capabilities in several areas, including civil and industrial engineering, nondestructive testing and evaluation, geophysical prospecting, and biomedical engineering. Microwave Imaging offers comprehensive descriptions of the most important techniques so far proposed for short-range microwave imaging-in

  1. Proof Image

    Science.gov (United States)

    Kidron, Ivy; Dreyfus, Tommy

    2014-01-01

    The emergence of a proof image is often an important stage in a learner's construction of a proof. In this paper, we introduce, characterize, and exemplify the notion of proof image. We also investigate how proof images emerge. Our approach starts from the learner's efforts to construct a justification without (or before) attempting any…

  2. Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

    Directory of Open Access Journals (Sweden)

    Xinyue Zhu

    2015-01-01

    Full Text Available A reaction-based two-photon (TP ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM.

  3. AccPbFRET: An ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images

    Directory of Open Access Journals (Sweden)

    Vereb György

    2008-08-01

    Full Text Available Abstract Background The acceptor photobleaching fluorescence resonance energy transfer (FRET method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios. Results AccPbFRET is a user-friendly, efficient ImageJ plugin which allows fully corrected, pixel-wise calculation and detailed, ROI (region of interest-based analysis of FRET efficiencies in microscopic images. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing FRET evaluation software. Conclusion Despite of the widespread applicability of the acceptor photobleaching FRET technique, this is the first paper where all possible sources of major errors of the measurement and analysis are considered, and AccPbFRET is the only program which provides the complete suite of corrections – for registering image pairs, for unwanted photobleaching of the donor, for cross-talk of the acceptor and/or its photoproduct to the donor channel and for partial photobleaching of the acceptor. The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.

  4. Multifunctional in vivo vascular imaging using near-infrared II fluorescence

    CERN Document Server

    Hong, Guosong; Robinson, Joshua T; Raaz, Uwe; Xie, Liming; Huang, Ngan F; Cooke, John P; Dai, Hongjie

    2012-01-01

    In vivo real-time epifluorescence imaging of mouse hindlimb vasculatures in the second near-infrared region (NIR-II, 1.1~1.4 microns) is performed using single-walled carbon nanotubes (SWNTs) as fluorophores. Both high spatial resolution (~30 microns) and temporal resolution (<200 ms/frame) for small vessel imaging are achieved 1-3 mm deep in the tissue owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I, 0.75~0.9 microns) or microscopic computed tomography (micro-CT), while the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique based on their distinct hemodynamics. Further, the deep tissue penetration, high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femo...

  5. Biological imaging without autofluorescence in the second near-infrared region

    Institute of Scientific and Technical Information of China (English)

    Shuo Diao[1; Guosong Hong[1; Alexander L. Antaris[1; Jeffrey L. Blackbum[2; Kai Cheng[3; Zhen Cheng[3; Hongjie Dai[1

    2015-01-01

    Fluorescence imaging is capable of acquiring anatomical and functional infor- mation with high spatial and temporal resolution. This imaging technique has been indispensable in biological research and disease detection/diagnosis. Imaging in the visible and to a lesser degree, in the near-infrared (NIR) regions below 900 nm, suffers from autofluorescence arising from endogenous fluorescent molecules in biological tissues. This autofluorescence interferes with fluorescent molecules of interest, causing a high background and low detection sensitivity. Here, we report that fluorescence imaging in the 1,500-1,700-nm region (termed "NIR-IIb") under 808-nm excitation results in nearly zero tissue autofluorescence, allowing for background-free imaging of fluorescent species in otherwise notoriously autofluorescent biological tissues, including liver. Imaging of the intrinsic fluorescence of individual fluorophores, such as a single carbon nanotube, can be readily achieved with high sensitivity and without autofluorescence background in mouse liver within the 1,500-1,700-nm wavelength region.

  6. The Image

    DEFF Research Database (Denmark)

    Fausing, Bent

    2015-01-01

    We pick up the world and form it through the images we shape and seek out. Internal and external images intervene. We go out into the world with our images and the world enters us through its images. The body is the intermediate link for the images, which come to and from us. We are not only...... sensing subjects; we are constantly symbolizing and getting hold of our sensoric affects through imagery. To see and sense is in itself a way of articulating. We cannot stop it. The images must come out – in dreams, in concrete pictures, constructed or not, in phantasms, in sound images, in the metaphors...... of words, in bodily expressions, in sensations of textures, and in synesthetic sensing. The world is perceived from the inside though the body and we have the possibility to look at it from the outside through the images, which are made from this material. The Image-Currency, The Indefinite Image, The Self-Image...

  7. Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

    Science.gov (United States)

    Sholokh, Marianna; Zamotaiev, Oleksandr M; Das, Ranjan; Postupalenko, Viktoriia Y; Richert, Ludovic; Dujardin, Denis; Zaporozhets, Olga A; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2015-02-12

    Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

  8. Up-converting NaYF4:0.1%Tm3+, 20%Yb3+nanoparticles as luminescent labels for deep-tissue optical imaging

    Institute of Scientific and Technical Information of China (English)

    A. Gnach; K. Prorok; M. Misiak; B. Cichy; A. Bednarkiewicz

    2014-01-01

    Optical imaging plays an important role in biomedical research being extremely useful for early detection, screening and image-guided therapy. Lanthanide-doped up-converting nanoparticles were ideally suited for bioimaging because they could be ex-cited in near infrared (NIR) and emit in NIR or visible (VIS). Here, we compared lanthanide doped up-converting NaYF4 and organic fluorophores for application in deep-tissue imaging. For that purpose-tissue phantoms mimicking the natural properties of light scat-tering by living tissues were prepared. The studies allowed to quantitatively compare optical resolution of different fluorescent com-pounds, revealing that the NIR photoexcitation was favorable over conventional UV photoexcitation.

  9. PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs.

    Science.gov (United States)

    Feige, Jérôme N; Sage, Daniel; Wahli, Walter; Desvergne, Béatrice; Gelman, Laurent

    2005-09-01

    Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed-throughs (SBTs), which requires to calculate the ratios between the intensities in the FRET and in the donor or acceptor settings, when only the donor or acceptor are present. Theoretically, SBT ratios should be constant. However, experimentally, these ratios can vary as a function of fluorophore intensity, and assuming constant values may hinder precise FRET calculation. One possible cause for such a variation is the use of a microscope set-up with different photomultipliers for the donor and FRET channels, a set-up allowing higher speed acquisitions on very dynamic fluorescent molecules in living cells. Herein, we show that the bias introduced by the differential response of the two PMTs can be circumvented by a simple modeling of the SBT ratios as a function of fluorophore intensity. Another important issue when performing FRET is the localization of FRET within the cell or a population of cells. We hence developed a freely available ImageJ plug-in, called PixFRET, that allows a simple and rapid determination of SBT parameters and the display of normalized FRET images. The usefulness of this modeling and of the plug-in are exemplified by the study of FRET in a system where two interacting nuclear receptors labeled with ECFP and EYFP are coexpressed in living cells.

  10. Albumin nanoshell encapsulation of near-infrared-excitable rare-Earth nanoparticles enhances biocompatibility and enables targeted cell imaging.

    Science.gov (United States)

    Naczynski, Dominik J; Andelman, Tamar; Pal, David; Chen, Suzie; Riman, Richard E; Roth, Charles M; Moghe, Prabhas V

    2010-08-02

    The use of traditional fluorophores for in vivo imaging applications is limited by poor quantum yield, poor tissue penetration of the excitation light, and excessive tissue autofluorescence, while the use of inorganic fluorescent particles that offer a high quantum yield is frequently limited due to particle toxicity. Rare-earth-doped nanoparticles that utilize near-infrared upconversion overcome the optical limitations of traditional fluorophores, but are not typically suitable for biological application due to their insolubility in aqueous solution, lack of functional surface groups for conjugation of biomolecules, and potential cytotoxicity. A new approach to establish highly biocompatible and biologically targetable nanoshell complexes of luminescent rare-earth-doped NaYF(4) nanoparticles (REs) excitable with 920-980 nm near-infrared light for biomedical imaging applications is reported. The approach involves the encapsulation of NaYF(4) nanoparticles doped with Yb and Er within human serum albumin nanoshells to create water-dispersible, biologically functionalizable composite particles. These particles exhibit narrow size distributions around 200 nm and are stable in aqueous solution for over 4 weeks. The albumin shell confers cytoprotection and significantly enhances the biocompatibility of REs even at concentrations above 200 microg REs mL(-1). Composite particles conjugated with cyclic arginine-glycine-aspartic acid (cRGD) specifically target both human glioblastoma cell lines and melanoma cells expressing alpha(v)beta(3) integrin receptors. These findings highlight the promise of albumin-encapsulated rare-earth nanoparticles for imaging cancer cells in vitro and the potential for targeted imaging of disease sites in vivo.

  11. Quantitative mapping of aqueous microfluidic temperature with sub-degree resolution using fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Graham, Emmelyn M; Iwai, Kaoru; Uchiyama, Seiichi; de Silva, A Prasanna; Magennis, Steven W; Jones, Anita C

    2010-05-21

    The use of a water-soluble, thermo-responsive polymer as a highly sensitive fluorescence-lifetime probe of microfluidic temperature is demonstrated. The fluorescence lifetime of poly(N-isopropylacrylamide) labelled with a benzofurazan fluorophore is shown to have a steep dependence on temperature around the polymer phase transition and the photophysical origin of this response is established. The use of this unusual fluorescent probe in conjunction with fluorescence lifetime imaging microscopy (FLIM) enables the spatial variation of temperature in a microfluidic device to be mapped, on the micron scale, with a resolution of less than 0.1 degrees C. This represents an increase in temperature resolution of an order of magnitude over that achieved previously by FLIM of temperature-sensitive dyes.

  12. Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy

    CERN Document Server

    Chmyrov, Andriy; Grotjohann, Tim; Schoenle, Andreas; Keller-Findeisen, Jan; Kastrup, Lars; Jakobs, Stefan; Donnert, Gerald; Sahl, Steffen J; Hell, Stefan W

    2016-01-01

    Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between on and off states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells at 60-80 nm resolution for 100 times 100 square micrometers fields of vie...

  13. Maximum precision closed-form solution for localizing diffraction-limited spots in noisy images.

    Science.gov (United States)

    Larkin, Joshua D; Cook, Peter R

    2012-07-30

    Super-resolution techniques like PALM and STORM require accurate localization of single fluorophores detected using a CCD. Popular localization algorithms inefficiently assume each photon registered by a pixel can only come from an area in the specimen corresponding to that pixel (not from neighboring areas), before iteratively (slowly) fitting a Gaussian to pixel intensity; they fail with noisy images. We present an alternative; a probability distribution extending over many pixels is assigned to each photon, and independent distributions are joined to describe emitter location. We compare algorithms, and recommend which serves best under different conditions. At low signal-to-noise ratios, ours is 2-fold more precise than others, and 2 orders of magnitude faster; at high ratios, it closely approximates the maximum likelihood estimate.

  14. Imaging Biomarkers or Biomarker Imaging?

    Directory of Open Access Journals (Sweden)

    Markus Mitterhauser

    2014-06-01

    Full Text Available Since biomarker imaging is traditionally understood as imaging of molecular probes, we highly recommend to avoid any confusion with the previously defined term “imaging biomarkers” and, therefore, only use “molecular probe imaging (MPI” in that context. Molecular probes (MPs comprise all kinds of molecules administered to an organism which inherently carry a signalling moiety. This review highlights the basic concepts and differences of molecular probe imaging using specific biomarkers. In particular, PET radiopharmaceuticals are discussed in more detail. Specific radiochemical and radiopharmacological aspects as well as some legal issues are presented.

  15. Acoustical Imaging

    CERN Document Server

    Litniewski, Jerzy; Kujawska, Tamara; 31st International Symposium on Acoustical Imaging

    2012-01-01

    The International Symposium on Acoustical Imaging is a unique forum for advanced research, covering new technologies, developments, methods and theories in all areas of acoustics. This interdisciplinary Symposium has been taking place continuously since 1968. In the course of the years the proceedings volumes in the Acoustical Imaging Series have become a reference for cutting-edge research in the field. In 2011 the 31st International Symposium on Acoustical Imaging was held in Warsaw, Poland, April 10-13. Offering both a broad perspective on the state-of-the-art as well as  in-depth research contributions by the specialists in the field, this Volume 31 in the Series contains an excellent collection of papers in six major categories: Biological and Medical Imaging Physics and Mathematics of Acoustical Imaging Acoustic Microscopy Transducers and Arrays Nondestructive Evaluation and Industrial Applications Underwater Imaging

  16. Image Processing

    Science.gov (United States)

    1993-01-01

    Electronic Imagery, Inc.'s ImageScale Plus software, developed through a Small Business Innovation Research (SBIR) contract with Kennedy Space Flight Center for use on space shuttle Orbiter in 1991, enables astronauts to conduct image processing, prepare electronic still camera images in orbit, display them and downlink images to ground based scientists for evaluation. Electronic Imagery, Inc.'s ImageCount, a spin-off product of ImageScale Plus, is used to count trees in Florida orange groves. Other applications include x-ray and MRI imagery, textile designs and special effects for movies. As of 1/28/98, company could not be located, therefore contact/product information is no longer valid.

  17. Neuroblastoma imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ley, Sebastian; Ley-Zaporozhan, J.; Schenk, J.P. [Univ. Hospital Heidelberg (Germany). Div. of Pediatric Radiology; Guenther, P. [Univ. Heidelberg (Germany). Division of Pediatric Surgery; Deubzer, H.E.; Witt, O. [Children' s Hospital Heidelberg (Germany). Dept. of Pediatric Oncology; German Cancer Research Center Heidelberg (Germany). Clinical Cooperation Unit Pediatric Oncology

    2011-03-15

    Neuroblastoma is an embryonic tumor of the sympathetic nervous system which represents one of the most common malignancies in early childhood. Its clinical and biological behavior show a remarkable heterogeneity, ranging from spontaneous regression to inexorable progression with a fatal outcome. This review summarizes the clinical risk stratification and treatment options. An extensive overview of the role of imaging during the course of the disease and typical imaging findings in all imaging modalities are demonstrated. (orig.)

  18. Cerenkov Imaging

    OpenAIRE

    Das, Sudeep; Thorek, Daniel L.J.; Grimm, Jan

    2014-01-01

    Cerenkov luminescence (CL) has been used recently in a plethora of medical applications like imaging and therapy with clinically relevant medical isotopes. The range of medical isotopes used is fairly large and expanding. The generation of in vivo light is useful since it circumvents depth limitations for excitation light. Cerenkov luminescence imaging (CLI) is much cheaper in terms of infrastructure than positron emission tomography (PET) and is particularly useful for imaging of superficial...

  19. Inherently fluorescent polystyrene microspheres for coating, sensing and cellular imaging.

    Science.gov (United States)

    Qu, Jian-Bo; Xu, Yu-Liang; Liu, Yu; Wang, Yanan; Sui, Yuanhong; Liu, Jian-Guo; Wang, Xiaojuan

    2017-04-01

    Commercially available polystyrene (PS) fluorescent microspheres are widely used in biological field for tracing, in vivo imaging and calibration of flow cytometry, among other applications. However, these particles do suffer from some drawbacks such as the leakage and photobleaching of organic dyes within them. In the present study, inherently fluorescent properties of PS based microspheres have been explored for the first time. Here we find that a simple chloromethylation reaction endows the polystyrene particles with inherent fluorescence without any subsequent conjugation of an external fluorophore. A possible mechanism for fluorescence is elucidated by synthesizing and investigating p-ethylbenzyl chloride, a compound with similar structure. Significantly, no photobleaching or leaking issues were observed owing to the stable structure of the microspheres. Chloromethylated PS (CMPS) microspheres can keep their perpetual blue fluorescence even in dry powder state making them attractive as a potential coating material. Furthermore, the chloromethyl groups on CMPS microspheres make them very convenient for further functionalization. Poly(ethylene glycol) (PEG) grafted microspheres showed good biocompatibility and negligible cytotoxicity, and could be used to image intracellular Fe(3+) due to the selective fluorescence quenching effect of aqueous Fe(3+) in cytoplasm.

  20. Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

    Science.gov (United States)

    Monroy-Contreras, Ricardo; Vaca, Luis

    2011-01-01

    Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs) are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore) at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area. PMID:21876785

  1. Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

    Directory of Open Access Journals (Sweden)

    Ricardo Monroy-Contreras

    2011-01-01

    Full Text Available Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area.

  2. Normalized fluorescence lifetime imaging for tumor identification and margin delineation

    Science.gov (United States)

    Sherman, Adria J.; Papour, Asael; Bhargava, Siddharth; Taylor, Zach; Grundfest, Warren S.; Stafsudd, Oscar M.

    2013-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is a technique that has been proven to produce quantitative and qualitative differentiation and identification of substances with good specificity and sensitivity based on lifetime extracted information. This technique has shown the ability to also differentiate between a wide range of tissue types to identify malignant from benign tissue in vivo and ex vivo. However, the complexity, long duration and effort required to generate this information has limited the adoption of these techniques in a clinical setting. Our group has developed a time-resolved imaging system (patent pending) that does not require the extraction of lifetimes or use of complex curve fitting algorithms to display the needed information. The technique, entitled Lifetime Fluorescence Imaging (LFI, or NoFYI), converts fluorescence lifetime decay information directly into visual contrast. Initial studies using Fluorescein and Rhodamine-B demonstrated the feasibility of this approach. Subsequent studies demonstrated the ability to separate collagen and elastin powders. The technique uses nanosecond pulsed UV LEDs at 375 nm for average illumination intensities of ~4.5 μW on the tissue surface with detection by a gated CCD camera. To date, we have imaged 11 surgical head and neck squamous cell carcinoma and brain cancer biopsy specimens including 5 normal and 6 malignant samples. Images at multiple wavelengths clearly demonstrate differentiation between benign and malignant tissue, which was later confirmed by histology. Contrast was obtained between fluorophores with 35 μm spatial resolution and an SNR of ~30 dB allowing us to clearly define tumor margins in these highly invasive cancers. This method is capable of providing both anatomical and chemical information for the pathologist and the surgeon. These results suggest that this technology has a possible role in identifying tumors in tissue specimens and detecting tumor margins during procedures.

  3. Hyperspectral imaging of endogenous fluorescent metabolic molecules to identify pain states in central nervous system tissue

    Science.gov (United States)

    Staikopoulos, Vasiliki; Gosnell, Martin E.; Anwer, Ayad G.; Mustafa, Sanam; Hutchinson, Mark R.; Goldys, Ewa M.

    2016-12-01

    Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

  4. Fluorescent metal nanoshell and CK19 detection on single cell image.

    Science.gov (United States)

    Zhang, Jian; Fu, Yi; Li, Ge; Lakowicz, Joseph R; Zhao, Richard Y

    2011-09-16

    In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.

  5. Quantitative assessment of p-glycoprotein expression and function using confocal image analysis.

    Science.gov (United States)

    Hamrang, Zahra; Arthanari, Yamini; Clarke, David; Pluen, Alain

    2014-10-01

    P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

  6. Improved Excitation Light Rejection Enhances Small-Animal Fluorescent Optical Imaging

    Directory of Open Access Journals (Sweden)

    Kildong Hwang

    2005-07-01

    Full Text Available Small-animal fluorescence-enhanced imaging involves the detection of weak fluorescent signals emanating from nanomolar to picomolar concentrations of exogenous or endogenously produced fluorophore concurrent with the rejection of an overwhelmingly large component of backscattered excitation light. The elimination of the back-reflected excitation light of the collected signal remains a major and often unrecognized challenge for further reducing the noise floor and increasing sensitivity of small-animal fluorescence imaging. Herein, we show that the combination of three-cavity interference and holographic super notch filters with appropriate imaging lenses to collimate light improves rejection of excitation light, enabling more accurate imaging. To assess excitation leakage, the “out-of-band (S(Λx” to “in-band (S(Λm–S(Λx” signal ratio from phantom studies and the target-to-background ratio (TBR from in vivo animal imaging was acquired with and without collimating optics. The addition of collimating optics resulted in a 51% to 75% reduction in the ratio of (S(Λx/(S(Λm–S(Λx for the phantom studies and an improvement of TBR from 11% to 31% and of signal-to-noise ratio from 11% to 142% for an integrin-targeting conjugate in human glioma xenografts.

  7. Near-infrared fluorescence image quality test methods for standardized performance evaluation

    Science.gov (United States)

    Kanniyappan, Udayakumar; Wang, Bohan; Yang, Charles; Ghassemi, Pejhman; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

    2017-03-01

    Near-infrared fluorescence (NIRF) imaging has gained much attention as a clinical method for enhancing visualization of cancers, perfusion and biological structures in surgical applications where a fluorescent dye is monitored by an imaging system. In order to address the emerging need for standardization of this innovative technology, it is necessary to develop and validate test methods suitable for objective, quantitative assessment of device performance. Towards this goal, we develop target-based test methods and investigate best practices for key NIRF imaging system performance characteristics including spatial resolution, depth of field and sensitivity. Characterization of fluorescence properties was performed by generating excitation-emission matrix properties of indocyanine green and quantum dots in biological solutions and matrix materials. A turbid, fluorophore-doped target was used, along with a resolution target for assessing image sharpness. Multi-well plates filled with either liquid or solid targets were generated to explore best practices for evaluating detection sensitivity. Overall, our results demonstrate the utility of objective, quantitative, target-based testing approaches as well as the need to consider a wide range of factors in establishing standardized approaches for NIRF imaging system performance.

  8. Comparative evaluation of methylene blue and demeclocycline for enhancing optical contrast of gliomas in optical images

    Science.gov (United States)

    Wirth, Dennis; Snuderl, Matija; Curry, William; Yaroslavsky, Anna

    2014-09-01

    Contrast agents have shown to be useful in the detection of cancers. The goal of this study was to compare enhancement of brain cancer contrast using reflectance and fluorescence confocal imaging of two fluorophores, methylene blue (MB) and demeclocycline (DMN). MB absorbs light in the red spectral range and fluoresces in the near-infrared. It is safe for in vivo staining of human skin and breast tissue. However, its safety for staining human brain is questionable. Thus, DMN, which absorbs light in the violet spectral range and fluoresces between 470 and 570 nm, could provide a safer alternative to MB. Fresh human gliomas, obtained from surgeries, were cut in half and stained with aqueous solutions of MB and DMN, respectively. Stained tissues were imaged using multimodal confocal microscopy. Resulting reflectance and fluorescence optical images were compared with hematoxylin and eosin histopathology, processed from each imaged tissue. Results indicate that images of tissues stained with either stain exhibit comparable contrast and resolution of morphological detail. Further studies are required to establish the safety and efficacy of these contrast agents for use in human brain.

  9. Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

    Science.gov (United States)

    Adur, J.; Pelegati, V. B.; de Thomaz, A. A.; Bottcher-Luiz, F.; Andrade, L. A. L. A.; Almeida, D. B.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

  10. Imaging Genetics

    Science.gov (United States)

    Munoz, Karen E.; Hyde, Luke W.; Hariri, Ahmad R.

    2009-01-01

    Imaging genetics is an experimental strategy that integrates molecular genetics and neuroimaging technology to examine biological mechanisms that mediate differences in behavior and the risks for psychiatric disorder. The basic principles in imaging genetics and the development of the field are discussed.

  11. Imaging Genetics

    Science.gov (United States)

    Munoz, Karen E.; Hyde, Luke W.; Hariri, Ahmad R.

    2009-01-01

    Imaging genetics is an experimental strategy that integrates molecular genetics and neuroimaging technology to examine biological mechanisms that mediate differences in behavior and the risks for psychiatric disorder. The basic principles in imaging genetics and the development of the field are discussed.

  12. Cerenkov imaging.

    Science.gov (United States)

    Das, Sudeep; Thorek, Daniel L J; Grimm, Jan

    2014-01-01

    Cerenkov luminescence (CL) has been used recently in a plethora of medical applications like imaging and therapy with clinically relevant medical isotopes. The range of medical isotopes used is fairly large and expanding. The generation of in vivo light is useful since it circumvents depth limitations for excitation light. Cerenkov luminescence imaging (CLI) is much cheaper in terms of infrastructure than positron emission tomography (PET) and is particularly useful for imaging of superficial structures. Imaging can basically be done using a sensitive camera optimized for low-light conditions, and it has a better resolution than any other nuclear imaging modality. CLI has been shown to effectively diagnose disease with regularly used PET isotope ((18)F-FDG) in clinical setting. Cerenkov luminescence tomography, Cerenkov luminescence endoscopy, and intraoperative Cerenkov imaging have also been explored with positive conclusions expanding the current range of applications. Cerenkov has also been used to improve PET imaging resolution since the source of both is the radioisotope being used. Smart imaging agents have been designed based on modulation of the Cerenkov signal using small molecules and nanoparticles giving better insight of the tumor biology. © 2014 Elsevier Inc. All rights reserved.

  13. Star Imager

    DEFF Research Database (Denmark)

    Madsen, Peter Buch; Jørgensen, John Leif; Thuesen, Gøsta;

    1997-01-01

    The version of the star imager developed for Astrid II is described. All functions and features are described as well as the operations and the software protocol.......The version of the star imager developed for Astrid II is described. All functions and features are described as well as the operations and the software protocol....

  14. Image Inpainting

    Directory of Open Access Journals (Sweden)

    A. A. Kondekar

    2014-09-01

    Full Text Available Inpainting refers to the art of restoring lost parts of image and reconstructing them based on the background information i.e Image inpainting is the process of reconstructing lost or deteriorated parts of images using information from surrounding areas. In fine art museums, inpainting of degraded paintings is traditionally carried out by professional artists and usually very time consuming.The purpose of inpainting is to reconstruct missing regions in a visually plausible manner so that it seems reasonable to the human eye. There have been several approaches proposed for the same. This paper gives an overview of different Techniques of Image Inpainting.The proposed work includes the overview of PDE based inpainting algorithm and Texture synthesis based inpainting algorithm. This paper presents a brief survey on comparative study of these two techniques used for Image Inpainting.

  15. Acoustical Imaging

    CERN Document Server

    Akiyama, Iwaki

    2009-01-01

    The 29th International Symposium on Acoustical Imaging was held in Shonan Village, Kanagawa, Japan, April 15-18, 2007. This interdisciplinary Symposium has been taking place every two years since 1968 and forms a unique forum for advanced research, covering new technologies, developments, methods and theories in all areas of acoustics. In the course of the years the volumes in the Acoustical Imaging Series have developed and become well-known and appreciated reference works. Offering both a broad perspective on the state-of-the-art in the field as well as an in-depth look at its leading edge research, this Volume 29 in the Series contains again an excellent collection of seventy papers presented in nine major categories: Strain Imaging Biological and Medical Applications Acoustic Microscopy Non-Destructive Evaluation and Industrial Applications Components and Systems Geophysics and Underwater Imaging Physics and Mathematics Medical Image Analysis FDTD method and Other Numerical Simulations Audience Researcher...

  16. Medical imaging

    CERN Document Server

    Townsend, David W

    1996-01-01

    Since the introduction of the X-ray scanner into radiology almost 25 years ago, non-invasive imaging has become firmly established as an essential tool in the diagnosis of disease. Fully three-dimensional imaging of internal organs is now possible, b and for studies which explore the functional status of the body. Powerful techniques to correlate anatomy and function are available, and scanners which combine anatomical and functional imaging in a single device are under development. Such techniques have been made possible through r ecent technological and mathematical advances. This series of lectures will review both the physical basis of medical imaging techniques using X-rays, gamma and positron emitting radiosiotopes, and nuclear magnetic resonance, and the mathematical methods used to reconstruct three-dimentional distributions from projection data. The lectures will trace the development of medical imaging from simple radiographs to the present-day non-invasive measurement of in vivo biochemistry. They ...

  17. Photobleaching correction in fluorescence microscopy images

    Energy Technology Data Exchange (ETDEWEB)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H [Microscopy Laboratory, School of Engineering - Bioengineering, National University of Entre Rios (UNER), Ruta 11, Km 10 (3101), Oro Verde, Entre Rios (Argentina)

    2007-11-15

    Fluorophores are used to detect molecular expression by highly specific antigen-antibody reactions in fluorescence microscopy techniques. A portion of the fluorophore emits fluorescence when irradiated with electromagnetic waves of particular wavelengths, enabling its detection. Photobleaching irreversibly destroys fluorophores stimulated by radiation within the excitation spectrum, thus eliminating potentially useful information. Since this process may not be completely prevented, techniques have been developed to slow it down or to correct resulting alterations (mainly, the decrease in fluorescent signal). In the present work, the correction by photobleaching curve was studied using E-cadherin (a cell-cell adhesion molecule) expression in Bufo arenarum embryos. Significant improvements were observed when applying this simple, inexpensive and fast technique.

  18. Hypericin from St. John's Wort (hypericum perforatum) as a novel natural fluorophore for chemiluminescence reaction of bis (2,4,6-trichlorophenyl) oxalate-H{sub 2}O{sub 2}-imidazole and quenching effect of some natural lipophilic hydrogen peroxide scavengers

    Energy Technology Data Exchange (ETDEWEB)

    Kazemi, Sayed Yahya [Department of Basic Sciences, Sari Agricultural Sciences and Natural Resources University, P.O. Box 578, Sari (Iran, Islamic Republic of); Abedirad, Seyed Mohammad, E-mail: mabedirad@yahoo.com [Department of Basic Sciences, Sari Agricultural Sciences and Natural Resources University, P.O. Box 578, Sari (Iran, Islamic Republic of); Zali, Seyed Hassan; Amiri, Mohadeseh [Department of Range and Watershed Management, Sari Agricultural Sciences and Natural Resources University, P.O. Box 578, Sari (Iran, Islamic Republic of)

    2012-05-15

    Hypericin (HYP) molecule is a natural photoactive pigment, which plays a role as an effective photoreceptor in some plants of the Hypericum species (the most common of which is Saint John's Wort) and some insect species. The present work deals with the first attempt to the study of peroxyoxalate chemiluminescence (POCL) system in the presense of HYP as a natural fluorophore. Reaction of bis (2,4,6-trichlorophenyl) oxalate(TCPO)-H{sub 2}O{sub 2}-imidazole can transfer energy to a HYP via formation of dioxetane through the chemically initiated electron exchange luminescence (CIEEL) mechanism and can emits a very intense red light. The effects of HYP, hydrogen peroxide, TCPO and imidazole concentrations on kinetic chemiluminescence parameters were also studied. These parameters including rise and fall rate constant for the chemiluminescence burst, theoretical and experimental maximum intensity, theoretical and experimental time to reach maximum intensity and total light yield emission were evaluated by using a pooled intermediate model for a non-linear least-squares curve fitting program, KINFIT. Moreover, quenching effect of two lipophilic natural antioxidant, Quercetin and {beta}-carotene on it system was also investigated. The measurable concentration range of 7 Multiplication-Sign 10{sup -6} M to 7.5 Multiplication-Sign 10{sup -5} M of antioxidants were evaluated from the proper Stern-Volmer plots with satisfactory RSD% and corresponding detection limits of 2.2 Multiplication-Sign 10{sup -6} and 3.7 Multiplication-Sign 10{sup -6} for {beta}-carotene and quercetin respectively. - Highlights: Black-Right-Pointing-Pointer Red fluorophores may therefore chemiluminescence more intensely than other commonly chemiluminophores and emits light in longer wavelengths. Black-Right-Pointing-Pointer Hypericin from St. John's wort (hypericum perforatum) as natural red fluorophore for peroxyoxalate chemiluminescence was introduced. Black-Right-Pointing-Pointer Quenching

  19. Quantitative whole body biodistribution of fluorescent-labeled agents by non-invasive tomographic imaging.

    Directory of Open Access Journals (Sweden)

    Kristine O Vasquez

    Full Text Available When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR or near infrared (NIR imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D fluorescence reflectance imaging of excised intact organs (r²  =  0.996 and 0.969, respectively. Dynamic FMT imaging (multiple times from 0 to 24 h performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20-50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor

  20. Retinal imaging and image analysis

    NARCIS (Netherlands)

    Abramoff, M.D.; Garvin, Mona K.; Sonka, Milan

    2010-01-01

    Many important eye diseases as well as systemic diseases manifest themselves in the retina. While a number of other anatomical structures contribute to the process of vision, this review focuses on retinal imaging and image analysis. Following a brief overview of the most prevalent causes of blindne

  1. COLOR IMAGES

    Directory of Open Access Journals (Sweden)

    Dominique Lafon

    2011-05-01

    Full Text Available The goal of this article is to present specific capabilities and limitations of the use of color digital images in a characterization process. The whole process is investigated, from the acquisition of digital color images to the analysis of the information relevant to various applications in the field of material characterization. A digital color image can be considered as a matrix of pixels with values expressed in a vector-space (commonly 3 dimensional space whose specificity, compared to grey-scale images, is to ensure a coding and a representation of the output image (visualisation printing that fits the human visual reality. In a characterization process, it is interesting to regard color image attnbutes as a set of visual aspect measurements on a material surface. Color measurement systems (spectrocolorimeters, colorimeters and radiometers and cameras use the same type of light detectors: most of them use Charge Coupled Devices sensors. The difference between the two types of color data acquisition systems is that color measurement systems provide a global information of the observed surface (average aspect of the surface: the color texture is not taken into account. Thus, it seems interesting to use imaging systems as measuring instruments for the quantitative characterization of the color texture.

  2. Image registration

    CERN Document Server

    Goshtasby, A Ardeshir

    2012-01-01

    This book presents a thorough and detailed guide to image registration, outlining the principles and reviewing state-of-the-art tools and methods. The book begins by identifying the components of a general image registration system, and then describes the design of each component using various image analysis tools. The text reviews a vast array of tools and methods, not only describing the principles behind each tool and method, but also measuring and comparing their performances using synthetic and real data. Features: discusses similarity/dissimilarity measures, point detectors, feature extr

  3. Image retrieval

    DEFF Research Database (Denmark)

    Ørnager, Susanne

    1997-01-01

    The paper touches upon indexing and retrieval for effective searches of digitized images. Different conceptions of what subject indexing means are described as a basis for defining an operational subject indexing strategy for images. The methodology is based on the art historian Erwin Panofsky...... of signs and connotation as meanings relating to feelings or associations. A joint methodology is suggested between the two researchers and the methodology is implemented in analyzing press photos. Fields of application discussed include the messages in an image and the linking between information running....... The empirical analysis demonstrates how the results can be applied as the foundation for a semantic model....

  4. Image retrieval

    DEFF Research Database (Denmark)

    Ørnager, Susanne

    1997-01-01

    The paper touches upon indexing and retrieval for effective searches of digitized images. Different conceptions of what subject indexing means are described as a basis for defining an operational subject indexing strategy for images. The methodology is based on the art historian Erwin Panofsky...... of signs and connotation as meanings relating to feelings or associations. A joint methodology is suggested between the two researchers and the methodology is implemented in analyzing press photos. Fields of application discussed include the messages in an image and the linking between information running...

  5. IMAGE ENHANCEMENT USING IMAGE FUSION AND IMAGE PROCESSING TECHNIQUES

    OpenAIRE

    Arjun Nelikanti

    2015-01-01

    Principle objective of Image enhancement is to process an image so that result is more suitable than original image for specific application. Digital image enhancement techniques provide a multitude of choices for improving the visual quality of images. Appropriate choice of such techniques is greatly influenced by the imaging modality, task at hand and viewing conditions. This paper will provide a combination of two concepts, image fusion by DWT and digital image processing techniques. The e...

  6. DNA as sensors and imaging agents for metal ions.

    Science.gov (United States)

    Xiang, Yu; Lu, Yi

    2014-02-17

    Increasing interest in detecting metal ions in many chemical and biomedical fields has created demands for developing sensors and imaging agents for metal ions with high sensitivity and selectivity. This review covers recent progress in DNA-based sensors and imaging agents for metal ions. Through both combinatorial selection and rational design, a number of metal-ion-dependent DNAzymes and metal-ion-binding DNA structures that can selectively recognize specific metal ions have been obtained. By attachment of these DNA molecules with signal reporters such as fluorophores, chromophores, electrochemical tags, and Raman tags, a number of DNA-based sensors for both diamagnetic and paramagnetic metal ions have been developed for fluorescent, colorimetric, electrochemical, and surface Raman detection. These sensors are highly sensitive (with a detection limit down to 11 ppt) and selective (with selectivity up to millions-fold) toward specific metal ions. In addition, through further development to simplify the operation, such as the use of "dipstick tests", portable fluorometers, computer-readable disks, and widely available glucose meters, these sensors have been applied for on-site and real-time environmental monitoring and point-of-care medical diagnostics. The use of these sensors for in situ cellular imaging has also been reported. The generality of the combinatorial selection to obtain DNAzymes for almost any metal ion in any oxidation state and the ease of modification of the DNA with different signal reporters make DNA an emerging and promising class of molecules for metal-ion sensing and imaging in many fields of applications.

  7. Body Imaging

    Science.gov (United States)

    2001-01-01

    The high-tech art of digital signal processing (DSP) was pioneered at NASA's Jet Propulsion Laboratory (JPL) in the mid-1960s for use in the Apollo Lunar Landing Program. Designed to computer enhance pictures of the Moon, this technology became the basis for the Landsat Earth resources satellites and subsequently has been incorporated into a broad range of Earthbound medical and diagnostic tools. DSP is employed in advanced body imaging techniques including Computer-Aided Tomography, also known as CT and CATScan, and Magnetic Resonance Imaging (MRI). CT images are collected by irradiating a thin slice of the body with a fan-shaped x-ray beam from a number of directions around the body's perimeter. A tomographic (slice-like) picture is reconstructed from these multiple views by a computer. MRI employs a magnetic field and radio waves, rather than x-rays, to create images.

  8. Imaging System

    Science.gov (United States)

    1995-01-01

    The 1100C Virtual Window is based on technology developed under NASA Small Business Innovation (SBIR) contracts to Ames Research Center. For example, under one contract Dimension Technologies, Inc. developed a large autostereoscopic display for scientific visualization applications. The Virtual Window employs an innovative illumination system to deliver the depth and color of true 3D imaging. Its applications include surgery and Magnetic Resonance Imaging scans, viewing for teleoperated robots, training, and in aviation cockpit displays.

  9. Image Security

    Science.gov (United States)

    2007-11-02

    popularity, contemplates the cru- cial needs for protecting intellectual property rights on multimedia content like images, video, audio , and oth- ers...protection for still images, audio , video, and multimedia products.’ The networking environment of the future will require tools that provide m secure and fast...technique known as steganography ? Steganography , or “covered writing,” George Voyatzis and Ioannis Pitas University of Thessaloniki has a long

  10. Heterocyclic quinol-type fluorophores: synthesis, X-ray crystal structures, and solid-state photophysical properties of novel 5-hydroxy-5-substituent-benzo[b]naphtho[1,2-d]furan-6-one and 3-hydroxy-3-substituent-benzo[kl]xanthen-2-one derivatives.

    Science.gov (United States)

    Ooyama, Yousuke; Okamoto, Tomohiro; Yamaguchi, Takahiro; Suzuki, Toshihisa; Hayashi, Akiko; Yoshida, Katsuhira

    2006-10-16

    Novel heterocyclic quinol-type fluorophores (4 a-c) and (5 a-c) that contain substituents (R = Me, Bu, Ph) with nonconjugated linkages to the chromophore skeleton have been synthesized and their photophysical properties have been investigated in solution and in the solid state. Considerable differences in the absorption and fluorescence spectra were observed between the two states. Quinols 4 a-c and 5 a-c exhibited almost the same absorption and fluorescence spectra in solution; however, their solid-state fluorescence excitation and emission spectra in the crystalline state were quite different. We performed X-ray crystallographic analyses to elucidate the dramatic effect of the substituents of the nonconjugated linkage on the solid-state fluorescence excitation and emission spectra. The relationships between the solid-state photophysical properties and the chemical and crystal structures of 4 a-c and 5 a-c are discussed on the basis of the X-ray crystal structures.

  11. Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

    Science.gov (United States)

    Plaunt, Adam J; Harmatys, Kara M; Wolter, William R; Suckow, Mark A; Smith, Bradley D

    2014-04-16

    Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

  12. Traceable working standards with SI units of radiance for characterizing the measurement performance of investigational clinical NIRF imaging devices

    Science.gov (United States)

    Zhu, Banghe; Rasmussen, John C.; Litorja, Maritoni; Sevick-Muraca, Eva M.

    2017-03-01

    All medical devices for Food and Drug market approval require specifications of performance based upon International System of Units (SI) or units derived from SI for reasons of traceability. Recently, near-infrared fluorescence (NIRF) imaging devices of a variety of designs have emerged on the market and in investigational clinical studies. Yet the design of devices used in the clinical studies vary widely, suggesting variable device performance. Device performance depends upon optimal excitation of NIRF imaging agents, rejection of backscattered excitation and ambient light, and selective collection of fluorescence emanating from the fluorophore. There remains no traceable working standards with SI units of radiance to enable prediction that a given molecular imaging agent can be detected in humans by a given NIRF imaging device. Furthermore, as technologies evolve and as NIRF imaging device components change, there remains no standardized means to track device improvements over time and establish clinical performance without involving clinical trials, often costly. In this study, we deployed a methodology to calibrate luminescent radiance of a stable, solid phantom in SI units of mW/cm2/sr for characterizing the measurement performance of ICCD and IsCMOS camera based NIRF imaging devices, such as signal-to-noise ratio (SNR) and contrast. The methodology allowed determination of superior SNR of the ICCD over the IsCMOS system; comparable contrast of ICCD and IsCMOS depending upon binning strategies.

  13. Label-free SHG imaging and spectral FLIM of corneas using a sub-15 fs laser microscope

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Seitz, Berthold; Morgado, António Miguel; König, Karsten

    2014-02-01

    Alterations to the corneal cell metabolism or to the structural organization of collagen fibrils occur in several corneal and systemic pathologies. In this work we resort to multiphoton microscopy corneal imaging to achieve a characterization of the corneal state. Using fluorescence lifetime imaging microscopy (FLIM) the assessment of the metabolic state of corneal cells is possible, whereas second harmonic generation (SHG) imaging can be used to assess corneal structural alterations. A sub-15 fs near-infrared laser source with a broad excitation spectrum was used for SHG imaging and FLIM. The broad spectrum allows simultaneous excitation of both metabolic co-factors. The signals were collected by a photomultiplier tubes (PMT) detector with 16 simultaneous recording channels, which allowed the separation of fluorophores autofluorescence based on their emission wavelengths. We were able to successfully image ex-vivo human and porcine cornea at multiple depths. Simultaneous NADH and flavin autofluorescence, SHG of collagen fibrils, and stroma autofluorescence imaging was performed which may in future allow an improved characterization of the metabolic and structural alterations of the corneal tissue due to pathophysiological conditions. This would be an important step towards a better understanding of corneal dystrophies and systemic metabolic disorders.

  14. Emerging images

    KAUST Repository

    Mitra, Niloy J.

    2009-01-01

    Emergence refers to the unique human ability to aggregate information from seemingly meaningless pieces, and to perceive a whole that is meaningful. This special skill of humans can constitute an effective scheme to tell humans and machines apart. This paper presents a synthesis technique to generate images of 3D objects that are detectable by humans, but difficult for an automatic algorithm to recognize. The technique allows generating an infinite number of images with emerging figures. Our algorithm is designed so that locally the synthesized images divulge little useful information or cues to assist any segmentation or recognition procedure. Therefore, as we demonstrate, computer vision algorithms are incapable of effectively processing such images. However, when a human observer is presented with an emergence image, synthesized using an object she is familiar with, the figure emerges when observed as a whole. We can control the difficulty level of perceiving the emergence effect through a limited set of parameters. A procedure that synthesizes emergence images can be an effective tool for exploring and understanding the factors affecting computer vision techniques. © 2009 ACM.

  15. Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

    Science.gov (United States)

    Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

    2014-02-01

    Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

  16. Image reconstruction for diagnosis and prognosis of breast cancer using fluorescence measurements: phantom studies

    Science.gov (United States)

    Roy, R.; Godavarty, A.; Thompson, A. B., Jr.; Sevick-Muraca, E. M.

    2005-04-01

    Fluorescence-enhance optical tomography is performed using (i) point illumination and point collection and (ii) area illumination and area collection geometrics in 3D. In both measurement techniques, an image-intensified charge-coupled (ICCD) imaging system is used in the frequency-domain to image near-infrared contrast agents. The experimental measurements are compared to diffusion model predictions in least squares form in the inverse problem. For image recovery for both area and point illumination geometries, an efficient gradient-based optimization technique based on the Penalty/modified barrier function (PMBF) method and the constrained truncated Newton with trust region (CONTN) method is developed. Targets in 3D were reconstructed from experimental data under two conditions of (i) perfect uptake (1:0, target to background ratio) and (ii) imperfect uptake (100:1, target to background ratio). Parameters of absorption cross section due to fluorophore and lifetimes are reconstructed. The present work demonstrates that 3D fluorescence enhanced optical tomography reconstructions can be successfully performed from both point/area illumination and collection experimental measurements of the time-dependent light propagation on clinically relevant tissue phantoms using a gain-modulated ICCD camera.

  17. Quantitative Imaging of Cell-Permeable Magnetic Resonance Contrast Agents Using X-Ray Fluorescence

    Directory of Open Access Journals (Sweden)

    Paul J. Endres

    2006-10-01

    Full Text Available The inability to transduce cellular membranes is a limitation of current magnetic resonance imaging probes used in biologic and clinical settings. This constraint confines contrast agents to extracellular and vascular regions of the body, drastically reducing their viability for investigating processes and cycles in developmental biology. Conversely, a contrast agent with the ability to permeate cell membranes could be used in visualizing cell patterning, cell fate mapping, gene therapy, and, eventually, noninvasive cancer diagnosis. Therefore, we describe the synthesis and quantitative imaging of four contrast agents with the capability to cross cell membranes in sufficient quantity for detection. Each agent is based on the conjugation of a Gd(III chelator with a cellular transduction moiety. Specifically, we coupled Gd(III–diethylenetriaminepentaacetic acid DTPA and Gd(III–1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with an 8–amino acid polyarginine oligomer and an amphipathic stilbene molecule, 4-amino-4'-(N,N-dimethylaminostilbene. The imaging modality that provided the best sensitivity and spatial resolution for direct detection of the contrast agents is synchrotron radiation x-ray fluorescence (SR-XRF. Unlike optical microscopy, SR-XRF provides two-dimensional images with resolution 103 better than 153Gd gamma counting, without altering the agent by organic fluorophore conjugation. The transduction efficiency of the intracellular agents was evaluated by T1 analysis and inductively coupled plasma mass spectrometry to determine the efficacy of each chelate-transporter combination.

  18. Using multiphoton fluorescence lifetime imaging to characterize liver damage and fluorescein disposition in liver in vivo

    Science.gov (United States)

    Thorling, Camilla A.; Studier, Hauke; Crawford, Darrell; Roberts, Michael S.

    2016-03-01

    Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.

  19. New light on ion channel imaging by total internal reflection fluorescence (TIRF microscopy

    Directory of Open Access Journals (Sweden)

    Hisao Yamamura

    2015-05-01

    Full Text Available Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca2+ events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca2+ signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels.

  20. An all-fiber-optic endoscopy platform for simultaneous OCT and fluorescence imaging.

    Science.gov (United States)

    Mavadia, Jessica; Xi, Jiefeng; Chen, Yongping; Li, Xingde

    2012-11-01

    We present an all-fiber-optically based endoscope platform for simultaneous optical coherence tomography (OCT) and fluorescence imaging. This design entails the use of double-clad fiber (DCF) in the endoscope for delivery of OCT source and fluorescence excitation light while collecting the backscattered OCT signal through the single-mode core and fluorescence emission through the large inner cladding of the DCF. Circumferential beam scanning was performed by rotating a 45° reflector using a miniature DC motor at the distal end of the endoscope. Additionally, a custom DCF coupler and a wavelength division multiplexer (WDM) were utilized to seamlessly integrate both imaging modalities to achieve an entirely fiber-optically based dual-modality imaging system. We demonstrated simultaneous intraluminal 3D OCT and 2D (surface) fluorescence imaging in ex vivo rabbit esophagus using the dual-modal endomicroscopy system. Structural morphologies (provided by OCT) and fluorophore distribution (provided by the fluorescence module) could be clearly visualized, suggesting the potential of the dual-modality system for future in vivo and clinical applications.

  1. Synthetic strategies for controlling inter- and intramolecular interactions: Applications in single-molecule fluorescence imaging, bioluminescence imaging, and palladium catalysis

    Science.gov (United States)

    Conley, Nicholas R.

    The field of synthetic organic chemistry has reached such maturity that, with sufficient effort and resources, the synthesis of virtually any small molecule which exhibits reasonable stability at room temperature can be realized. While representing a monumental achievement for the field, the ability to exert precise control over molecular structure is just a means to an end, and it is frequently the responsibility of the synthetic chemist to determine which molecules should actually be synthesized. For better or worse, there exists no competitive free market in academia for new molecules, and as a result, the decision of which compounds should be synthesized is seldom driven by the forces of supply and demand; rather, it is guided by the synthetic chemist's interest in an anticipated structure-function relationship or in the properties of a previously unstudied class of molecules. As a consequence, there exists a pervasive need for chemists with synthetic expertise in fields (e.g., molecular imaging) and subdisciplines of chemistry (e.g., physical chemistry) in which the identification of promising synthetic targets dramatically outpaces the synthetic output in that field or subdiscipline, and ample opportunities are available for synthetic chemists who choose to pursue such cross-disciplinary research. This thesis describes synthetic efforts that leverage these opportunities to realize applications in biological imaging and in palladium catalysis. In Part I, the synthesis and characterization of three novel luminophores and their imaging applications are discussed. The first is a molecular beacon that utilizes a fluorophorefluorophore pair which exhibits H-dimer quenching in the closed conformation. This probe offers several advantages over conventional fluorophore-quencher molecular beacons in the detection of oligonucleotides, both in bulk and at the single-molecule level. Secondly, a fluorescent, Cy3-Cy5 covalent heterodimer is reported, which on account of the

  2. Versatile quantitative phase imaging system applied to high-speed, low noise and multimodal imaging (Conference Presentation)

    Science.gov (United States)

    Federici, Antoine; Aknoun, Sherazade; Savatier, Julien; Wattellier, Benoit F.

    2017-02-01

    Quadriwave lateral shearing interferometry (QWLSI) is a well-established quantitative phase imaging (QPI) technique based on the analysis of interference patterns of four diffraction orders by an optical grating set in front of an array detector [1]. As a QPI modality, this is a non-invasive imaging technique which allow to measure the optical path difference (OPD) of semi-transparent samples. We present a system enabling QWLSI with high-performance sCMOS cameras [2] and apply it to perform high-speed imaging, low noise as well as multimodal imaging. This modified QWLSI system contains a versatile optomechanical device which images the optical grating near the detector plane. Such a device is coupled with any kind of camera by varying its magnification. In this paper, we study the use of a sCMOS Zyla5.5 camera from Andor along with our modified QWLSI system. We will present high-speed live cell imaging, up to 200Hz frame rate, in order to follow intracellular fast motions while measuring the quantitative phase information. The structural and density information extracted from the OPD signal is complementary to the specific and localized fluorescence signal [2]. In addition, QPI detects cells even when the fluorophore is not expressed. This is very useful to follow a protein expression with time. The 10 µm spatial pixel resolution of our modified QWLSI associated to the high sensitivity of the Zyla5.5 enabling to perform high quality fluorescence imaging, we have carried out multimodal imaging revealing fine structures cells, like actin filaments, merged with the morphological information of the phase. References [1]. P. Bon, G. Maucort, B. Wattellier, and S. Monneret, "Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells," Opt. Express, vol. 17, pp. 13080-13094, 2009. [2] P. Bon, S. Lécart, E. Fort and S. Lévêque-Fort, "Fast label-free cytoskeletal network imaging in living mammalian cells," Biophysical journal, 106

  3. Time-Resolved Spectroscopy and Near Infrared Imaging for Prostate Cancer Detection: Receptor-targeted and Native Biomarker

    Science.gov (United States)

    Pu, Yang

    Optical spectroscopy and imaging using near-infrared (NIR) light provides powerful tools for non-invasive detection of cancer in tissue. Optical techniques are capable of quantitative reconstructions maps of tissue absorption and scattering properties, thus can map in vivo the differences in the content of certain marker chromophores and/or fluorophores in normal and cancerous tissues (for example: water, tryptophan, collagen and NADH contents). Potential clinical applications of optical spectroscopy and imaging include functional tumor detection and photothermal therapeutics. Optical spectroscopy and imaging apply contrasts from intrinsic tissue chromophores such as water, collagen and NADH, and extrinsic optical contrast agents such as Indocyanine Green (ICG) to distinguish disease tissue from the normal one. Fluorescence spectroscopy and imaging also gives high sensitivity and specificity for biomedical diagnosis. Recent developments on specific-targeting fluorophores such as small receptor-targeted dye-peptide conjugate contrast agent offer high contrast between normal and cancerous tissues hence provide promising future for early tumour detection. This thesis focus on a study to distinguish the cancerous prostate tissue from the normal prostate tissues with enhancement of specific receptor-targeted prostate cancer contrast agents using optical spectroscopy and imaging techniques. The scattering and absorption coefficients, and anisotropy factor of cancerous and normal prostate tissues were investigated first as the basis for the biomedical diagnostic and optical imaging. Understanding the receptors over-expressed prostate cancer cells and molecular target mechanism of ligand, two small ICG-derivative dye-peptides, namely Cypate-Bombesin Peptide Analogue Conjugate (Cybesin) and Cypate-Octreotate Peptide Conjugate (Cytate), were applied to study their clinical potential for human prostate cancer detection. In this work, the steady-state and time

  4. Boron dipyrromethene (BODIPY) functionalized carbon nano-onions for high resolution cellular imaging

    Science.gov (United States)

    Bartelmess, Juergen; de Luca, Elisa; Signorelli, Angelo; Baldrighi, Michele; Becce, Michele; Brescia, Rosaria; Nardone, Valentina; Parisini, Emilio; Echegoyen, Luis; Pompa, Pier Paolo; Giordani, Silvia

    2014-10-01

    Carbon nano-onions (CNOs) are an exciting class of carbon nanomaterials, which have recently demonstrated a facile cell-penetration capability. In the present work, highly fluorescent boron dipyrromethene (BODIPY) dyes were covalently attached to the surface of CNOs. The introduction of this new carbon nanomaterial-based imaging platform, made of CNOs and BODIPY fluorophores, allows for the exploration of synergetic effects between the two building blocks and for the elucidation of its performance in biological applications. The high fluorescence intensity exhibited by the functionalized CNOs translates into an excellent in vitro probe for the high resolution imaging of MCF-7 human breast cancer cells. It was also found that the CNOs, internalized by the cells by endocytosis, localized in the lysosomes and did not show any cytotoxic effects. The presented results highlight CNOs as excellent platforms for biological and biomedical studies due to their low toxicity, efficient cellular uptake and low fluorescence quenching of attached probes.Carbon nano-onions (CNOs) are an exciting class of carbon nanomaterials, which have recently demonstrated a facile cell-penetration capability. In the present work, highly fluorescent boron dipyrromethene (BODIPY) dyes were covalently attached to the surface of CNOs. The introduction of this new carbon nanomaterial-based imaging platform, made of CNOs and BODIPY fluorophores, allows for the exploration of synergetic effects between the two building blocks and for the elucidation of its performance in biological applications. The high fluorescence intensity exhibited by the functionalized CNOs translates into an excellent in vitro probe for the high resolution imaging of MCF-7 human breast cancer cells. It was also found that the CNOs, internalized by the cells by endocytosis, localized in the lysosomes and did not show any cytotoxic effects. The presented results highlight CNOs as excellent platforms for biological and biomedical

  5. Urothelial Tumors and Dual-Band Imaging: A New Concept in Confocal Laser Endomicroscopy.

    Science.gov (United States)

    Marien, Arnaud; Rock, Aurélien; Maadarani, Khaled El; Francois, Catherine; Gosset, Pierre; Mauroy, Brigitte; Bonnal, Jean-Louis

    2017-05-01

    Confocal laser endomicroscopy (CLE) uses a low-energy laser light source to obtain microscopic histology images of bladder tissue exposed to a fluorescent dye. To evaluate the feasibility of using CLE with two fluorophores: fluorescein (FLUO) and hexylaminolevulinate (HAL) to determine histologic and cytologic bladder cancer criteria. Patients eligible for HAL-photodynamic diagnosis-assisted transurethral resection of bladder tumor were included. The procedures were performed with the patient under regional or general anesthesia (60-90 minutes) after bladder instillation of HAL (50 mL, 8 mmol/L; Hexvix(®); Ipsen, France). Resected tissue was examined ex vivo using CLE either with Cellvizio(®) system (CVI) single laser (488 nm) or with Cellvizio Dual system (CVII) double laser (488, 660 nm). Twenty-one patients were included, 12 examined by CVI and 9 by CVII. Sample examination on CVI after HAL-CLE-only histologic analysis was not possible because HAL is mostly cytoplasmic and gives poor details on cellular architecture. On the contrary, FLUO-CLE gives good extracellular architecture and not clear information of nucleocytoplasmic abnormality. Samples on CVII for seven out of nine patients clearly showed cytoplasm of suspect cells and nuclei. In real time, fluorescence observed on bandwidth (673-800 nm) with HAL and FLUO was associated with the presence of cancer, with a sensibility and specificity of 80% and 100%, respectively. Real-time cytodetection was feasible using two fluorophores (FLUO and HAL) and the new system of CVII. This technology was useful to observe cytoplasm, nuclei, and nucleocytoplasmic abnormality, but an improved system is necessary (to overcome the overlapping of fluorescence) to increase the specificity.

  6. Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

    Directory of Open Access Journals (Sweden)

    Zaccolo Manuela

    2008-06-01

    Full Text Available Abstract Background A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. Results The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor. Conclusion The novel analog combines good membrane permeability- comparable to 8-Br-cAMP – with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.

  7. Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging.

    Science.gov (United States)

    Omer, Travis; Intes, Xavier; Hahn, Juergen

    2015-01-01

    Fluorescence lifetime imaging (FLIM) when paired with Förster resonance energy transfer (FLIM-FRET) enables the monitoring of nanoscale interactions in living biological samples. FLIM-FRET model-based estimation methods allow the quantitative retrieval of parameters such as the quenched (interacting) and unquenched (non-interacting) fractional populations of the donor fluorophore and/or the distance of the interactions. The quantitative accuracy of such model-based approaches is dependent on multiple factors such as signal-to-noise ratio and number of temporal points acquired when sampling the fluorescence decays. For high-throughput or in vivo applications of FLIM-FRET, it is desirable to acquire a limited number of temporal points for fast acquisition times. Yet, it is critical to acquire temporal data sets with sufficient information content to allow for accurate FLIM-FRET parameter estimation. Herein, an optimal experimental design approach based upon sensitivity analysis is presented in order to identify the time points that provide the best quantitative estimates of the parameters for a determined number of temporal sampling points. More specifically, the D-optimality criterion is employed to identify, within a sparse temporal data set, the set of time points leading to optimal estimations of the quenched fractional population of the donor fluorophore. Overall, a reduced set of 10 time points (compared to a typical complete set of 90 time points) was identified to have minimal impact on parameter estimation accuracy (≈5%), with in silico and in vivo experiment validations. This reduction of the number of needed time points by almost an order of magnitude allows the use of FLIM-FRET for certain high-throughput applications which would be infeasible if the entire number of time sampling points were used.

  8. Potential applications of near infrared auto-fluorescence spectral polarized imaging for assessment of food quality

    Science.gov (United States)

    Zhou, Kenneth J.; Chen, Jun

    2016-03-01

    The current growing of food industry for low production costs and high efficiency needs for maintenance of high-quality standards and assurance of food safety while avoiding liability issues. Quality and safety of food depend on physical (texture, color, tenderness etc.), chemical (fat content, moisture, protein content, pH, etc.), and biological (total bacterial count etc.) features. There is a need for a rapid (less than a few minutes) and accurate detection system in order to optimize quality and assure safety of food. However, the fluorescence ranges for known fluorophores are limited to ultraviolet emission bands, which are not in the tissue near infrared (NIR) "optical window". Biological tissues excited by far-red or NIR light would exhibit strong emission in spectral range of 650-1,100 nm although no characteristic peaks show the emission from which known fluorophores. The characteristics of the auto-fluorescence emission of different types of tissues were found to be different between different tissue components such as fat, high quality muscle food. In this paper, NIR auto-fluorescence emission from different types of muscle food and fat was measured. The differences of fluorescence intensities of the different types of muscle food and fat emissions were observed. These can be explained by the change of the microscopic structure of physical, chemical, and biological features in meat. The difference of emission intensities of fat and lean meat tissues was applied to monitor food quality and safety using spectral polarized imaging, which can be detect deep depth fat under the muscle food up to several centimeter.

  9. Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging.

    Science.gov (United States)

    Han, Meng; Giese, Guenter; Schmitz-Valckenberg, Steffen; Bindewald-Wittich, Almut; Holz, Frank G; Yu, Jiayi; Bille, Josef F; Niemz, Markolf H

    2007-01-01

    The intensive metabolism of photoreceptors is delicately maintained by the retinal pigment epithelium (RPE) and the choroid. Dysfunction of either the RPE or choroid may lead to severe damage to the retina. Two-photon excited autofluorescence (TPEF) from endogenous fluorophores in the human retina provides a novel opportunity to reveal age-related structural abnormalities in the retina-choroid complex prior to apparent pathological manifestations of age-related retinal diseases. In the photoreceptor layer, the regularity of the macular photoreceptor mosaic is preserved during aging. In the RPE, enlarged lipofuscin granules demonstrate significantly blue-shifted autofluorescence, which coincides with the depletion of melanin pigments. Prominent fibrillar structures in elderly Bruch's membrane and choriocapillaries represent choroidal structure and permeability alterations. Requiring neither slicing nor labeling, TPEF imaging is an elegant and highly efficient tool to delineate the thick, fragile, and opaque retina-choroid complex, and may provide clues to the trigger events of age-related macular degeneration.

  10. Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

    Science.gov (United States)

    DSouza, Alisha V.; Lin, Huiyun; Henderson, Eric R.; Samkoe, Kimberley S.; Pogue, Brian W.

    2016-08-01

    There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here.

  11. Indocyanine green-encapsulating calcium phosphosilicate nanoparticles: Bifunctional theranostic vectors for near infrared diagnostic imaging and photodynamic therapy

    Science.gov (United States)

    Altinoglu, Erhan I.

    The synthesis, laundering, and properties of calcium phosphosilicate nanoparticles (CPSNPs) that encapsulate the NIR fluorophore indocyanine green (ICG) related to multifunctional fluorescent photosensitization is presented. Imaging with transmission electron microscopy (TEM) revealed the well dispersed state of the nanoparticles, the spherical morphology, and the log normal mean particle diameter of 16 nm. Electron energy loss spectroscopy (EELS) mapping identified a Ca:P:Si ratio of 1:1.72:0.41 and a homogeneous composition without evidence of an element rich or deficient architecture. Zeta potential of the as-synthesized, citrate-functionalized CPSNPs was -29 +/-3 mV. A theoretical solids loading of 1.9 x 1013 CPSNP/mL was calculated for a standard suspension. The mean ICG content per suspension is 2 x 10 -6 M, which equates to approximately 63 fluorophore molecules encapsulated per CPSNP. For imaging and diagnostic considerations, the doped CPSNPs exhibited significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore. The quantum efficiency of the fluorescent agent is 200% greater at 0.053+/-0.003 over the free fluorophore in PBS. Also, photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. These performance enhancements are attributed to the matrix shielding effect of the NP around the internalized fluorophore molecules. The in vivo emission signal stability from ICG-CPSNPs was compared to the free fluorophore by whole animal NIR imaging. The duration of fluorescent signal from the ICG-CPSPNPs was extended to up to four days post-injection, highlighting the potential for long-term imaging and sensitive tracking applications using ICG when encapsulated within the protective matrix of CPSNPs. The surfaces of the ICG-CPSNPs were covalently bound with polyethylene glycol (PEG). The pharmacokinetic behavior of the

  12. Fluorescence-Doped Particles for Simultaneous Temperature and Velocity Imaging

    Science.gov (United States)

    Danehy, Paul M.; Tiemsin, Pacita I.; Wohl, Chrostopher J.; Verkamp, Max; Lowe, T.; Maisto, P.; Byun, G.; Simpson, R.

    2012-01-01

    Polystyrene latex microspheres (PSLs) have been used for particle image velocimetry (PIV) and laser Doppler velocimetry (LDV) measurements for several decades. With advances in laser technologies, instrumentation, and data processing, the capability to collect more information about fluid flow beyond velocity is possible using new seed materials. To provide additional measurement capability, PSLs were synthesized with temperature-sensitive fluorescent dyes incorporated within the particle. These multifunctional PSLs would have the greatest impact if they could be used in large scale facilities with minimal modification to the facilities or the existing instrumentation. Consequently, several potential dyes were identified that were amenable to existing laser systems currently utilized in wind tunnels at NASA Langley Research Center as well as other wind and fluid (water) tunnels. PSLs incorporated with Rhodamine B, dichlorofluorescein (DCF, also known as fluorescein 548 or fluorescein 27) and other dyes were synthesized and characterized for morphology and spectral properties. The resulting particles were demonstrated to exhibit fluorescent emission, which would enable determination of both fluid velocity and temperature. They also would allow near-wall velocity measurements whereas laser scatter from surfaces currently prevents near-wall measurements using undoped seed materials. Preliminary results in a wind tunnel facility located at Virginia Polytechnic Institute and State University (Virginia Tech) have verified fluorescent signal detection and temperature sensitivity of fluorophore-doped PSLs.

  13. Application of hyperspectral fluorescence lifetime imaging to tissue autofluorescence: arthritis

    Science.gov (United States)

    Talbot, C. B.; Benninger, R. K. P.; de Beule, P.; Requejo-Isidro, J.; Elson, D. S.; Dunsby, C.; Munro, I.; Neil, M. A.; Sandison, A.; Sofat, N.; Nagase, H.; French, P. M. W.; Lever, M. J.

    2005-08-01

    Tissue contains many natural fluorophores and therefore by exploiting autofluorescence, we can obtain information from tissue with less interference than conventional histological techniques. However, conventional intensity imaging is prone to artifacts since it is an absolute measurement. Fluorescence lifetime and spectral measurements are relative measurements and therefore allow for better measurements. We have applied FLIM and hyperspectral FLIM to the study of articular cartilage and its disease arthritis. We have analyzed normal human articular cartilage and cartilage which was in the early stages of disease. In this case, it was found that FLIM was able to detect changes in the diseased tissue that were not detectable with the conventional diagnosis. Specifically, the fluorescence lifetimes (FL) of the cells were different between the two samples. We have also applied hyperspectral FLIM to degraded cartilage through treatment with interleukin-1. In this case, it was found that there was a shift in the emission spectrum with treatment and that the lifetime had also increased. We also showed that there was greater contrast between the cells and the extracellular matrix (ECM) at longer wavelengths.

  14. Cryo-imaging of fluorescently labeled single cells in a mouse

    Science.gov (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    -scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

  15. Imaging Scatterometry

    CERN Document Server

    Madsen, Morten Hannibal

    2015-01-01

    We present an optical metrology system for characterization of topography of micro/nano-structures on a surface or embedded in a semi-transparent material. Based on the principles of scatterometry, where the intensity of scattered light is used as a 'fingerprint' to reconstruct a surface, this new imaging scatterometer can easily find areas of interest on the cm scale and measure multiple segments simultaneously. The imaging scatterometer measures structural features, such as height, width, and sidewall angle of a grating locally on few um2 areas with nm resolution. We demonstrate two imaging scatterometers, one built into an optical microscope and one in a split configuration. The two scatterometers are targeted characterization of mm2 and cm2 areas, respectively, and both setups are validated using nano-textured samples.

  16. Image Analysis

    DEFF Research Database (Denmark)

    The 19th Scandinavian Conference on Image Analysis was held at the IT University of Copenhagen in Denmark during June 15-17, 2015. The SCIA conference series has been an ongoing biannual event for more than 30 years and over the years it has nurtured a world-class regional research and development....... The topics of the accepted papers range from novel applications of vision systems, pattern recognition, machine learning, feature extraction, segmentation, 3D vision, to medical and biomedical image analysis. The papers originate from all the Scandinavian countries and several other European countries...

  17. Imaging sciences workshop

    Energy Technology Data Exchange (ETDEWEB)

    Candy, J.V.

    1994-11-15

    This workshop on the Imaging Sciences sponsored by Lawrence Livermore National Laboratory contains short abstracts/articles submitted by speakers. The topic areas covered include the following: Astronomical Imaging; biomedical imaging; vision/image display; imaging hardware; imaging software; Acoustic/oceanic imaging; microwave/acoustic imaging; computed tomography; physical imaging; imaging algorithms. Selected papers are indexed separately for inclusion in the Energy Science and Technology Database.

  18. Development of optical phantoms for use in fluorescence-based imaging

    Science.gov (United States)

    Noiseux, Isabelle; Fortin, Michel; Leclair, Sébastien; Osouf, Jocelyne; Mermut, Ozzy

    2010-02-01

    We fabricated permanent solid polyurethane-based phantoms in which fluorophores were homogeneously incorporated. For this study, fluorophores of three different families were used: Cyanines, Alexa Fluor and Quantum Dots. The goal of this study was to evaluate the impact of casting the fluorophores in a polyurethane matrix on their optical properties, more specifically the absorbance, molecular extinction coefficient, emission of fluorescence and the resultant fluorescence intensity. All measurements were carried out with 5 concentrations of each fluorophores embedded in polyurethane and in solution. Stability over time was also monitored for a three months period. The casting of fluorophores affects the optical properties of the three dyes under study. The max absorbance, the fluorescence emission and intensity along with the molar extinction coefficient were all affected. Quantum dots behave differently to the cyanine and Alexa Fluor dyes. It was also observed that the incorporation of dyes enables long-term stability of the fluorescence signal.

  19. Open Images

    DEFF Research Database (Denmark)

    Sanderhoff, Merete

    2013-01-01

    , museums have a unique opportunity to broaden the scope of their public mission to serve and educate the public on 21st century media terms. What could be controversial about that? Art museums have a long legacy of restricting access to high quality images of artworks in order to protect them from improper...

  20. Forest Imaging

    Science.gov (United States)

    1992-01-01

    NASA's Technology Applications Center, with other government and academic agencies, provided technology for improved resources management to the Cibola National Forest. Landsat satellite images enabled vegetation over a large area to be classified for purposes of timber analysis, wildlife habitat, range measurement and development of general vegetation maps.

  1. Geriatric imaging

    Energy Technology Data Exchange (ETDEWEB)

    Guglielmi, Giuseppe [Scientific Institute Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo (Italy). Dept. of Radiology; Peh, Wilfred C.G. [Khoo Teck Puat Hospital, Singapore (Singapore). Dept. of Diagnostic Radiology; Guermazi, Ali (eds.) [Boston Univ. School of Medicine, Boston, MA (United States). Dept. of Radiology

    2013-08-01

    Considers all aspect of geriatric imaging. Explains clearly how to distinguish the healthy elderly from those in need of treatment. Superbly illustrated. Written by recognized experts in field. In the elderly, the coexistence of various diseases, the presence of involutional and degenerative changes, and the occurrence of both physical and cognitive problems represent ''the norm.'' It is therefore important to know how to distinguish the healthy elderly from those in need of treatment as a sound basis for avoiding overdiagnosis and overtreatment. This aspect is a central theme in Geriatric Imaging, which covers a wide range of applications of different imaging techniques and clearly explains both the potential and the limitations of diagnostic imaging in geriatric patients. Individual sections are devoted to each major region or system of the body, and a concluding section focuses specifically on interventional procedures. The book, written by recognized experts in the field, is superbly illustrated and will be an ideal resource for geriatricians, radiologists, and trainees.

  2. Neuroperformance Imaging

    Science.gov (United States)

    2014-10-01

    SUPPLEMENTARY NOTES 14. ABSTRACT We have designed an appropriate PET imaging protocol that will allow us to differentiate regional metabolic activity...host of neurological problems and limiting their ability to perform the challenging mental tasks that their missions require. Further, the effects... computes such as magneto- and electrophysiology. Should additional funded become available, we will explore the effects of sleep quality on

  3. Doppler imaging

    Energy Technology Data Exchange (ETDEWEB)

    Piskunov, N [Department of Physics and Astronomy, Uppsala University, Box 515, S-75120 Uppsala (Sweden)], E-mail: piskunov@fysast.uu.se

    2008-12-15

    In this paper, I present a short review of the history and modern status of Doppler imaging techniques, highlighting their dependence on the knowledge of the fundamental stellar parameters, the quality of stellar atmospheric models and the accuracy of spectral synthesis.

  4. Irving Langmuir Prize Talk: Single-Molecule Fluorescence Imaging: Nanoscale Emitters with Photoinduced Switching Enable Superresolution.

    Science.gov (United States)

    Moerner, W. E.

    2009-03-01

    In the two decades since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. 62, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. The early years concentrated on high-resolution spectroscopy in solids, which provided observations of lifetime-limited spectra, optical saturation, spectral diffusion, optical switching, vibrational spectra, and magnetic resonance of a single molecular spin. In the mid-1990's, much of the field moved to room temperature, where a wide variety of biophysical effects were subsequently explored, but it is worth noting that several features from the low-temperature studies have analogs at high temperature. For example, in our first studies of yellow-emitting variants of green fluorescent protein (EYFP) in the water-filled pores of a gel (Nature 388, 355 (1997)), optically induced switching of the emission was observed, a room-temperature analog of the earlier low-temperature behavior. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. Recent work has allowed measurement of the shape of single filaments in a living cell simply by allowing a single molecule to move through the filament (PNAS 103, 10929 (2006)). The additional use of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (superresolution) by several novel approaches proposed by different researchers. For example, using photoswitchable EYFP, a novel protein superstructure can now be directly imaged in a living bacterial cell at

  5. Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope

    Science.gov (United States)

    Meyer, Stephanie A.; Ozbay, Baris N.; Potcoava, Mariana; Salcedo, Ernesto; Restrepo, Diego; Gibson, Emily A.

    2016-06-01

    We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ˜81 and ˜44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

  6. Fluorescent metal nanoshell and CK19 detection on single cell image

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jian, E-mail: jian@cfs.biomet.umaryland.edu [Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201 (United States); Fu, Yi [Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201 (United States); Li, Ge [Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States); Lakowicz, Joseph R. [Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201 (United States); Zhao, Richard Y., E-mail: rzhao@som.umaryland.edu [Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States); Department of Microbiology-Immunology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States); Institute of Human Virology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States)

    2011-09-16

    Highlights: {yields} Novel metal nanoshell as fluorescence imaging agent. {yields} Fluorescent mAb-metal complex with enhanced intensity and shortened lifetime. {yields} Immuno-interactions of mAb-metal complexes with CK19 molecules on CNCAP and HeLa cell surfaces. {yields} Isolation of conjugated mAb-metal complexes from cellular autofluorescence on cell image. -- Abstract: In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.

  7. A comparison between a time domain and continuous wave small animal optical imaging system.

    Science.gov (United States)

    Keren, S; Gheysens, O; Levin, C S; Gambhir, S S

    2008-01-01

    We present a phantom study to evaluate the performance of the eXplore Optix (Advanced Research Technologies-GE Healthcare), the first commercially available time-domain tomography system for small animal fluorescence imaging, and compare its capabilities with the widely used IVIS 200 (Xenogen Corporation-Caliper) continuous wave planar imaging system. The eXplore Optix, based on point-wise illumination and collection scheme, is found to be a log order more sensitive with significantly higher detection depth and spatial resolution as compared with the wide-area illumination IVIS 200 under the conditions tested. A time-resolved detection system allows the eXplore Optix to measure the arrival time distribution of fluorescence photons. This enables fluorescence lifetime measurement, absorption mapping, and estimation of fluorescent inclusion depth, which in turn is used by a reconstruction algorithm to calculate the volumetric distribution of the fluorophore concentration. An increased acquisition time and lack of ability to image multiple animals simultaneously are the main drawbacks of the eXplore Optix as compared with the IVIS 200.

  8. Advances in two photon scanning and scanless microscopy technologies for functional neural circuit imaging.

    Science.gov (United States)

    Schultz, Simon R; Copeland, Caroline S; Foust, Amanda J; Quicke, Peter; Schuck, Renaud

    2017-01-01

    Recent years have seen substantial developments in technology for imaging neural circuits, raising the prospect of large scale imaging studies of neural populations involved in information processing, with the potential to lead to step changes in our understanding of brain function and dysfunction. In this article we will review some key recent advances: improved fluorophores for single cell resolution functional neuroimaging using a two photon microscope; improved approaches to the problem of scanning active circuits; and the prospect of scanless microscopes which overcome some of the bandwidth limitations of current imaging techniques. These advances in technology for experimental neuroscience have in themselves led to technical challenges, such as the need for the development of novel signal processing and data analysis tools in order to make the most of the new experimental tools. We review recent work in some active topics, such as region of interest segmentation algorithms capable of demixing overlapping signals, and new highly accurate algorithms for calcium transient detection. These advances motivate the development of new data analysis tools capable of dealing with spatial or spatiotemporal patterns of neural activity, that scale well with pattern size.

  9. Intraoperative targeted optical imaging: a guide towards tumor-free margins in cancer surgery.

    Science.gov (United States)

    Orbay, Hakan; Bean, Jero; Zhang, Yin; Cai, Weibo

    2013-01-01

    Over the last several decades, development of various imaging techniques such as computed tomography, magnetic resonance imaging, and positron emission tomography greatly facilitated the early detection of cancer. Another important aspect that is closely related to the survival of cancer patients is complete tumor removal during surgical resection. The major obstacle in achieving this goal is to distinguish between tumor tissue and normal tissue during surgery. Currently, tumor margins are typically assessed by visual assessment and palpation of the tumor intraoperatively. However, the possibility of microinvasion to the surrounding tissues makes it difficult to determine an adequate tumor-free excision margin, often forcing the surgeons to perform wide excisions including the healthy tissue that may contain vital structures. It would be ideal to remove the tumor completely, with minimal safety margins, if surgeons could see precise tumor margins during the operation. Molecular imaging with optical techniques can visualize the tumors via fluorophore conjugated probes targeting tumor markers such as proteins and enzymes that are upregulated during malignant transformation. Intraoperative use of this technique may facilitate complete excision of the tumor and tumor micromasses located beyond the visual capacity of the naked eye, ultimately improving the clinical outcome and survival rates of cancer patients.

  10. Multiphoton fluorescence imaging of NADH to quantify metabolic changes in epileptic tissue in vitro

    Science.gov (United States)

    Chia, Thomas H.; Zinter, Joseph; Spencer, Dennis D.; Williamson, Anne; Levene, Michael J.

    2007-02-01

    A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD +; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

  11. Siloxane Nanoprobes for Labeling and Dual Modality Functional Imaging of Neural Stem Cells.

    Science.gov (United States)

    Addington, Caroline P; Cusick, Alex; Shankar, Rohini Vidya; Agarwal, Shubhangi; Stabenfeldt, Sarah E; Kodibagkar, Vikram D

    2016-03-01

    Cell therapy represents a promising therapeutic for a myriad of medical conditions, including cancer, traumatic brain injury, and cardiovascular disease among others. A thorough understanding of the efficacy and cellular dynamics of these therapies necessitates the ability to non-invasively track cells in vivo. Magnetic resonance imaging (MRI) provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. We recently reported a new nanoprobe platform for cell labeling and imaging using fluorophore doped siloxane core nanoemulsions as dual modality ((1)H MRI/Fluorescence), dual-functional (oximetry/detection) nanoprobes. Here, we successfully demonstrate the labeling, dual-modality imaging, and oximetry of neural progenitor/stem cells (NPSCs) in vitro using this platform. Labeling at a concentration of 10 μL/10(4) cells with a 40%v/v polydimethylsiloxane core nanoemulsion, doped with rhodamine, had minimal effect on viability, no effect on migration, proliferation and differentiation of NPSCs and allowed for unambiguous visualization of labeled NPSCs by (1)H MR and fluorescence and local pO2 reporting by labeled NPSCs. This new approach for cell labeling with a positive contrast (1)H MR probe has the potential to improve mechanistic knowledge of current therapies, and guide the design of future cell therapies due to its clinical translatability.

  12. Super-aperture metrology: overcoming a fundamental limit in imaging smooth highly curved surfaces.

    Science.gov (United States)

    Liu, J; Liu, C; Tan, J; Yang, B; Wilson, T

    2016-03-01

    The imaging of smooth, highly curved or tilted surfaces is widely recognized as one of the most challenging and unsolved problems in optical imaging and metrology today. The reason is that even when such surfaces are imaged using high aperture microscope objectives the steepness of the features causes the light to be reflected in such a way that it is not captured by the lens. This is true even in the limiting case of unity numerical aperture since the illuminating light may also be reflected in the forward direction. In order to overcome this fundamental problem we have developed a method whereby such specimens are covered with a readily removable organic fluorescent film thereby creating an isotropic scattering surface. We show that we are readily able to detect slopes with angles close 90° using a 0.75 NA objective--an 82% improvement over the theoretical aperture limit. Issues of variation in film thickness deposition are shown to be readily accommodated. This approach may be used with other fluorophore materials, organic or inorganic, since there is no need for biocompatibility in this application.

  13. Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

    Science.gov (United States)

    Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

    2017-07-01

    The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

  14. Two-photon microscopy for imaging germinal centers and T follicular helper cells.

    Science.gov (United States)

    Clatworthy, Menna R

    2015-01-01

    One of the principle features of immune cells is their dynamic nature. Lymphocytes circulate in the blood between secondary lymphoid organs and tissues in an effort to maximize the likelihood of a rapid and appropriate immune response to invading pathogens and tissue damage. Conventional experimental techniques such as histology and flow cytometry have greatly increased our understanding of immune cells, but in the last decade, two-photon microscopy has revolutionized our ability to interrogate the dynamic behavior of immune cells, a facet so critical to their function. Two-photon microscopy relies on the excitation of fluorophores by simultaneous application of two photons of longer wavelength light. This allows a greater depth of imaging with minimal photodamage. Thus, living tissues can be imaged, including immune cells in lymph nodes. This technique has been used to interrogate the events occurring in a germinal center response and the interactions between cells in the germinal center, including T follicular helper cells (Tfh), germinal center B cells, and follicular dendritic cells (FDC). Herein, a method is described by which the interactions between Tfh and B cells within a germinal center in a popliteal lymph node can be imaged in a live mouse.

  15. Fiber based in-vivo imaging of epithelial FAD fluorescence: experiments and simulations

    Science.gov (United States)

    Kanakaraj, Bala Nivetha; Narayanan Unni, Sujatha

    2015-03-01

    Fluorescence from endogenous fluorophores has been emerging as a promising biomarker for tissue discrimination resulting a noninvasive screening methodology to understand the biochemical and morphological variations in tissues associated with cancer development. We have developed a scan based fiber optic probe system to image increased flavin adenine dinucleotide (FAD) fluorescence from epithelial tissues under conditions mimicking dysplasia surrounded by normal tissues. Experiments were conducted on optical phantoms mimicking epithelial tissues excited by 450nm LED source. The spectral emission from the sample is collected via optical fibers and the imaging is performed by scanning the sample using a translation stage at desired resolution. Monte Carlo simulations were also performed by devising an optical model corresponding to epithelial tissue and the results were correlated with experimental fluorescence measurements. This whole field imaging approach could be useful for in vivo assessment of tissue pathologies based on auto fluorescence and can give a better quantitative approach for estimation of tissue properties by correlating the experimental and simulated data.

  16. Imaging dementias

    Energy Technology Data Exchange (ETDEWEB)

    Savoiardo, M.; Grisoli, M. [Dept. of Neuroradiology, Istituto Nazionale Neurologico, Milan (Italy)

    2001-03-01

    Dementia is the progressive loss of intellectual functions due to involvement of cortical or subcortical areas. Specific involvement of certain brain areas in the different diseases leads to impairment of different functions, e. g., memory, language, visuospatial abilities, and behavior. Magnetic resonance imaging and other neuroradiological studies may indicate which structures are mainly or selectively involved in a demented patient, thus allowing clinical-radiological correlations. Clinical presentation and evolution of the disease, supported by imaging studies, may lead to a highly probable diagnosis. The most common disorders, or the most relevant from the neuroradiological point of view, such as Alzheimer's disease, frontotemporal dementia, vascular dementias, dementia associated with parkinsonism, Huntington's disease, Creutzfeldt-Jakob disease, and normal-pressure hydrocephalus, are briefly discussed. (orig.)

  17. IMAGE IMPROVEMENT

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    After five years of development, the Shanghai Cooperation Organization says it has become a player in maintaining regional security and promoting economic cooperation. On June 15, leaders of SCO member countries will gather in Shanghai, birthplace of the SCO, to discuss the future development of the organization. SCO Secretary General Zhang Deguang fielded questions from reporters on the organization's development and international image at a press conference on June 6 in Beijing. The following are excer...

  18. [Molecular imaging].

    Science.gov (United States)

    Turetschek, K; Wunderbaldinger, P

    2002-01-01

    The disclosure of the human genoma, the progress in understanding of diseases on molecular and cellular levels, the discovery of new disease-specific targets, and the development of new medications will revolutionize our understanding of the etiology and the treatment of many disease entities. Radiologists are faced with a paradigm shift from unspecific to specific molecular imaging techniques as well as with enormous speed in the development of new methods and should be enrolled actively in this field of medicine.

  19. Imaging stress.

    Science.gov (United States)

    Brielle, Shlomi; Gura, Rotem; Kaganovich, Daniel

    2015-11-01

    Recent innovations in cell biology and imaging approaches are changing the way we study cellular stress, protein misfolding, and aggregation. Studies have begun to show that stress responses are even more variegated and dynamic than previously thought, encompassing nano-scale reorganization of cytosolic machinery that occurs almost instantaneously, much faster than transcriptional responses. Moreover, protein and mRNA quality control is often organized into highly dynamic macromolecular assemblies, or dynamic droplets, which could easily be mistaken for dysfunctional "aggregates," but which are, in fact, regulated functional compartments. The nano-scale architecture of stress-response ranges from diffraction-limited structures like stress granules, P-bodies, and stress foci to slightly larger quality control inclusions like juxta nuclear quality control compartment (JUNQ) and insoluble protein deposit compartment (IPOD), as well as others. Examining the biochemical and physical properties of these dynamic structures necessitates live cell imaging at high spatial and temporal resolution, and techniques to make quantitative measurements with respect to movement, localization, and mobility. Hence, it is important to note some of the most recent observations, while casting an eye towards new imaging approaches that offer the possibility of collecting entirely new kinds of data from living cells.

  20. Telepathology and imaging spectroscopy as a new modality in histopathology.

    Science.gov (United States)

    Vari, S G; Müller, G; Lerner, J M; Naber, R D

    1999-01-01

    Telemedicine started in the late 1950's by transmitting data on patients' pulse and heart rates. In the 1980's it expanded to radiology and orthopedics. The technology is now expanding to other specialties that can digitally gather patient data. Telepathology comprises the transmission of microscopic images via telecommunication network. Image compression and multiplexing technologies enabled high-resolution telepathology as well as real-time video consultations over international telephone lines. Organ transplantation has become a viable treatment and offers new life to an increasing number of patients suffering from chronic end stage diseases and from irreversible organ failure. Rejection is still a major problem in kidney, liver, and heart transplantation. To gain further insight into the complex interactions within the components of the immune system, it has become increasingly necessary to develop rapid and simple methods to monitor the status of the immune system in patients. Clinical signs suggest organ rejection and abnormal laboratory test results, but only histological signs on biopsy specimens are adequately specific. The financial cost of organ transplant makes it imperative to develop tools for the early identification and treatment of organ rejection. An increasingly sensitive and accurate way of localizing key structures and abnormalities is through spectroscopy of either H&E stained samples or with a fluorescent tag (fluorophore) or by relying on natural fluorescence. The system is based on a unique Prism and Mirror Imaging Spectroscopy System ("PARISS), spectrometer originally designed and implemented for remote Earth monitoring from space and aircraft and astronomical imaging spectroscopy. Compact and lightweight both the mirror and prism are presently constructed in inexpensive glass but can also be injection molded in plastic. Any number of vendors anywhere in the world can produce all parts of the assembly. This greatly enhances the chances of

  1. Image processing of 2D crystal images.

    Science.gov (United States)

    Arheit, Marcel; Castaño-Díez, Daniel; Thierry, Raphaël; Gipson, Bryant R; Zeng, Xiangyan; Stahlberg, Henning

    2013-01-01

    Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to image frozen-hydrated 2D crystals. The processing of recorded images exploits the periodic arrangement of the structures in the images to extract the amplitudes and phases of diffraction spots in Fourier space. However, image imperfections require a crystal unbending procedure to be applied to the image before evaluation in Fourier space. We here describe the process of 2D crystal image unbending, using the 2dx software system.

  2. Analyzing blinking effects in super resolution localization microscopy with single-photon SPAD imagers

    Science.gov (United States)

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron; Charbon, Edoardo

    2016-02-01

    For many scientific applications, electron multiplying charge coupled devices (EMCCDs) have been the sensor of choice because of their high quantum efficiency and built-in electron amplification. Lately, many researchers introduced scientific complementary metal-oxide semiconductor (sCMOS) imagers in their instrumentation, so as to take advantage of faster readout and the absence of excess noise. Alternatively, single-photon avalanche diode (SPAD) imagers can provide even faster frame rates and zero readout noise. SwissSPAD is a 1-bit 512×128 SPAD imager, one of the largest of its kind, featuring a frame duration of 6.4 μs. Additionally, a gating mechanism enables photosensitive windows as short as 5 ns with a skew better than 150 ps across the entire array. The SwissSPAD photon detection efficiency (PDE) uniformity is very high, thanks on one side to a photon-to-digital conversion and on the other to a reduced fraction of "hot pixels" or "screamers", which would pollute the image with noise. A low native fill factor was recovered to a large extent using a microlens array, leading to a maximum PDE increase of 12×. This enabled us to detect single fluorophores, as required by ground state depletion followed by individual molecule return imaging microscopy (GSDIM). We show the first super resolution results obtained with a SPAD imager, with an estimated localization uncertainty of 30 nm and resolution of 100 nm. The high time resolution of 6.4 μs can be utilized to explore the dye's photophysics or for dye optimization. We also present the methodology for the blinking analysis on experimental data.

  3. Real-time imaging of renin release in vitro.

    Science.gov (United States)

    Peti-Peterdi, János; Fintha, Attila; Fuson, Amanda L; Tousson, Albert; Chow, Robert H

    2004-08-01

    Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin, we isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold increase in the number of both individual granules and renin-positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 microM isoproterenol. There appeared to be a quantal release of the granular contents; i.e., an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further classic signs of granule exocytosis, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS)-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool for the further study of the mechanism, regulation, and dynamics of renin release.

  4. Breast imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kopans, D.B.; Meyer, J.E.; Sadowsky, N.

    1984-04-12

    The majority of information available today indiates that the most efficient and accurate method of screening women to detect early-stage breast cancer is an aggressive program of patient self-examination, physical examination by well-trained, motivated personnel, and high-quality x-ray mammography. There are two important factors in the implementation of mammographic screening. The first is the availability of facilities to perform high-quality, low-dose mammography, which is directly related to the second factor: the expense to society for support of this large-scale effort. Cost-benefit analysis is beyond the scope of this review. In 1979 Moskowitz and Fox attempted to address this issue, using data from the Breast Cancer Detection Demonstration Project in Cincinnati, but additional analysis is required. The cost for each ''curable'' cancer that is detected must be compared with the psychological, social, and personal losses that accrue, as well as the numerous medical expenses incurred, in a frequently protracted death from breast cancer. All other imaging techniques that have been reviewed should be regarded as adjuncts to rather than replacements for mammographic screening. Ultrasound and computerized tomography are helpful when the physical examination and mammogram are equivocal. Other techniques, such as transillumination, thermography, and magnetic-resonance imaging, should be considered experimental. In patients with clinically evident lesions, x-ray mammography is helpful to evaluate the suspicious area, as well as to ''screen'' the remaining tissue in both breasts and to search for multicentric or bilateral lesions. Mammography is the only imaging technique that has been proved effective for screening.

  5. Special issue on high-resolution optical imaging

    Science.gov (United States)

    Smith, Peter J. S.; Davis, Ilan; Galbraith, Catherine G.; Stemmer, Andreas

    2013-09-01

    , such as the combination of light sheet and structured illumination microscopy (SIM), which efficiently utilize photon budget and avoid illuminating regions of the specimen not currently being imaged, hold the greatest promise for future biological applications. Therefore, the combined setup for SIM and single molecule localization microscopy (SMLM) described by Rossberger et al [3] will be very helpful and stimulating to advanced microscopists in further modifying their setups. The SIM image helps in identifying artefacts in SMLM reconstruction, e.g. when two active fluorophores are close together and get rejected as 'out-of-focus'. This combined setup is another way to facilitate imaging live samples. The article by Thomas et al [4] presents another advance for biological super-resolution imaging with a new approach to reconstruct optically sectioned images using structured illumination. The method produces images with higher spatial resolution and greater signal to noise compared to existing approaches. This algorithm demonstrates great promise for reconstructing biological images where the signal intensities are inherently lower. Shevchuk et al [5] present a non-optic near field approach to imaging with a review of scanning ion-conductance microscopy. This is a powerful alternative approach for examining the surface dynamics of living cells including exo and endocytosis, unlabelled, and at the level of the single event. Here they present the first data on combining this approach with fluorescence confocal microscopy—adding that extra dimension. Different approaches to label-free live cell imaging are presented in the papers by Patel et al [6], Mehta and Oldenbourg [7], as well as Rogers and Zheludev [8]. All three papers bring home the excitement of looking at live cell dynamics without reporters—Patel et al [6] review both the potential of coherent anti-Stokes Raman scattering and biological applications, where specific biomolecules are detected on the basis of

  6. Electronic Imaging

    Science.gov (United States)

    1997-10-01

    08/154311 (Filed 1993). [6] M.I. Sezan and A.M. Tekalp, IEEE Trans. Acoust, Speech, Signal Proc. 38 (1990) 181. [7] G. Arfken , Mathematical methods ...has been to make contributions in imaS^JS^SV!^!^ SS22? ? ^ Tl:mS C°mbining °ptical ima^ Pn^ensors, and digtellmputers AAtSotUSTT mathematical ...or speckle-like psf. This novel method for controlled blurring appears to be useful in secure transmission and in image compression; and this psf

  7. Open Images

    DEFF Research Database (Denmark)

    Sanderhoff, Merete

    2013-01-01

    Museums around the world hold enormous troves of public domain artworks. In digitized form, they can be powerful tools for research and learning, as well as building blocks, in the hands of students, teachers, scholars, developers, and creative people. By opening up their digitized assets for reuse......, museums have a unique opportunity to broaden the scope of their public mission to serve and educate the public on 21st century media terms. What could be controversial about that? Art museums have a long legacy of restricting access to high quality images of artworks in order to protect them from improper...

  8. MRI (Magnetic Resonance Imaging)

    Science.gov (United States)

    ... and Procedures Medical Imaging MRI (Magnetic Resonance Imaging) MRI (Magnetic Resonance Imaging) Share Tweet Linkedin Pin it More sharing options ... usually given through an IV in the arm. MRI Research Programs at FDA Magnetic Resonance Imaging (MRI) ...

  9. Molecular photoacoustic imaging

    OpenAIRE

    Frogh Jafarian Dehkordi; Ali Mahmoud Pashazadeh; Majid Assadi

    2015-01-01

    Background: Hybrid imaging modalities which simultaneously benefit from capabilities of combined modalities provides an opportunity to modify quality of the images which can be obtained by each of the combined imaging systems. One of the imaging modalities, emerged in medical research area as a hybrid of ultrasound imaging and optical imaging, is photoacoustic imaging which apply ultrasound wave generated by tissue, after receiving laser pulse, to produce medical images. Materials and Methods...

  10. Sensitive near-infrared fluorescent probes for thiols based on Se-N bond cleavage: imaging in living cells and tissues.

    Science.gov (United States)

    Wang, Rui; Chen, Lingxin; Liu, Ping; Zhang, Qin; Wang, Yunqing

    2012-09-03

    Cy-NiSe and Cy-TfSe were designed and synthesized as sensitive near-infrared (NIR) fluorescent probes for detecting thiols on the basis of Se-N bond cleavage both in cells and in tissues. Since a donor-excited photoinduced electron transfer (d-PET) process occurs between the modulator and the fluorophore, Cy-NiSe and Cy-TfSe have weak fluorescence. On titration with glutathione, the free dye exhibits significant fluorescence enhancement. The two probes are sensitive and selective for thiols over other relevant biological species. They can function rapidly at pH 7.4, and their emission lies in the NIR region. Confocal imaging confirms that Cy-NiSe and Cy-TfSe can be used for detecting thiols in living cells and tissues. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

    Directory of Open Access Journals (Sweden)

    Beth L. Haas

    2014-08-01

    Full Text Available Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.

  12. Indexing Images: Testing an Image Description Template.

    Science.gov (United States)

    Jorgensen, Corinne

    1996-01-01

    A template for pictorial image description to be used by novice image searchers in recording their descriptions of images was tested; image attribute classes derived in previous research were used to model the template. Results indicated that users may need training and/or more guidance to correctly assign descriptors to higher-level classes.…

  13. Image processing with ImageJ

    NARCIS (Netherlands)

    Abramoff, M.D.; Magalhães, Paulo J.; Ram, Sunanda J.

    2004-01-01

    Wayne Rasband of NIH has created ImageJ, an open source Java-written program that is now at version 1.31 and is used for many imaging applications, including those that that span the gamut from skin analysis to neuroscience. ImageJ is in the public domain and runs on any operating system (OS). Image

  14. Speckle imaging algorithms for planetary imaging

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, E. [Lawrence Livermore National Lab., CA (United States)

    1994-11-15

    I will discuss the speckle imaging algorithms used to process images of the impact sites of the collision of comet Shoemaker-Levy 9 with Jupiter. The algorithms use a phase retrieval process based on the average bispectrum of the speckle image data. High resolution images are produced by estimating the Fourier magnitude and Fourier phase of the image separately, then combining them and inverse transforming to achieve the final result. I will show raw speckle image data and high-resolution image reconstructions from our recent experiment at Lick Observatory.

  15. Hybrid ultrasound imaging techniques (fusion imaging).

    Science.gov (United States)

    Sandulescu, Daniela Larisa; Dumitrescu, Daniela; Rogoveanu, Ion; Saftoiu, Adrian

    2011-01-07

    Visualization of tumor angiogenesis can facilitate non-invasive evaluation of tumor vascular characteristics to supplement the conventional diagnostic imaging goals of depicting tumor location, size, and morphology. Hybrid imaging techniques combine anatomic [ultrasound, computed tomography (CT), and/or magnetic resonance imaging (MRI)] and molecular (single photon emission CT and positron emission tomography) imaging modalities. One example is real-time virtual sonography, which combines ultrasound (grayscale, colour Doppler, or dynamic contrast harmonic imaging) with contrast-enhanced CT/MRI. The benefits of fusion imaging include an increased diagnostic confidence, direct comparison of the lesions using different imaging modalities, more precise monitoring of interventional procedures, and reduced radiation exposure.

  16. Detection of Leukotriene Receptor CysLT1R in Inflammatory Diseases by Molecular Imaging with Near-Infrared Fluorescence-Based Contrast Agents

    Directory of Open Access Journals (Sweden)

    Corinna Busch

    2011-03-01

    Full Text Available As leukotriene D4 receptor CysLT1R upregulation is an early event in inflammatory processes, specific detection of CysLT1R via molecular imaging might be a promising diagnostic tool for inflammatory diseases. We coupled a specific anti-CysLT1R IgG antibody to near-infrared (NIR hemicyanine fluorophore DY-734. The fluorophore was also coupled to unspecific rabbit-IgG antibody or corresponding Fab fragments. Expression of CysLT1R in HL-60 human promyelocytic leukemia cells in vitro could be proven by reverse transcriptase—polymerase chain reaction (PCR, real-time PCR, and flow cytometry. Detection of the probes by flow cytometry showed that CysLT1R*DY-734 probe binds distinctly stronger to HL-60 cells than IgG*DY-734. Induction of ear edema in mice was conducted to test signaling of the synthesized probes in vivo. A markedly higher fluorescence intensity was observed in the edematous region than in the healthy region by a whole-body imaging system. Semiquantitative analysis showed that CysLT1R*DY-734 and Fab-CysLT1R*DY-734 probes bind 1.9- and 1.2-fold stronger, respectively, than the unspecific probes. Biodistribution studies revealed an enrichment of full-length IgG probes in liver and spleen, whereas Fab-containing probes are mostly found in liver and kidneys. Taken together, we present an approach that might improve early diagnosis of inflammatory diseases in the long term.

  17. Intraoperative fluorescence imaging for personalized brain tumor resection: Current state and future directions

    Directory of Open Access Journals (Sweden)

    Evgenii Belykh

    2016-10-01

    Full Text Available Introduction: Fluorescence-guided surgery is one of the rapidly emerging methods of surgical theranostics. In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients, as well as future applications of recent laboratory and translational studies.Methods: Review of the literature.Results: A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-ALA and ICG, less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine can be used for rapid tumor detection and pathological tissue examination. Other emerging agents such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed.Conclusion: We are standing on the doorstep of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.

  18. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.

    Directory of Open Access Journals (Sweden)

    Martin K Schwarz

    Full Text Available In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.

  19. Imaging the Population Dynamics of Bacterial Communities in the Zebrafish Gut

    Science.gov (United States)

    Jemielita, Matthew; Taormina, Michael; Burns, Adam; Zac Stephens, W.; Hampton, Jennifer; Guillemin, Karen; Parthasarathy, Raghuveer

    2013-03-01

    The vertebrate gut is home to a diverse microbial ecosystem whose composition has a strong influence on the development and health of the host organism. While researchers are increasingly able to identify the constituent members of the microbiome, very little is known about the spatial and temporal dynamics of commensal microbial communities, including the mechanisms by which communities nucleate, grow, and interact. We address these issues using a model organism: the larval zebrafish (Danio rerio) prepared microbe-free and inoculated with controlled compositions of fluorophore-expressing bacteria. Live imaging with light sheet fluorescence microscopy enables visualization of individual bacterial cells as well as growing colonies over the entire volume of the gut over periods up to 24 hours. We analyze the structure and dynamics of imaged bacterial communities, uncovering correlations between population size, growth rates, and the timing of inoculations that suggest the existence of active changes in the host environment induced by early bacterial exposure. Our data provide the first visualizations of gut microbiota development over an extended period of time in a vertebrate.

  20. Wide-field multispectral super-resolution imaging using spin-dependent fluorescence in nanodiamonds.

    Science.gov (United States)

    Chen, Edward H; Gaathon, Ophir; Trusheim, Matthew E; Englund, Dirk

    2013-05-08

    Recent advances in fluorescence microscopy have enabled spatial resolution below the diffraction limit by localizing multiple temporally or spectrally distinguishable fluorophores. Here, we introduce a super-resolution technique that deterministically controls the brightness of uniquely addressable, photostable emitters. We modulate the fluorescence brightness of negatively charged nitrogen-vacancy (NV(-)) centers in nanodiamonds through magnetic resonance techniques. Using a CCD camera, this "deterministic emitter switch microscopy" (DESM) technique enables super-resolution imaging with localization down to 12 nm across a 35 × 35 μm(2) area. DESM is particularly well suited for biological applications such as multispectral particle tracking since fluorescent nanodiamonds are not only cytocompatible but also nonbleaching and bright. We observe fluorescence count rates exceeding 1.5 × 10(6) photons per second from single NV(-) centers at saturation. When combined with emerging NV(-)-based techniques for sensing magnetic and electric fields, DESM opens the door to rapid, super-resolution imaging for tracking and sensing applications in the life and physical sciences.

  1. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.

    Science.gov (United States)

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-08-16

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore's fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.

  2. A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans.

    Science.gov (United States)

    Baranczak, Aleksandra; Liu, Yu; Connelly, Stephen; Du, Wen-Ge Han; Greiner, Erin R; Genereux, Joseph C; Wiseman, R Luke; Eisele, Yvonne S; Bradbury, Nadine C; Dong, Jiajia; Noodleman, Louis; Sharpless, K Barry; Wilson, Ian A; Encalada, Sandra E; Kelly, Jeffery W

    2015-06-17

    Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission maximum of probe 4 in comparison to its nonfluorosulfated analogue. We demonstrate that probe 4 is dark in aqueous buffers, whereas the TTR·4 complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR·4 fluorescence in macrophage-like coelomocytes. An analogue of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analogue suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.

  3. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

    Directory of Open Access Journals (Sweden)

    Alsu I. Zamaleeva

    2015-09-01

    Full Text Available Semiconductor nanocrystals (NCs or quantum dots (QDs are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxyethane-N,N,N′,N′-tetra-acetic acid for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging.

  4. Integrating Optical Tweezers, DNA Tightropes, and Single-Molecule Fluorescence Imaging: Pitfalls and Traps.

    Science.gov (United States)

    Wang, J; Barnett, J T; Pollard, M R; Kad, N M

    2017-01-01

    Fluorescence imaging is one of the cornerstone techniques for understanding how single molecules search for their targets on DNA. By tagging individual proteins, it is possible to track their position with high accuracy. However, to understand how proteins search for targets, it is necessary to elongate the DNA to avoid protein localization ambiguities. Such structures known as "DNA tightropes" are tremendously powerful for imaging target location; however, they lack information about how force and load affect protein behavior. The use of optically trapped microstructures offers the means to apply and measure force effects. Here we describe a system that we recently developed to enable individual proteins to be directly manipulated on DNA tightropes. Proteins bound to DNA can be conjugated with Qdot fluorophores for visualization and also directly manipulated by an optically trapped, manufactured microstructure. Together this offers a new approach to understanding the physical environment of molecules, and the combination with DNA tightropes presents opportunities to study complex biological phenomena. © 2017 Elsevier Inc. All rights reserved.

  5. Optical cryo-imaging of kidney mitochondrial redox state in diabetic mice models

    Science.gov (United States)

    Maleki, S.; Sepehr, R.; Staniszewski, K.; Sheibani, N.; Sorenson, C. M.; Ranji, M.

    2012-03-01

    Oxidative stress (OS), which increases during diabetes, exacerbates the development and progression of diabetes complications including renal vascular and proximal tubule cell dysfunction. The objective of this study was to investigate the changes in the metabolic state of the tissue in diabetic mice kidneys using fluorescence imaging. Mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH-2 (Flavin Adenine Dinucleotide) are autofluorescent and can be monitored without exogenous labels by optical techniques. The ratio of the fluorescence intensity of these fluorophores, (NADH/FAD), called the NADH redox ratio (RR), is a marker of metabolic state of a tissue. We examined mitochondrial redox states of kidneys from diabetic mice, Akita/+ and its control wild type (WT) for a group of 8- and 12-week-old mice. Average intensity and histogram of maximum projected images of FAD, NADH, and NADH RR were calculated for each kidney. Our results indicated a 17% decrease in the mean NADH RR of the kidney from 8-week-old mice compared with WT mice and, a 30% decrease in the mean NADH RR of kidney from12-week-old mice compared with WT mice. These results indicated an increase in OS in diabetic animals and its progression over time. Thus, NADH RR can be used as a hallmark of OS in diabetic kidney allowing temporal identification of oxidative state.

  6. Birefringence imaging

    CERN Document Server

    Geday, M A

    2001-01-01

    in tendons. Many transparent materials are optically anisotropic i.e. the refractive index varies with the polarisation orientation of the light. The variation, birefringence, can reveal the underlying anisotropy of the material, whether this anisotropy is caused by the structure of the material or by applied stress. Several methods have been developed to measure the birefringence, although only two imaging microscope techniques, the 'rotating polariser technique' and the 'Polscope', capable of separating the magnitude of the anisotropy (delta or vertical bar sin delta vertical bar), its orientation (phi) and its transmission (l sub 0) are in common use today. In this thesis the rotating polariser technique has been completely revised, with a new and easily accessible user interface as a result. Calibration routines and several analytical tools have been developed. The technique is now capable of measuring the change in birefringence during phase transitions to very high a degree of precision. Examples of the...

  7. Identifying Image Manipulation Software from Image Features

    Science.gov (United States)

    2015-03-26

    an overview of the DCT based encoding process [5]. When an image is processed by lossless compression, a file’s size is reduced while still...IDENTIFYING IMAGE MANIPULATION SOFTWARE FROM IMAGE FEATURES THESIS Devlin T. Boyter, CPT, USA AFIT-ENG-MS-15-M-051 DEPARTMENT OF THE AIR FORCE AIR...to copyright protection in the United States. AFIT-ENG-MS-15-M-051 IDENTIFYING IMAGE MANIPULATION SOFTWARE FROM IMAGE FEATURES THESIS Presented to

  8. Image processing techniques for acoustic images

    Science.gov (United States)

    Murphy, Brian P.

    1991-06-01

    The primary goal of this research is to test the effectiveness of various image processing techniques applied to acoustic images generated in MATLAB. The simulated acoustic images have the same characteristics as those generated by a computer model of a high resolution imaging sonar. Edge detection and segmentation are the two image processing techniques discussed in this study. The two methods tested are a modified version of the Kalman filtering and median filtering.

  9. Confocal Fluorescence Imaging Enables Noninvasive Quantitative Assessment of Host Cell Populations In Vivo Following Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Soumya Mitra, Oleg Mironov, Thomas H. Foster

    2012-01-01

    Full Text Available We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH-mediated photodynamic therapy (PDT of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

  10. Development of microscopic systems for high-speed dual-excitation ratiometric Ca2+ imaging.

    Science.gov (United States)

    Fukano, Takashi; Shimozono, Satoshi; Miyawaki, Atsushi

    2008-08-01

    For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems. (1) A system in which two laser beams are alternated on every scanning line, allowing us to obtain confocal images using dual-excitation ratiometric dyes. This system increases the rate at which ratio measurements can be made to 200 Hz and provides confocal images at 1-10 Hz depending on the image size. (2) A truly simultaneous dual-excitation ratiometry system that used linearly polarized excitation light and polarization detection, allowing us to obtain ratiometric images without any time lag. This system, however, is based on statistical features of the fluorescence polarization and is limited to samples that contain a large number of fluorophores. In addition, this method requires complicated calculations. (3) An efficient, nearly simultaneous dual-excitation ratiometry system that allows us to rapidly switch between two synchronized excitation-detection components by employing two high-power light-emitting diodes (LEDs) and two high-speed liquid crystal shutters. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity.

  11. Medical Imaging System

    Science.gov (United States)

    1991-01-01

    The MD Image System, a true-color image processing system that serves as a diagnostic aid and tool for storage and distribution of images, was developed by Medical Image Management Systems, Huntsville, AL, as a "spinoff from a spinoff." The original spinoff, Geostar 8800, developed by Crystal Image Technologies, Huntsville, incorporates advanced UNIX versions of ELAS (developed by NASA's Earth Resources Laboratory for analysis of Landsat images) for general purpose image processing. The MD Image System is an application of this technology to a medical system that aids in the diagnosis of cancer, and can accept, store and analyze images from other sources such as Magnetic Resonance Imaging.

  12. Manufacture of IRDye800CW-coupled Fe3O4 nanoparticles and their applications in cell labeling and in vivo imaging

    Directory of Open Access Journals (Sweden)

    Chen Zhongping

    2010-10-01

    Full Text Available Abstract Background In recent years, near-infrared fluorescence (NIRF-labeled iron nanoparticles have been synthesized and applied in a number of applications, including the labeling of human cells for monitoring the engraftment process, imaging tumors, sensoring the in vivo molecular environment surrounding nanoparticles and tracing their in vivo biodistribution. These studies demonstrate that NIRF-labeled iron nanoparticles provide an efficient probe for cell labeling. Furthermore, the in vivo imaging studies show excellent performance of the NIR fluorophores. However, there is a limited selection of NIRF-labeled iron nanoparticles with an optimal wavelength for imaging around 800 nm, where tissue autofluorescence is minimal. Therefore, it is necessary to develop additional alternative NIRF-labeled iron nanoparticles for application in this area. Results This study manufactured 12-nm DMSA-coated Fe3O4 nanoparticles labeled with a near-infrared fluorophore, IRDye800CW (excitation/emission, 774/789 nm, to investigate their applicability in cell labeling and in vivo imaging. The mouse macrophage RAW264.7 was labeled with IRDye800CW-labeled Fe3O4 nanoparticles at concentrations of 20, 30, 40, 50, 60, 80 and 100 μg/ml for 24 h. The results revealed that the cells were efficiently labeled by the nanoparticles, without any significant effect on cell viability. The nanoparticles were injected into the mouse via the tail vein, at dosages of 2 or 5 mg/kg body weight, and the mouse was discontinuously imaged for 24 h. The results demonstrated that the nanoparticles gradually accumulated in liver and kidney regions following injection, reaching maximum concentrations at 6 h post-injection, following which they were gradually removed from these regions. After tracing the nanoparticles throughout the body it was revealed that they mainly distributed in three organs, the liver, spleen and kidney. Real-time live-body imaging effectively reported the dynamic

  13. Medical imaging technology

    CERN Document Server

    Haidekker, Mark A

    2013-01-01

    Biomedical imaging is a relatively young discipline that started with Conrad Wilhelm Roentgen’s discovery of the x-ray in 1885. X-ray imaging was rapidly adopted in hospitals around the world. However, it was the advent of computerized data and image processing that made revolutionary new imaging modalities possible. Today, cross-sections and three-dimensional reconstructions of the organs inside the human body is possible with unprecedented speed, detail and quality. This book provides an introduction into the principles of image formation of key medical imaging modalities: X-ray projection imaging, x-ray computed tomography, magnetic resonance imaging, ultrasound imaging, and radionuclide imaging. Recent developments in optical imaging are also covered. For each imaging modality, the introduction into the physical principles and sources of contrast is provided, followed by the methods of image formation, engineering aspects of the imaging devices, and a discussion of strengths and limitations of the modal...

  14. Thermoacoustic imaging and spectroscopy for enhanced cancer diagnostics

    Science.gov (United States)

    Bauer, Daniel Ryan

    Early detection of cancer is paramount for improved patient survival. This dissertation presents work developing imaging techniques to improve cancer diagnostics and detection utilizing light and microwave induced thermoacoustic imaging. In the second chapter, the well-established pre-clinical mouse window chamber model is interfaced with simultaneously acquired high-resolution pulse echo (PE) ultrasound and photoacoustic (PA) imaging. Co-registered PE and PA imaging, coupled with developed image segmentation algorithms, are used to quantitatively track and monitor the size, shape, heterogeneity, and neovasculature of the tumor microenvironment during a month long study. Average volumetric growth was 5.35 mm3/day, which correlated well with two dimensional results from fluorescent imaging (R = 0.97, p imaging is also employed to probe the assumed oxygenation status of the tumor vasculature. The window chamber model combined with high-resolution PE and PA imaging could form a powerful testbed for characterizing cancers and evaluating new contrast and therapeutic agents. The third chapter utilizes a clinical ultrasound array to facilitate fast volumetric spectroscopic PA imaging to detect and discriminate endogenous absorbers (i.e. oxy/deoxygenated hemoglobin) as well as exogenous PA contrast agents (i.e. gold nanorods, fluorophores). In vivo spatiotemporal tracking of administered gold nanorods is presented, with the contrast agent augmenting the PA signal 18 dB. Furthermore, through the use of spectral unmixing algorithms, the relative concentrations of multiple endogenous and exogenous co-localized absorbers were reconstructed in tumor bearing mice. The concentration of Alexaflour647 was calculated to increase nearly 20 dB in the center of a prostate tumor after a tail-vein injection of the contrast agent. Additionally, after direct subcutaneous injections of two different gold nanorods into a breast tumor, the concentration of each nanoparticle was discriminated

  15. High-affinity Near-infrared Fluorescent Small-molecule Contrast Agents for In Vivo Imaging of Prostate-specific Membrane Antigen

    Directory of Open Access Journals (Sweden)

    Valerie Humblet

    2005-10-01

    Full Text Available Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM, single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

  16. Foundations of image science

    CERN Document Server

    Barrett, Harrison H

    2013-01-01

    Winner of the 2006 Joseph W. Goodman Book Writing Award! A comprehensive treatment of the principles, mathematics, and statistics of image science In today's visually oriented society, images play an important role in conveying messages. From seismic imaging to satellite images to medical images, our modern society would be lost without images to enhance our understanding of our health, our culture, and our world. Foundations of Image Science presents a comprehensive treatment of the principles, mathematics, and st

  17. Theranostic nanoshells: from probe design to imaging and treatment of cancer.

    Science.gov (United States)

    Bardhan, Rizia; Lal, Surbhi; Joshi, Amit; Halas, Naomi J

    2011-10-18

    Recent advances in nanoscience and biomedicine have expanded our ability to design and construct multifunctional nanoparticles that combine targeting, therapeutic, and diagnostic functions within a single nanoscale complex. The theranostic capabilities of gold nanoshells, spherical nanoparticles with silica cores and gold shells, have attracted tremendous attention over the past decade as nanoshells have emerged as a promising tool for cancer therapy and bioimaging enhancement. This Account examines the design and synthesis of nanoshell-based theranostic agents, their plasmon-derived optical properties, and their corresponding applications. We discuss the design and preparation of nanoshell complexes and their ability to enhance the photoluminescence of fluorophores while maintaining their properties as MR contrast agents. In this Account, we discuss the underlying physical principles that contribute to the photothermal response of nanoshells. We then elucidate the photophysical processes that induce nanoshells to enhance the fluorescence of weak near-infrared fluorophores. Nanoshells illuminated with resonant light are either strong optical absorbers or scatterers, properties that give rise to their unique capabilities. These physical processes have been harnessed to visualize and eliminate cancer cells. We describe the application of nanoshells as a contrast agent for optical coherence tomography of breast carcinoma cells in vivo. Our recent studies examine nanoshells as a multimodal theranostic probe, using these nanoparticles for near-infrared fluorescence and magnetic resonance imaging (MRI) and for the photothermal ablation of cancer cells. Multimodal nanoshells show theranostic potential for imaging subcutaneous breast cancer tumors in animal models and the distribution of tumors in various tissues. Nanoshells also show promise as light-triggered gene therapy vectors, adding temporal control to the spatial control characteristic of nanoparticle-based gene

  18. Superresolution images reconstructed from aliased images

    Science.gov (United States)

    Vandewalle, Patrick; Susstrunk, Sabine E.; Vetterli, Martin

    2003-06-01

    In this paper, we present a simple method to almost quadruple the spatial resolution of aliased images. From a set of four low resolution, undersampled and shifted images, a new image is constructed with almost twice the resolution in each dimension. The resulting image is aliasing-free. A small aliasing-free part of the frequency domain of the images is used to compute the exact subpixel shifts. When the relative image positions are known, a higher resolution image can be constructed using the Papoulis-Gerchberg algorithm. The proposed method is tested in a simulation where all simulation parameters are well controlled, and where the resulting image can be compared with its original. The algorithm is also applied to real, noisy images from a digital camera. Both experiments show very good results.

  19. COLOUR IMAGE REPRESENTION OF MULTISPECTRAL IMAGE FUSION

    Directory of Open Access Journals (Sweden)

    Preema Mole

    2016-07-01

    Full Text Available The availability of imaging sensors operating in multiple spectral bands has led to the requirement of image fusion algorithms that would combine the image from these sensors in an efficient way to give an image that is more perceptible to human eye. Multispectral Image fusion is the process of combining images optically acquired in more than one spectral band. In this paper, we present a pixel-level image fusion that combines four images from four different spectral bands namely near infrared(0.76-0.90um, mid infrared(1.55-1.75um,thermal- infrared(10.4-12.5um and mid infrared(2.08-2.35um to give a composite colour image. The work coalesces a fusion technique that involves linear transformation based on Cholesky decomposition of the covariance matrix of source data that converts multispectral source images which are in grayscale into colour image. This work is composed of different segments that includes estimation of covariance matrix of images, cholesky decomposition and transformation ones. Finally, the fused colour image is compared with the fused image obtained by PCA transformation.

  20. Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

    Science.gov (United States)

    Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

    2016-03-01

    Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

  1. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress.

    Science.gov (United States)

    Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R; Audi, Said; Ranji, Mahsa

    2012-04-01

    Ventilation with enhanced fractions of O(2) (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O(2)) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs.

  2. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress

    Science.gov (United States)

    Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R.; Audi, Said

    2012-01-01

    Abstract. Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs. PMID:22559688

  3. scikit-image: image processing in Python.

    Science.gov (United States)

    van der Walt, Stéfan; Schönberger, Johannes L; Nunez-Iglesias, Juan; Boulogne, François; Warner, Joshua D; Yager, Neil; Gouillart, Emmanuelle; Yu, Tony

    2014-01-01

    scikit-image is an image processing library that implements algorithms and utilities for use in research, education and industry applications. It is released under the liberal Modified BSD open source license, provides a well-documented API in the Python programming language, and is developed by an active, international team of collaborators. In this paper we highlight the advantages of open source to achieve the goals of the scikit-image library, and we showcase several real-world image processing applications that use scikit-image. More information can be found on the project homepage, http://scikit-image.org.

  4. scikit-image: image processing in Python

    Directory of Open Access Journals (Sweden)

    Stéfan van der Walt

    2014-06-01

    Full Text Available scikit-image is an image processing library that implements algorithms and utilities for use in research, education and industry applications. It is released under the liberal Modified BSD open source license, provides a well-documented API in the Python programming language, and is developed by an active, international team of collaborators. In this paper we highlight the advantages of open source to achieve the goals of the scikit-image library, and we showcase several real-world image processing applications that use scikit-image. More information can be found on the project homepage, http://scikit-image.org.

  5. Medical Image Fusion

    Directory of Open Access Journals (Sweden)

    Mitra Rafizadeh

    2007-08-01

    Full Text Available Technological advances in medical imaging in the past two decades have enable radiologists to create images of the human body with unprecedented resolution. MRI, PET,... imaging devices can quickly acquire 3D images. Image fusion establishes an anatomical correlation between corresponding images derived from different examination. This fusion is applied either to combine images of different modalities (CT, MRI or single modality (PET-PET."nImage fusion is performed in two steps:"n1 Registration: spatial modification (eg. translation of model image relative to reference image in order to arrive at an ideal matching of both images. Registration methods are feature-based and intensity-based approaches."n2 Visualization: the goal of it is to depict the spatial relationship between the model image and refer-ence image. We can point out its clinical application in nuclear medicine (PET/CT.

  6. Imaging systems and applications.

    Science.gov (United States)

    Catrysse, Peter B; Imai, Francisco H; Linne von Berg, Dale C; Sheridan, John T

    2013-03-01

    Imaging systems have numerous applications in industrial, military, consumer, and medical settings. Assembling a complete imaging system requires the integration of optics, sensing, image processing, and display rendering. This issue features original research ranging from fundamental theories to novel imaging modalities and provides a systems perspective to imaging.

  7. Smart Image Enhancement Process

    Science.gov (United States)

    Jobson, Daniel J. (Inventor); Rahman, Zia-ur (Inventor); Woodell, Glenn A. (Inventor)

    2012-01-01

    Contrast and lightness measures are used to first classify the image as being one of non-turbid and turbid. If turbid, the original image is enhanced to generate a first enhanced image. If non-turbid, the original image is classified in terms of a merged contrast/lightness score based on the contrast and lightness measures. The non-turbid image is enhanced to generate a second enhanced image when a poor contrast/lightness score is associated therewith. When the second enhanced image has a poor contrast/lightness score associated therewith, this image is enhanced to generate a third enhanced image. A sharpness measure is computed for one image that is selected from (i) the non-turbid image, (ii) the first enhanced image, (iii) the second enhanced image when a good contrast/lightness score is associated therewith, and (iv) the third enhanced image. If the selected image is not-sharp, it is sharpened to generate a sharpened image. The final image is selected from the selected image and the sharpened image.

  8. Image processing with ImageJ

    CERN Document Server

    Pascau, Javier

    2013-01-01

    The book will help readers discover the various facilities of ImageJ through a tutorial-based approach.This book is targeted at scientists, engineers, technicians, and managers, and anyone who wishes to master ImageJ for image viewing, processing, and analysis. If you are a developer, you will be able to code your own routines after you have finished reading this book. No prior knowledge of ImageJ is expected.

  9. Quantitative imaging of basic functions in renal (patho)physiology.

    Science.gov (United States)

    Kang, Jung Julie; Toma, Ildiko; Sipos, Arnold; McCulloch, Fiona; Peti-Peterdi, Janos

    2006-08-01

    Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 +/- 0.4 to 59.5 +/- 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased approximately 10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 +/- 0.4 mm/s) compared with peritubular vessels (4.7 +/- 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times ( approximately 45 s) to glomerular filtration, with a delay of approximately 5-10 and 25-30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology.

  10. Toward real-time tumor margin identification in image-guided robotic brain tumor resection

    Science.gov (United States)

    Hu, Danying; Jiang, Yang; Belykh, Evgenii; Gong, Yuanzheng; Preul, Mark C.; Hannaford, Blake; Seibel, Eric J.

    2017-03-01

    For patients with malignant brain tumors (glioblastomas), a safe maximal resection of tumor is critical for an increased survival rate. However, complete resection of the cancer is hard to achieve due to the invasive nature of these tumors, where the margins of the tumors become blurred from frank tumor to more normal brain tissue, but in which single cells or clusters of malignant cells may have invaded. Recent developments in fluorescence imaging techniques have shown great potential for improved surgical outcomes by providing surgeons intraoperative contrast-enhanced visual information of tumor in neurosurgery. The current near-infrared (NIR) fluorophores, such as indocyanine green (ICG), cyanine5.5 (Cy5.5), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), are showing clinical potential to be useful in targeting and guiding resections of such tumors. Real-time tumor margin identification in NIR imaging could be helpful to both surgeons and patients by reducing the operation time and space required by other imaging modalities such as intraoperative MRI, and has the potential to integrate with robotically assisted surgery. In this paper, a segmentation method based on the Chan-Vese model was developed for identifying the tumor boundaries in an ex-vivo mouse brain from relatively noisy fluorescence images acquired by a multimodal scanning fiber endoscope (mmSFE). Tumor contours were achieved iteratively by minimizing an energy function formed by a level set function and the segmentation model. Quantitative segmentation metrics based on tumor-to-background (T/B) ratio were evaluated. Results demonstrated feasibility in detecting the brain tumor margins at quasi-real-time and has the potential to yield improved precision brain tumor resection techniques or even robotic interventions in the future.

  11. Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy.

    Science.gov (United States)

    Mitra, Soumya; Modi, Kshitij D; Foster, Thomas H

    2013-10-01

    We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population.

  12. Outpatient Imaging Efficiency - State

    Data.gov (United States)

    U.S. Department of Health & Human Services — Use of medical imaging - state data. These measures give you information about hospitals' use of medical imaging tests for outpatients. Examples of medical imaging...

  13. General Ultrasound Imaging

    Medline Plus

    Full Text Available ... ultrasound. top of page How does the procedure work? Ultrasound imaging is based on the same principles ... to-use and less expensive than other imaging methods. Ultrasound imaging is extremely safe and does not ...

  14. Mold Image Library

    Science.gov (United States)

    The image library contains mold-related images in seven categories. There are also animated images that you can choose to view and download. These photos may be used for presentations and educational purposes without contacting EPA.

  15. General Ultrasound Imaging

    Science.gov (United States)

    ... Physician Resources Professions Site Index A-Z General Ultrasound Ultrasound imaging uses sound waves to produce pictures ... limitations of General Ultrasound Imaging? What is General Ultrasound Imaging? Ultrasound is safe and painless, and produces ...

  16. What Is an Image?

    Science.gov (United States)

    Gerber, Andrew J.; Peterson, Bradley S.

    2008-01-01

    The article helps to understand the interpretation of an image by presenting as to what constitutes an image. A common feature in all images is the basic physical structure that can be described with a common set of terms.

  17. Annotating images by mining image search results

    NARCIS (Netherlands)

    Wang, X.J.; Zhang, L.; Li, X.; Ma, W.Y.

    2008-01-01

    Although it has been studied for years by the computer vision and machine learning communities, image annotation is still far from practical. In this paper, we propose a novel attempt at model-free image annotation, which is a data-driven approach that annotates images by mining their search results

  18. Image Enhancement By Partially Coherent Imaging

    Science.gov (United States)

    Rhodes, William T.; Koizumi, M.

    1983-04-01

    A partially coherent Koehler-illumination imaging system equipped with complementary masks in source and pupil planes can be used to perform image enhancement operations such as directional or non-directional edge enhancement and emphasis of spatial bandpass features. With many objects the use of complementary masks results in high-contrast images. Underlying principles are explained and preliminary experimental results presented.

  19. To Image...or Not to Image?

    Science.gov (United States)

    Bruley, Karina

    1996-01-01

    Provides a checklist of considerations for installing document image processing with an electronic document management system. Other topics include scanning; indexing; the image file life cycle; benefits of imaging; document-driven workflow; and planning for workplace changes like postsorting, creating a scanning room, redeveloping job tasks and…

  20. Imaging oncogene expression

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Archana [Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)], E-mail: Archana.Mukherjee@jefferson.edu; Wickstrom, Eric [Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233S, 10th street, Philadelphia, PA 19107 (United States)], E-mail: eric@tesla.jci.tju.edu; Thakur, Mathew L. [Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)], E-mail: Mathew.Thakur@jefferson.edu

    2009-05-15

    This review briefly outlines the importance of molecular imaging, particularly imaging of endogenous gene expression for noninvasive genetic analysis of radiographic masses. The concept of antisense imaging agents and the advantages and challenges in the development of hybridization probes for in vivo imaging are described. An overview of the investigations on oncogene expression imaging is given. Finally, the need for further improvement in antisense-based imaging agents and directions to improve oncogene mRNA targeting is stated.