WorldWideScience

Sample records for biological fluorescence imaging

  1. Fluorescence confocal endomicroscopy in biological imaging

    Science.gov (United States)

    Delaney, Peter; Thomas, Steven; Allen, John; McLaren, Wendy; Murr, Elise; Harris, Martin

    2007-02-01

    In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular details could be readily visualised in vivo at high resolution. In

  2. Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

    Science.gov (United States)

    Müller, Francis; Fattinger, Christof

    Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal fluorophore. With fluorescence lifetimes in the microsecond range and fluorescence quantum yield of 0.4 some water soluble complexes of Ruthenium like modified Ru(sulfobathophenanthroline) complexes fulfill these properties. They are outstanding fluorescent labels for ultrasensitive assays as illustrated in two examples, in drug discovery and in point of care testing.We discuss the fundamentals and the state-of-the-art of the most sensitive time-gated fluorescence assays. We reflect on how the imaging devices currently employed for readout of these assays might evolve in the future. Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal

  3. Photon-counting H33D detector for biological fluorescence imaging

    Science.gov (United States)

    Michalet, X.; Siegmund, O. H. W.; Vallerga, J. V.; Jelinsky, P.; Millaud, J. E.; Weiss, S.

    2006-11-01

    We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross-delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions.

  4. Flexible imaging payload for real-time fluorescent biological imaging in parabolic, suborbital and space analog environments

    Science.gov (United States)

    Bamsey, Matthew T.; Paul, Anna-Lisa; Graham, Thomas; Ferl, Robert J.

    2014-10-01

    Fluorescent imaging offers the ability to monitor biological functions, in this case biological responses to space-related environments. For plants, fluorescent imaging can include general health indicators such as chlorophyll fluorescence as well as specific metabolic indicators such as engineered fluorescent reporters. This paper describes the Flex Imager a fluorescent imaging payload designed for Middeck Locker deployment and now tested on multiple flight and flight-related platforms. The Flex Imager and associated payload elements have been developed with a focus on 'flexibility' allowing for multiple imaging modalities and change-out of individual imaging or control components in the field. The imaging platform is contained within the standard Middeck Locker spaceflight form factor, with components affixed to a baseplate that permits easy rearrangement and fine adjustment of components. The Flex Imager utilizes standard software packages to simplify operation, operator training, and evaluation by flight provider flight test engineers, or by researchers processing the raw data. Images are obtained using a commercial cooled CCD image sensor, with light-emitting diodes for excitation and a suite of filters that allow biological samples to be imaged over wavelength bands of interest. Although baselined for the monitoring of green fluorescent protein and chlorophyll fluorescence from Arabidopsis samples, the Flex Imager payload permits imaging of any biological sample contained within a standard 10 cm by 10 cm square Petri plate. A sample holder was developed to secure sample plates under different flight profiles while permitting sample change-out should crewed operations be possible. In addition to crew-directed imaging, autonomous or telemetric operation of the payload is also a viable operational mode. An infrared camera has also been integrated into the Flex Imager payload to allow concurrent fluorescent and thermal imaging of samples. The Flex Imager has been

  5. Synthesis and Characterization of Water-Soluble Conjugated Oligoelectrolytes for Near-Infrared Fluorescence Biological Imaging.

    Science.gov (United States)

    Woo, Shin-Jae; Park, Sungmin; Jeong, Ji-Eun; Hong, Yoochan; Ku, Minhee; Kim, Bo Yun; Jang, Il Ho; Heo, Soon Chul; Wang, Taejun; Kim, Ki Hean; Yang, Jaemoon; Kim, Jae Ho; Woo, Han Young

    2016-06-29

    Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer. PMID:27267787

  6. Studying Biological Tissue with Fluorescence Lifetime Imaging: Microscopy, Endoscopy, and Complex Decay Profiles

    Science.gov (United States)

    Siegel, Jan; Elson, Daniel S.; Webb, Stephen E. D.; Lee, K. C. Benny; Vlandas, Alexis; Gambaruto, Giovanni L.; Léveque-Fort, Sandrine; Lever, M. John; Tadrous, Paul J.; Stamp, Gordon W. H.; Wallace, Andrew L.; Sandison, Ann; Watson, Tim F.; Alvarez, Fernando; French, Paul M. W.

    2003-06-01

    We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro , including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.

  7. Development of Two Color Fluorescent Imager and Integrated Fluidic System for Nanosatellite Biology Applications

    Science.gov (United States)

    Wu, Diana Terri; Ricco, Antonio Joseph; Lera, Matthew P.; Timucin, Linda R.; Parra, Macarena P.

    2012-01-01

    Nanosatellites offer frequent, low-cost space access as secondary payloads on launches of larger conventional satellites. We summarize the payload science and technology of the Microsatellite in-situ Space Technologies (MisST) nanosatellite for conducting automated biological experiments. The payload (two fused 10-cm cubes) includes 1) an integrated fluidics system that maintains organism viability and supports growth and 2) a fixed-focus imager with fluorescence and scattered-light imaging capabilities. The payload monitors temperature, pressure and relative humidity, and actively controls temperature. C. elegans (nematode, 50 m diameter x 1 mm long) was selected as a model organism due to previous space science experience, its completely sequenced genome, size, hardiness, and the variety of strains available. Three strains were chosen: two green GFP-tagged strains and one red tdTomato-tagged strain that label intestinal, nerve, and pharyngeal cells, respectively. The integrated fluidics system includes bioanalytical and reservoir modules. The former consists of four 150 L culture wells and a 4x5 mm imaging zone the latter includes two 8 mL fluid reservoirs for reagent and waste storage. The fluidic system is fabricated using multilayer polymer rapid prototyping: laser cutting, precision machining, die cutting, and pressure-sensitive adhesives it also includes eight solenoid-operated valves and one mini peristaltic pump. Young larval-state (L2) nematodes are loaded in C. elegans Maintenance Media (CeMM) in the bioanalytical module during pre-launch assembly. By the time orbit is established, the worms have grown to sufficient density to be imaged and are fed fresh CeMM. The strains are pumped sequentially into the imaging area, imaged, then pumped into waste. Reagent storage utilizes polymer bags under slight pressure to prevent bubble formation in wells or channels. The optical system images green and red fluorescence bands by excitation with blue (473 nm peak

  8. Robust overlay schemes for the fusion of fluorescence and color channels in biological imaging.

    Science.gov (United States)

    Glatz, Jürgen; Symvoulidis, Panagiotis; Garcia-Allende, P Beatriz; Ntziachristos, Vasilis

    2014-04-01

    Molecular fluorescence imaging is a commonly used method in various biomedical fields and is undergoing rapid translation toward clinical applications. Color images are commonly superimposed with fluorescence measurements to provide orientation, anatomical information, and molecular tissue properties in a single image. New adaptive methods that produce a more robust composite image than conventional lime green alpha blending are presented and demonstrated herein. Moreover, visualization through temporal changes is showcased as an alternative for real-time imaging systems.

  9. A Practical Approach to Quantitative Processing and Analysis of Small Biological Structures by Fluorescent Imaging

    Science.gov (United States)

    Noller, Crystal M.; Boulina, Maria; McNamara, George; Szeto, Angela; McCabe, Philip M.

    2016-01-01

    Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines. PMID:27182204

  10. Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine.

    Science.gov (United States)

    Sun, Shuting; Błażewska, Katarzyna M; Kadina, Anastasia P; Kashemirov, Boris A; Duan, Xuchen; Triffitt, James T; Dunford, James E; Russell, R Graham G; Ebetino, Frank H; Roelofs, Anke J; Coxon, Fraser P; Lundy, Mark W; McKenna, Charles E

    2016-02-17

    A bone imaging toolkit of 21 fluorescent probes with variable spectroscopic properties, bone mineral binding affinities, and antiprenylation activities has been created, including a novel linking strategy. The linking chemistry allows attachment of a diverse selection of dyes fluorescent in the visible to near-infrared range to any of the three clinically important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate, and minodronate or their analogues). The resultant suite of conjugates offers multiple options to "mix and match" parent drug structure, fluorescence emission wavelength, relative bone affinity, and presence or absence of antiprenylation activity, for bone-related imaging applications. PMID:26646666

  11. Correlated imaging of living biological cells with a soft X-ray microscope and a fluorescence microscope

    International Nuclear Information System (INIS)

    Soft X-ray microscope is a very powerful tool to observe cellular organelles of living biological. However the inner structures are very complicated and it is difficult to identify the organelles obtained with the soft X-ray microscopes. We have proposed a correlated imaging with a soft X-ray microscope and a fluorescence microscope that is to observe the same biological cells with the both microscopes at the same time. (author)

  12. Diffraction enhanced imaging and x-ray fluorescence microtomography for analyzing biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, H.S.; Pereira, G.R.; Lopes, R.T. [Laboratorio de Instrumentacao Nuclear-COPPE/UFRJ-RJ (Brazil); Anjos, M.J. [Instituto de Fisica-UERJ-RJ (Brazil); Faria, P. [Instituto Nacional do Cancer-INCa-RJ (Brazil); Kellermann, G.; Perez, C.A. [Laboratorio Nacional de Luz Sincrotron-Campinas-SP (Brazil); Tirao, G. [Faculdad de Mat. Astronomia y Fisica (FAMAF), UNC. Cordoba (Argentina); Mazzaro, I. [Laboratorio de Optica de Raios X e Instrumentacao-UFPR-Curitiba-PR (Brazil); Giles, C. [Laboratorio de Cristalografia Aplicada e Raios X-UNICAMP-Campinas-SP (Brazil)

    2007-07-15

    In this work, breast tissue samples were investigated in order to verify the distribution of certain elements by x-ray fluorescence computed tomography (XRFCT) correlated with the characteristics and pathology of each tissue observed by diffraction enhanced imaging (DEI). The DEI system can show details in low attenuation tissues. It is based on the contrast imaging obtained by extinction, diffraction and refraction characteristics and can improve reduction in false positive and false negative diagnoses. XRFCT allows mapping of all elements within the sample, since even a minute fluorescence signal can be detected. DEI imaging techniques revealed the complex structure of the disease, confirmed by the histological section, and showed microstructures in all planes of the sample. The XRFCT showed the distribution of Zn, Cu and Fe at higher concentration. (authors)

  13. Synthesis, and Fluorescence Properties of Coumarin and Benzocoumarin Derivatives Conjugated Pyrimidine Scaffolds for Biological Imaging Applications.

    Science.gov (United States)

    Al-Masoudi, Najim A; Al-Salihi, Niran J; Marich, Yossra A; Markus, Timo

    2015-11-01

    Series of coumarin and 5,6-benzomcomarin substituted pyrimidine derivatives 11-15 and 22-25 were synthesized, aiming to develop new imaging fluorescent agents. Analogously, treatment of 4-chloropyrimidine analog 16 with coumarin 3-carbohyrazide 5 under MWI condition followed by boiling with NH4OAc in HOAc furnished coumarin-1,2,4-triazolo-pyrimidine analog 18. The fluorescence property was investigated spectrophotometrically in MeOH with Rhodamine 6G as standard dye. All the compounds showed emission in the region between 331 and 495 nm. The quantum yield of all the compounds were found to be weak, except methyl benzocoumarin 3-carboxylate 22 which showed (ΦF = 0.98) in comparison to Rhodamine 6G as standard (ΦF = 0.95).

  14. Quinoxaline-Based Polymer Dots with Ultrabright Red to Near-Infrared Fluorescence for In Vivo Biological Imaging.

    Science.gov (United States)

    Liu, Hong-Yi; Wu, Pei-Jing; Kuo, Shih-Yu; Chen, Chuan-Pin; Chang, En-Hao; Wu, Chang-Yi; Chan, Yang-Hsiang

    2015-08-19

    This article describes the design and synthesis of quinoxaline-based semiconducting polymer dots (Pdots) that exhibit near-infrared fluorescence, ultrahigh brightness, large Stokes shifts, and excellent cellular targeting capability. We also introduced fluorine atoms and long alkyl chains into polymer backbones and systematically investigated their effect on the fluorescence quantum yields of Pdots. These new series of quinoxaline-based Pdots have a fluorescence quantum yield as high as 47% with a Stokes shift larger than 150 nm. Single-particle analysis reveals that the average per-particle brightness of the Pdots is at least 6 times higher than that of the commercially available quantum dots. We further demonstrated the use of this new class of quinoxaline-based Pdots for effective and specific cellular and subcellular labeling without any noticeable nonspecific binding. Moreover, the cytotoxicity of Pdots were evaluated on HeLa cells and zebrafish embryos to demonstrate their great biocompatibility. By taking advantage of their extreme brightness and minimal cytotoxicity, we performed, for the first time, in vivo microangiography imaging on living zebrafish embryos using Pdots. These quinoxaline-based NIR-fluorescent Pdots are anticipated to find broad use in a variety of in vitro and in vivo biological research.

  15. Fabrication of aggregation-induced emission based fluorescent nanoparticles and their biological imaging application: recent progress and perspectives

    Directory of Open Access Journals (Sweden)

    Bin Yang

    2016-06-01

    Full Text Available Aggregation-induced emission (AIE dyes have received wide-spread concern since their inception. Several types of AIE-based fluorescent nanoparticle (FNP have been developed, and the potential applications of these FNPs have also been explored. Recent studies of AIE-based FNPs in biological areas have suggested that they show promise as bio-materials for cell imaging and other biomedical applications. This article reviews recent progress in the synthesis of AIE-based FNPs via non-covalent, covalent and novel one-pot strategies, and the subsequent cell-imaging of those AIE-based FNPs. Many successes have been achieved, and there is still plenty of space for the development of AIE-based FNPs as new bio-materials.

  16. Fluorescent image tracking velocimeter

    Science.gov (United States)

    Shaffer, Franklin D.

    1994-01-01

    A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

  17. Fluorescence and Spectral Imaging

    Directory of Open Access Journals (Sweden)

    Ralph S. DaCosta

    2007-01-01

    Full Text Available Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots. This is an area of great promise, but still in its infancy, and preclinical studies are currently under way.

  18. Multi Spectral Fluorescence Imager (MSFI)

    Science.gov (United States)

    Caron, Allison

    2016-01-01

    Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

  19. Fluorescent Sensors for Biological Applications

    Directory of Open Access Journals (Sweden)

    Hui-wang Ai

    2014-09-01

    Full Text Available Fluorescence is one of the most important analytical methods used in biological studies. In the past decade or two, instrumentation in this field has greatly advanced, and now it is possible to detect single photons or fluorescent molecules [1,2], or break the Abbe diffraction limit to distinguish two points spaced less than 50 nm apart [3]. Concurrently, the development of improved fluorescent probes, which can be coupled with state-of-the-art instruments, has been equally important. This special issue on “fluorescent biosensors” in Sensors reports recent results from eight research groups in the field of sensor development. It includes three review articles, and six research articles reporting original results. [...

  20. Fluorescent Cell Imaging in Regenerative Medicine

    OpenAIRE

    Etai Sapoznik; Guoguang Niu; Yu Zhou; Murphy, Sean V.; Shay Soker

    2016-01-01

    Fluorescent protein imaging, a promising tool in biological research, incorporates numerous applications that can be of specific use in the field of regenerative medicine. To enhance tissue regeneration efforts, scientists have been developing new ways to monitor tissue development and maturation in vitro and in vivo. To that end, new imaging tools and novel fluorescent proteins have been developed for the purpose of performing deep-tissue high-resolution imaging. These new methods, such as i...

  1. Oligothiophenes as Fluorescent Markers for Biological Applications

    Directory of Open Access Journals (Sweden)

    Antonio Manetto

    2012-01-01

    Full Text Available This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (biomolecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

  2. Fluorescent Probes and Fluorescence (Microscopy Techniques — Illuminating Biological and Biomedical Research

    Directory of Open Access Journals (Sweden)

    Gregor P. C. Drummen

    2012-11-01

    Full Text Available Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo, to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein. Because fluorescent proteins are synthesized in vivo, integration of fluorescent detection methods into the biological system via genetic techniques now became feasible. Currently fluorescent proteins are available that virtually span the whole electromagnetic spectrum. Concomitantly, fluorescence imaging techniques were developed, and often progress in one field fueled innovation in the other. Impressively, the properties of fluorescence were utilized to develop new assays and imaging modalities, ranging from energy transfer to image molecular interactions to imaging beyond the diffraction limit with super-resolution microscopy. Here, an overview is provided of recent developments in both fluorescence imaging and fluorochrome engineering, which together constitute the “fluorescence toolbox” in life science research.

  3. Far-field fluorescence microscopy beyond the diffraction limit: Fluorescence imaging with ultrahigh resolution

    OpenAIRE

    Rice, James H.

    2007-01-01

    Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the f...

  4. Cancer detection by quantitative fluorescence image analysis.

    Science.gov (United States)

    Parry, W L; Hemstreet, G P

    1988-02-01

    Quantitative fluorescence image analysis is a rapidly evolving biophysical cytochemical technology with the potential for multiple clinical and basic research applications. We report the application of this technique for bladder cancer detection and discuss its potential usefulness as an adjunct to methods used currently by urologists for the diagnosis and management of bladder cancer. Quantitative fluorescence image analysis is a cytological method that incorporates 2 diagnostic techniques, quantitation of nuclear deoxyribonucleic acid and morphometric analysis, in a single semiautomated system to facilitate the identification of rare events, that is individual cancer cells. When compared to routine cytopathology for detection of bladder cancer in symptomatic patients, quantitative fluorescence image analysis demonstrated greater sensitivity (76 versus 33 per cent) for the detection of low grade transitional cell carcinoma. The specificity of quantitative fluorescence image analysis in a small control group was 94 per cent and with the manual method for quantitation of absolute nuclear fluorescence intensity in the screening of high risk asymptomatic subjects the specificity was 96.7 per cent. The more familiar flow cytometry is another fluorescence technique for measurement of nuclear deoxyribonucleic acid. However, rather than identifying individual cancer cells, flow cytometry identifies cellular pattern distributions, that is the ratio of normal to abnormal cells. Numerous studies by others have shown that flow cytometry is a sensitive method to monitor patients with diagnosed urological disease. Based upon results in separate quantitative fluorescence image analysis and flow cytometry studies, it appears that these 2 fluorescence techniques may be complementary tools for urological screening, diagnosis and management, and that they also may be useful separately or in combination to elucidate the oncogenic process, determine the biological potential of tumors

  5. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    OpenAIRE

    Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    International audience In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent prote...

  6. Assessing Photosynthesis by Fluorescence Imaging

    Science.gov (United States)

    Saura, Pedro; Quiles, Maria Jose

    2011-01-01

    This practical paper describes a novel fluorescence imaging experiment to study the three processes of photochemistry, fluorescence and thermal energy dissipation, which compete during the dissipation of excitation energy in photosynthesis. The technique represents a non-invasive tool for revealing and understanding the spatial heterogeneity in…

  7. [Development of Zn(2+) selective fluorescent probes for biological applications].

    Science.gov (United States)

    Hagimori, Masayori

    2013-01-01

    Zn(2+) is an essential element for life and is known to play important roles in biological processes including gene expression, apoptosis, enzyme regulation, immune system and neurotransmission. To investigate physiological roles of free or chelatable Zn(2+) in living cells, Zn(2+)-selective fluorescent probes are valuable tools. A variety of fluorescent probes based on quinoline, BF2 chelated dipyrromethene, fluorescein, etc. has been developed recently. In principle, such tools can provide useful information about zinc biology. However, most of the fluorescent probes presented so far possess a fluorescent core and a separate part for binding to Zn(2+) within the molecule, so that the molecular weight is usually large and the molecules are hydrophobic. As a result, the applications of such molecules in biological systems often face difficulties. Therefore, we need to develop a new class of fluorescent probes for Zn(2+) with improved molecular characteristics. If the initial core structure is small enough, the fluorescent probes may still be molecular weight below 500 with desirable physico-chemical properties, even after the modifications. In this review, we described novel low-molecular-weight fluorescent probes for Zn(2+) based on pyridine-pyridone. Small modification of pyridine-pyridone core structure brought about a marked improvement such as aqueous solubility, affinity toward Zn(2+), and fluorescence ON/OFF switching. Fluorescence images of Zn(2+) in cells showed that the pyridine-pyridone probe can be used in biological applications.

  8. Fluorescent Quantum Dots for Biological Labeling

    Science.gov (United States)

    McDonald, Gene; Nadeau, Jay; Nealson, Kenneth; Storrie-Lomardi, Michael; Bhartia, Rohit

    2003-01-01

    Fluorescent semiconductor quantum dots that can serve as "on/off" labels for bacteria and other living cells are undergoing development. The "on/off" characterization of these quantum dots refers to the fact that, when properly designed and manufactured, they do not fluoresce until and unless they come into contact with viable cells of biological species that one seeks to detect. In comparison with prior fluorescence-based means of detecting biological species, fluorescent quantum dots show promise for greater speed, less complexity, greater sensitivity, and greater selectivity for species of interest. There are numerous potential applications in medicine, environmental monitoring, and detection of bioterrorism.

  9. Phase-sensitive fluorescent imaging with coherent reconstruction

    CERN Document Server

    Field, Jeffrey J; Bartels, Randy A

    2015-01-01

    Optical imaging plays a critical role in advancing our understanding of three dimensional dynamics of biological systems. Coherent imaging (CI) methods exploit spatial phase information, encoded through propagation of coherent signal light emerging from a specimen, to extract a three-dimensional representation of the object from a single high-speed measurement. Until now, CI methods could not be applied to incoherent light, severely limiting their ability to image the most powerful biological probes available - fluorescent molecules - with sufficient speed and volume to observe important processes, such as neural processing in live specimens. We introduce a new imaging technique that transfers the spatial propagation phase of coherent illumination light to incoherent fluorescent light emission. The transfer of propagation phase allows CI techniques to be applied to fluorescent light imaging, and leads to large increases in imaging speed and depth of field. With this advance, biological imaging of fluorescent ...

  10. Spectrally resolved fluorescent lifetime imaging

    OpenAIRE

    Hanley, Quentin S.

    2008-01-01

    Placing an imaging spectrograph or related components capable of generating a spectrum between a microscope and the image intensifier of a conventional fluorescence lifetime imaging (FLIM) system creates a spectrally resolved FLIM (SFLIM). This arrangement provides a number of opportunities not readily available to conventional systems using bandpass filters. The examples include: simultaneous viewing of multiple fluorophores; tracking of both the donor and acceptor; and observation of a rang...

  11. Two-photon fluorescence anisotropy imaging

    Science.gov (United States)

    Li, Wei; Wang, Yi; Shao, Hanrong; He, Yonghong; Ma, Hui

    2006-09-01

    We have developed a novel method for imaging the fluorescence intensity and anisotropy by two-photon fluorescence microscopy and tested its capability in biological application. This method is applied to model sample including FITC and FITC-CD44 antibody solution and also FITC-CD44 stained cells. The fluorescence anisotropy (FA) of FITC-CD44ab solution is higher than the FITC solution with the same concentration. The fluorescence in cell sample has even higher FA than in solution because the rotation diffusion is restrained in membrane. The method is employed to study the effect of berberine a kind of Chinese medicine, on tumor metastasis. The results indicated that tumor cell membrane fluidity is decreasing with increasing the concentration of berberine in culture medium.

  12. Fluorescence lidar imaging of historical monuments.

    Science.gov (United States)

    Weibring, P; Johansson, T; Edner, H; Svanberg, S; Sundnér, B; Raimondi, V; Cecchi, G; Pantani, L

    2001-11-20

    What is believed to be the first fluorescence imaging of the facades of a historical building, which was accomplished with a scanning fluorescence lidar system, is reported. The mobile system was placed at a distance of ~60 m from the medieval Lund Cathedral (Sweden), and a 355-nm pulsed laser beam was swept over the stone facades row by row while spectrally resolved fluorescence signals of each measurement point were recorded. By multispectral image processing, either by formation of simple spectral-band ratios or by use of multivariate techniques, areas with different spectral signatures were classified. In particular, biological growth was observed and different stone types were distinguished. The technique can yield data for use in facade status assessment and restoration planning. PMID:18364910

  13. Biological applications of confocal fluorescence polarization microscopy

    Science.gov (United States)

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

  14. Nanoprobes for super-resolution fluorescence imaging at the nanoscale

    Institute of Scientific and Technical Information of China (English)

    HOU ShangGuo; LIANG Le; DENG SuHui; CHEN JianFang; HUANG Qing; CHENG Ya; FAN ChunHai

    2014-01-01

    Compared with other imaging techniques,fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells.Owing to the resolution limit set by the diffraction of light,fluorescence microscopy could not resolve the nanostructures in the range of〈200 nm.Recently,many techniques have been emerged to overcome the diffraction barrier,providing nanometer spatial resolution.In the course of development,the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy.Here,we describe the contributions of the fluorescent probes to far-field super resolution imaging,focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes,like photoswitching,bleaching and blinking.Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods.

  15. Multispectral fluorescence imaging techniques for nondestructive food safety inspection

    Science.gov (United States)

    Kim, Moon S.; Lefcourt, Alan M.; Chen, Yud-Ren

    2004-03-01

    The use of spectral sensing has gained acceptance as a rapid means for nondestructive inspection of postharvest food produce. Current technologies generally use color or a single wavelength camera technology. The applicability and sensitivity of these techniques can be expanded through the use of multiple wavelengths. Reflectance in the Vis/NIR is the prevalent spectral technique. Fluorescence, compared to reflectance, is regarded as a more sensitive technique due to its dynamic responses to subtle changes in biological entities. Our laboratory has been exploring fluorescence as a potential means for detection of quality and wholesomeness of food products. Applications of fluorescence sensing require an understanding of the spectral characteristics emanating from constituents and potential contaminants. A number of factors affecting fluorescence emission characteristics are discussed. Because of relatively low fluorescence quantum yield from biological samples, a system with a powerful pulse light source such as a laser coupled with a gated detection device is used to harvest fluorescence, in the presence of ambient light. Several fluorescence sensor platforms developed in our laboratory, including hyperspectral imaging, and laser-induced fluorescence (LIF) and steady-state fluorescence imaging systems with multispectral capabilities are presented. We demonstrate the potential uses of recently developed fluorescence imaging platforms in food safety inspection of apples contaminated with animal feces.

  16. Monitoring biological aerosols using UV fluorescence

    Science.gov (United States)

    Eversole, Jay D.; Roselle, Dominick; Seaver, Mark E.

    1999-01-01

    An apparatus has been designed and constructed to continuously monitor the number density, size, and fluorescent emission of ambient aerosol particles. The application of fluorescence to biological particles suspended in the atmosphere requires laser excitation in the UV spectral region. In this study, a Nd:YAG laser is quadrupled to provide a 266 nm wavelength to excite emission from single micrometer-sized particles in air. Fluorescent emission is used to continuously identify aerosol particles of biological origin. For calibration, biological samples of Bacillus subtilis spores and vegetative cells, Esherichia coli, Bacillus thuringiensis and Erwinia herbicola vegetative cells were prepared as suspensions in water and nebulized to produce aerosols. Detection of single aerosol particles, provides elastic scattering response as well as fluorescent emission in two spectral bands simultaneously. Our efforts have focuses on empirical characterization of the emission and scattering characteristics of various bacterial samples to determine the feasibility of optical discrimination between different cell types. Preliminary spectroscopic evidence suggest that different samples can be distinguished as separate bio-aerosol groups. In addition to controlled sample results, we will also discuss the most recent result on the effectiveness of detection outdoor releases and variations in environmental backgrounds.

  17. Entangled-photon coincidence fluorescence imaging

    OpenAIRE

    Scarcelli, Giuliano; Yun, Seok H.

    2008-01-01

    We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations.

  18. Fluorescence lifetime imaging of skin cancer

    Science.gov (United States)

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

    2011-03-01

    Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

  19. Near-Infrared Fluorescent Materials for Sensing of Biological Targets

    Directory of Open Access Journals (Sweden)

    Julia Xiaojun Zhao

    2008-05-01

    Full Text Available Near-infrared fluorescent (NIRF materials are promising labeling reagents for sensitive determination and imaging of biological targets. In the near-infrared region biological samples have low background fluorescence signals, providing high signal to noise ratio. Meanwhile, near-infrared radiation can penetrate into sample matrices deeply due to low light scattering. Thus, in vivo and in vitro imaging of biological samples can be achieved by employing the NIRF probes. To take full advantage of NIRF materials in the biological and biomedical field, one of the key issues is to develop intense and biocompatible NIRF probes. In this review, a number of NIRF materials are discussed including traditional NIRF dye molecules, newly developed NIRF quantum dots and single-walled carbon nanotubes, as well as rare earth metal compounds. The use of some NIRF materials in various nanostructures is illustrated. The enhancement of NIRF using metal nanostructures is covered as well. The fluorescence mechanism and bioapplications of each type of the NIRF materials are discussed in details.

  20. ZnO nanocomposites modified by hydrophobic and hydrophilic silanes with dramatically enhanced tunable fluorescence and aqueous ultrastability toward biological imaging applications.

    Science.gov (United States)

    Li, Shuying; Sun, Zongzhao; Li, Rui; Dong, Minmin; Zhang, Liyan; Qi, Wei; Zhang, Xuelin; Wang, Hua

    2015-01-01

    Multicolor ZnO quantum dots (QDs) were synthesized and further modified with hydrophobic hexadecyltrimethoxysilane (HDS) and then hydrophilic aminopropyltriethoxysilane (APS) bilayers, resulting in amine-functionalized ZnO@HDS@APS nanocomposites with tunable fluorescence from blue to green yellow. Systematic investigations verify that the resulting ZnO@HDS@APS could display extremely high stability in aqueous media and unexpectedly, dramatically-enhanced fluorescence intensities, which are about 10-fold higher than those of bare ZnO QDs. The feasibility of the as-prepared ZnO nanocomposites for blood, cell, and tissue imaging was preliminarily demonstrated, promising the wide bio-applications for cell or tissue imaging, proteome analysis, drug delivery, and molecular labeling.

  1. Intrinsic fluorescence of selenium nanoparticles for cellular imaging applications

    Science.gov (United States)

    Khalid, A.; Tran, Phong A.; Norello, Romina; Simpson, David A.; O'Connor, Andrea J.; Tomljenovic-Hanic, Snjezana

    2016-02-01

    Nanoparticles hold great potential in contributing to high-resolution bioimaging as well as for biomedical applications. Although, selenium (Se) nanoparticles (NPs) have been investigated owing to their potential roles in therapeutics, the imaging capability of these NPs has never been explored. This manuscript identifies the intrinsic fluorescence of Se NPs, which is highly beneficial for nanoscale imaging of biological structures. The emission of individual NPs and its evolution with time is explored. The photoluminescence spectra has revealed visible to near infrared emission for Se NPs. The work finally reflects on the role of this intrinsic fluorescence for in vitro imaging and tracking in fibroblast cells, without the need of any additional tags. This technique would overcome the limitations of the conventionally used methods of imaging with tagged fluorescent proteins and dyes, preventing possible adverse cellular effects or phototoxicity caused by the added fluorescent moieties.Nanoparticles hold great potential in contributing to high-resolution bioimaging as well as for biomedical applications. Although, selenium (Se) nanoparticles (NPs) have been investigated owing to their potential roles in therapeutics, the imaging capability of these NPs has never been explored. This manuscript identifies the intrinsic fluorescence of Se NPs, which is highly beneficial for nanoscale imaging of biological structures. The emission of individual NPs and its evolution with time is explored. The photoluminescence spectra has revealed visible to near infrared emission for Se NPs. The work finally reflects on the role of this intrinsic fluorescence for in vitro imaging and tracking in fibroblast cells, without the need of any additional tags. This technique would overcome the limitations of the conventionally used methods of imaging with tagged fluorescent proteins and dyes, preventing possible adverse cellular effects or phototoxicity caused by the added fluorescent

  2. APD detectors for biological fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mazeres, S. [Institut de Pharmacologie et de Biologie Structurale, IPBS-CNRS, 205 route de Narbonne 31077 Toulouse Cedex 4 (France)]. E-mail: serge.mazeres@ipbs.fr; Borrel, V. [GIATHE/CESR, 9 avenue du Colonel Roche BP 4346, 31029 Toulouse Cedex (France); Magenc, C. [GIATHE/CESR, 9 avenue du Colonel Roche BP 4346, 31029 Toulouse Cedex (France); Courrech, J.L. [GIATHE/CESR, 9 avenue du Colonel Roche BP 4346, 31029 Toulouse Cedex (France); Bazer-Bachi, R. [GIATHE/CESR, 9 avenue du Colonel Roche BP 4346, 31029 Toulouse Cedex (France)

    2006-11-01

    Fluorescence spectroscopy is a very convenient and widely used method for studying the molecular background of biological processes [L. Salome, J.L. Cazeil, A. Lopez, J.F. Tocanne, Eur. Biophys. J. 27 (1998) 391-402]. Chromophores are included in the structure under study and a flash of laser light induces fluorescence (Fluorescence Recovery After Photo-bleaching), the decay of which yields information on the polarity, the speed of rotation, and the speed of diffusion as well as on the temporal and spatial evolution of interactions between molecular species. The method can even be used to study living cells [J.F. Tocanne, L. Cezanne, A. Lopez, Prog. Lipid Res. 33 (1994) 203-237, L. Cezanne, A. Lopez, F. Loste, G. Parnaud, O. Saurel, P. Demange, J.F. Tocanne, Biochemistry 38 (1999) 2779-2786]. This is classically performed with a PM-based system. For biological reasons a decrease of the excitation of the cells is highly desirable. Because the fluorescence response then becomes fainter a significant improvement in detector capability would be welcome. We present here results obtained with an Avalanche Photo Diode (APD)-based system. The small sensitive area of detection allows a very significant improvement in signal/noise ratio, improvement in gain, and the opening-up of a new parameter space. With these new detectors we can begin the study of information transmission between cells through morphine receptors. This work involves both electronics engineers and biophysicists, so results and techniques in both fields will be presented here.

  3. In vivo cellular imaging using fluorescent proteins - Methods and Protocols

    Directory of Open Access Journals (Sweden)

    M. Monti

    2012-12-01

    Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

  4. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    CERN Document Server

    Cognet, Laurent; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells.

  5. Development of an oxidative dehydrogenation-based fluorescent probe for Cu{sup 2+} and its biological imaging in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan Jiangli, E-mail: fanjl@dlut.edu.cn [State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024 (China); Liu Xiaojian; Hu Mingming; Zhu Hao; Song Fengling [State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024 (China); Peng Xiaojun, E-mail: pengxj@dlut.edu.cn [State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer The fluorescent probe contains an N, O and S tridentate ligand. Black-Right-Pointing-Pointer The probe is simple but highly sensitive and selective toward Cu{sup 2+}. Black-Right-Pointing-Pointer The mechanism is based on the Cu{sup 2+}-promoted dehydrogenation of amine in different organic and aqueous solutions. Black-Right-Pointing-Pointer It was successfully applied to visualize Cu{sup 2+} in living cells. - Abstract: Based on a boron dipyrromethene (BODIPY) derivative containing an N, O and S tridentate ligand, a Cu{sup 2+} fluorescent probe BTCu was developed. The detection mechanism was verified as Cu{sup 2+}-promoted oxidative dehydrogenation of an amine moiety, leading to a formation of a fluorescent Cu{sup +}-Schiff base complex. Free BTCu exhibited a maximum absorption wavelength at 496 nm, and a very weak maximum emission at 511 nm. Upon addition of various metals ions, it showed large fluorescence enhancement toward Cu{sup 2+} (417-fold in MeCN and 103-fold in MeCN/HEPES solution, respectively) with high selectivity. The detection limits are as low as 1.74 Multiplication-Sign 10{sup -8} M and 4.96 Multiplication-Sign 10{sup -8} M in the two different solutions, respectively. And BTCu could work in a wide pH range with an extraordinary low pK{sub a} of 1.21 {+-} 0.06. Using fluorescence microscopy, the probe was shown to be capable of penetrating into living cells and imaging intracellular Cu{sup 2+} changes.

  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. Optical imaging of biological tissues

    Science.gov (United States)

    Bouza Dominguez, Jorge

    In this thesis, a new time-dependent model for describing light propagation in biological media is proposed. The model is based on the simplified spherical harmonics approximation and is represented by a set of coupled parabolic partial differential equations (TD-pSPN equations). In addition, the model is extended for modeling the time-dependent response of fluorescent agents in biological tissues and the ensuing time-domain propagation of light therein. In a comparison with Monte Carlo simulations, it is shown that the TD-pSPN equations present unique features in its derivation that makes it a more accurate alternative to the diffusion equation (DE). The TD-pSPN model (for orders N > 1) outperforms the DE in the description of the propagation of light in near-nondiffusive media and in all the physical situations where DE fails. Often, only small orders of the SP N approximation are needed to obtain accurate results. A diffuse optical tomography (DOT) algorithm is also implemented based on the TD-pSPN equations as the forward model using constrained optimization methods. The algorithm uses time-dependent (TD) data directly. Such an approach is benefited from both the accuracy of the SPN models and the richness of TD data. In the calculation of the gradient of the objective function, a time-dependent adjoint differentiation method is introduced that reduces computation time. Several numerical experiments are performed for small geometry media with embedded inclusions that mimic small animal imaging. In these experiments, the values of the optical coefficients are varied within realistic bounds that are representative of those found in the range of the near-infrared spectrum, including high absorption values. Single and multi-parameter reconstructions (absorption and diffusion coefficients) are performed. The reconstructed images based on the TD-pSPN equations (N > 1) give better estimates of the optical properties of the media than the DE. On the other hand

  8. Fast x-ray fluorescence microtomography of hydrated biological samples.

    Directory of Open Access Journals (Sweden)

    Enzo Lombi

    Full Text Available Metals and metalloids play a key role in plant and other biological systems as some of them are essential to living organisms and all can be toxic at high concentrations. It is therefore important to understand how they are accumulated, complexed and transported within plants. In situ imaging of metal distribution at physiological relevant concentrations in highly hydrated biological systems is technically challenging. In the case of roots, this is mainly due to the possibility of artifacts arising during sample preparation such as cross sectioning. Synchrotron x-ray fluorescence microtomography has been used to obtain virtual cross sections of elemental distributions. However, traditionally this technique requires long data acquisition times. This has prohibited its application to highly hydrated biological samples which suffer both radiation damage and dehydration during extended analysis. However, recent advances in fast detectors coupled with powerful data acquisition approaches and suitable sample preparation methods can circumvent this problem. We demonstrate the heightened potential of this technique by imaging the distribution of nickel and zinc in hydrated plant roots. Although 3D tomography was still impeded by radiation damage, we successfully collected 2D tomograms of hydrated plant roots exposed to environmentally relevant metal concentrations for short periods of time. To our knowledge, this is the first published example of the possibilities offered by a new generation of fast fluorescence detectors to investigate metal and metalloid distribution in radiation-sensitive, biological samples.

  9. Hadamard-transform fluorescence-lifetime imaging.

    Science.gov (United States)

    Mizuno, Takahiko; Iwata, Tetsuo

    2016-04-18

    We discuss a Hadamard-transform-based fluorescence-lifetime-imaging (HT-FLI) technique for fluorescence-lifetime-imaging microscopy (FLIM). The HT-FLI uses a Fourier-transform phase-modulation fluorometer (FT-PMF) for fluorescence-lifetime measurements, where the modulation frequency of the excitation light is swept linearly in frequency from zero to a specific maximum during a fixed duration of time. Thereafter, fluorescence lifetimes are derived through Fourier transforms for the fluorescence and reference waveforms. The FT-PMF enables the analysis of multi-component samples simultaneously. HT imaging uses electronic exchange of HT illumination mask patterns, and a high-speed, high-sensitivity photomultiplier, to eliminate frame-rate issues that accompany two-dimensional image detectors. PMID:27137259

  10. High frame rate fluorescence lifetime imaging

    Energy Technology Data Exchange (ETDEWEB)

    Agronskaia, A V [Department of Molecular Biophysics, Debye Institute, Utrecht University, PO Box 80000, Princetonplein 1, 3508 TA Utrecht(Netherlands); Tertoolen, L [KNAW Hubrecht Laboratory, Netherlands Institute for Developmental Biology (NIOB), Uppsalalaan 8, 3584 CT Utrecht (Netherlands); Gerritsen, H C [Department of Molecular Biophysics, Debye Institute, Utrecht University, PO Box 80000, Princetonplein 1, 3508 TA Utrecht (Netherlands)

    2003-07-21

    A fast time-domain based fluorescence lifetime imaging (FLIM) microscope is presented that can operate at frame rates of hundreds of frames per second. A beam splitter in the detection path of a wide-field fluorescence microscope divides the fluorescence in two parts. One part is optically delayed with respect to the other. Both parts are viewed with a single time-gated intensified CCD camera with a gate width of 5 ns. The fluorescence lifetime image is obtained from the ratio of these two images. The fluorescence lifetime resolution of the FLIM microscope is verified both with dye solutions and fluorescent latex beads. The fluorescence lifetimes obtained from the reference specimens are in good agreement with values obtained from time correlated single photon counting measurements on the same specimens. The acquisition speed of the FLIM system is evaluated with a measurement of the calcium fluxes in neonatal rat myocytes stained with the calcium probe Oregon Green 488-Bapta. Fluorescence lifetime images of the calcium fluxes related to the beating of the myocytes are acquired with frame rates of up to 100 Hz.

  11. In Vivo Metal Ion Imaging Using Fluorescent Sensors.

    Science.gov (United States)

    Van de Bittner, Genevieve C; Hirayama, Tasuku

    2016-01-01

    In vivo imaging in living animals provides the ability to monitor alterations of signaling molecules, ions, and other biological components during various life stages and in disease. The data gained from in vivo imaging can be used for biological discovery or to determine elements of disease progression and can inform the development and translation of therapeutics. Herein, we present theories behind small-molecule, fluorescent, metal ion sensors as well as the methods for their successful application to in vivo metal ion imaging, including ex vivo validation. PMID:27283424

  12. In Vivo Metal Ion Imaging Using Fluorescent Sensors.

    Science.gov (United States)

    Van de Bittner, Genevieve C; Hirayama, Tasuku

    2016-01-01

    In vivo imaging in living animals provides the ability to monitor alterations of signaling molecules, ions, and other biological components during various life stages and in disease. The data gained from in vivo imaging can be used for biological discovery or to determine elements of disease progression and can inform the development and translation of therapeutics. Herein, we present theories behind small-molecule, fluorescent, metal ion sensors as well as the methods for their successful application to in vivo metal ion imaging, including ex vivo validation.

  13. Imaging an atomic beam using fluorescence

    Institute of Scientific and Technical Information of China (English)

    Ming He(何明); Jin Wang(王谨); Mingsheng Zhan(詹明生)

    2003-01-01

    A fluorescence detection scheme is applied to image an atomic beam. Using two laser diodes as the sources of detection light and pumping light respectively, the fluorescence image of the atomic beam is then observed by a commercial CCD-camera, which is corresponding to the atomic state and velocity distribution. The detection scheme has a great utilization in the experiments of cold atoms and atomic optics.

  14. Reflectance and fluorescence hyperspectral elastic image registration

    Science.gov (United States)

    Lange, Holger; Baker, Ross; Hakansson, Johan; Gustafsson, Ulf P.

    2004-05-01

    Science and Technology International (STI) presents a novel multi-modal elastic image registration approach for a new hyperspectral medical imaging modality. STI's HyperSpectral Diagnostic Imaging (HSDI) cervical instrument is used for the early detection of uterine cervical cancer. A Computer-Aided-Diagnostic (CAD) system is being developed to aid the physician with the diagnosis of pre-cancerous and cancerous tissue regions. The CAD system uses the fusion of multiple data sources to optimize its performance. The key enabling technology for the data fusion is image registration. The difficulty lies in the image registration of fluorescence and reflectance hyperspectral data due to the occurrence of soft tissue movement and the limited resemblance of these types of imagery. The presented approach is based on embedding a reflectance image in the fluorescence hyperspectral imagery. Having a reflectance image in both data sets resolves the resemblance problem and thereby enables the use of elastic image registration algorithms required to compensate for soft tissue movements. Several methods of embedding the reflectance image in the fluorescence hyperspectral imagery are described. Initial experiments with human subject data are presented where a reflectance image is embedded in the fluorescence hyperspectral imagery.

  15. Multiparameter fluorescence spectroscopic imaging of cell function

    Science.gov (United States)

    Bright, Gary R.

    1994-08-01

    The ability to quantitate physiological parameters in single living cells using fluorescence spectroscopic imaging has expanded our understanding of many cell regulatory processes. Previous studies have focussed on the measurement of single parameters, such as the concentration of calcium, and more recently two parameters, such as calcium and pH using fluorescence ratio imaging. The complexity of the interrelationships among cell biochemical reactions suggests a need to extend the measurement scheme to several parameters. Expansion of the number of parameters involves several complexities associated with fluorescent probe selection and instrumentation design as well as the processing and management of the data. A system has been assembled which provides maximum flexibility in multiparameter fluorescence imaging measurements. The system provides multiple combinations of excitation, dichroic mirror, and emission wavelengths. It has automatic acquisition of any number of parameters. The number of parameters is primarily limited by the selection of fluorescent probes with nonoverlapping spectra. We demonstrate the utility of the system by the coordinated monitoring of stimulated changes in the concentrations of calcium, magnesium, and pH using fluorescence ratio imaging coupled with a conventional transmitted light image of single smooth muscle cells. The results demonstrate coordinated changes in some instances but uncoordinated changes in others.

  16. Hyperspectral Fluorescence and Reflectance Imaging Instrument

    Science.gov (United States)

    Ryan, Robert E.; O'Neal, S. Duane; Lanoue, Mark; Russell, Jeffrey

    2008-01-01

    The system is a single hyperspectral imaging instrument that has the unique capability to acquire both fluorescence and reflectance high-spatial-resolution data that is inherently spatially and spectrally registered. Potential uses of this instrument include plant stress monitoring, counterfeit document detection, biomedical imaging, forensic imaging, and general materials identification. Until now, reflectance and fluorescence spectral imaging have been performed by separate instruments. Neither a reflectance spectral image nor a fluorescence spectral image alone yields as much information about a target surface as does a combination of the two modalities. Before this system was developed, to benefit from this combination, analysts needed to perform time-consuming post-processing efforts to co-register the reflective and fluorescence information. With this instrument, the inherent spatial and spectral registration of the reflectance and fluorescence images minimizes the need for this post-processing step. The main challenge for this technology is to detect the fluorescence signal in the presence of a much stronger reflectance signal. To meet this challenge, the instrument modulates artificial light sources from ultraviolet through the visible to the near-infrared part of the spectrum; in this way, both the reflective and fluorescence signals can be measured through differencing processes to optimize fluorescence and reflectance spectra as needed. The main functional components of the instrument are a hyperspectral imager, an illumination system, and an image-plane scanner. The hyperspectral imager is a one-dimensional (line) imaging spectrometer that includes a spectrally dispersive element and a two-dimensional focal plane detector array. The spectral range of the current imaging spectrometer is between 400 to 1,000 nm, and the wavelength resolution is approximately 3 nm. The illumination system consists of narrowband blue, ultraviolet, and other discrete

  17. Imaging and quantification of trace metals in thin biological specimens using microprobe techniques: synchrotron induced X-ray fluorescence microprobe and nuclear microprobe

    International Nuclear Information System (INIS)

    The combination of Synchrotron X-Ray Fluorescence microprobe (SXRF) with other micro-analytical techniques based on accelerated particle beams, such as Rutherford Backscattering Spectrometry (RBS), and Particle Induced X-ray Emission (PIXE) enable to quantify SXRF results of thin biological specimens. The same specimens were analysed by both techniques. The average thickness calculated by RBS of discrete tissue regions in equivalent sample sections was used to normalize SXRF data. Analysing the same sample region with PIXE technique, simultaneously to RBS, can validate the methodology once it will enable to compare concentrations obtained for elements that are detected by both SXRF and PIXE. Major departures were found for the quantification of Ca and Ti. For Cr and Fe a good agreement was found between both micro-analytical techniques. Differences below 20% were obtained for the majority of the elements and samples analysed. The procedure can be applied to produce quantitative elemental maps and it will be advantageous to quantify toxic elements present at low concentrations in tissues, such as Cr, Ni and Ph, which can only be efficiently assessed by SXRF. (authors)

  18. An amide-containing metal-organic tetrahedron responding to a spin-trapping reaction in a fluorescent enhancement manner for biological imaging of NO in living cells.

    Science.gov (United States)

    Wang, Jian; He, Cheng; Wu, Pengyan; Wang, Jing; Duan, Chunying

    2011-08-17

    Metal-organic polyhedra represent a unique class of functional molecular containers that display interesting molecular recognition properties and fascinating reactivity reminiscent of the natural enzymes. By incorporating a triphenylamine moiety as a bright blue emitter, a robust cerium-based tetrahedron was developed as a luminescent detector of nitronyl nitroxide (PTIO), a specific spin-labeling nitric oxide (NO) trapper. The tetrahedron encapsulates molecules of NO and PTIO within the cavity to prompt the spin-trapping reaction and transforms the normal EPR responses into a more sensitively luminescent signaling system with the limit of detection improved to 5 nM. Twelve-fold amide groups are also functionalized within the tetrahedron to modify the hydrophilic/lipophilic environment, ensuring the successful application of biological imaging in living cells.

  19. Nonlinear spectral imaging of biological tissues

    Science.gov (United States)

    Palero, J. A.

    2007-07-01

    The work presented in this thesis demonstrates live high resolution 3D imaging of tissue in its native state and environment. The nonlinear interaction between focussed femtosecond light pulses and the biological tissue results in the emission of natural autofluorescence and second-harmonic signal. Because biological intrinsic emission is generally very weak and extends from the ultraviolet to the visible spectral range, a broad-spectral range and high sensitivity 3D spectral imaging system is developed. Imaging the spectral characteristics of the biological intrinsic emission reveals the structure and biochemistry of the cells and extra-cellular components. By using different methods in visualizing the spectral images, discrimination between different tissue structures is achieved without the use of any stain or fluorescent label. For instance, RGB real color spectral images of the intrinsic emission of mouse skin tissues show blue cells, green hair follicles, and purple collagen fibers. The color signature of each tissue component is directly related to its characteristic emission spectrum. The results of this study show that skin tissue nonlinear intrinsic emission is mainly due to the autofluorescence of reduced nicotinamide adenine dinucleotide (phosphate), flavins, keratin, melanin, phospholipids, elastin and collagen and nonlinear Raman scattering and second-harmonic generation in Type I collagen. In vivo time-lapse spectral imaging is implemented to study metabolic changes in epidermal cells in tissues. Optical scattering in tissues, a key factor in determining the maximum achievable imaging depth, is also investigated in this work.

  20. Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Lesoine, Michael; Bose, Sayantan; Petrich, Jacob; Smith, Emily

    2012-06-13

    Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging.

  1. Dynamic fluorescence lifetime imaging based on acousto-optic deflectors

    Science.gov (United States)

    Yan, Wei; Peng, Xiao; Qi, Jing; Gao, Jian; Fan, Shunping; Wang, Qi; Qu, Junle; Niu, Hanben

    2014-11-01

    We report a dynamic fluorescence lifetime imaging (D-FLIM) system that is based on a pair of acousto-optic deflectors for the random regions of interest (ROI) study in the sample. The two-dimensional acousto-optic deflector devices are used to rapidly scan the femtosecond excitation laser beam across the sample, providing specific random access to the ROI. Our experimental results using standard fluorescent dyes in live cancer cells demonstrate that the D-FLIM system can dynamically monitor the changing process of the microenvironment in the ROI in live biological samples.

  2. 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.

  3. Nonnegative matrix factorization: a blind spectra separation method for in vivo fluorescent optical imaging.

    Science.gov (United States)

    Montcuquet, Anne-Sophie; Hervé, Lionel; Navarro, Fabrice; Dinten, Jean-Marc; Mars, Jérôme I

    2010-01-01

    Fluorescence imaging in diffusive media is an emerging imaging modality for medical applications that uses injected fluorescent markers that bind to specific targets, e.g., carcinoma. The region of interest is illuminated with near-IR light and the emitted back fluorescence is analyzed to localize the fluorescence sources. To investigate a thick medium, as the fluorescence signal decreases with the light travel distance, any disturbing signal, such as biological tissues intrinsic fluorescence (called autofluorescence) is a limiting factor. Several specific markers may also be simultaneously injected to bind to different molecules, and one may want to isolate each specific fluorescent signal from the others. To remove the unwanted fluorescence contributions or separate different specific markers, a spectroscopic approach is explored. The nonnegative matrix factorization (NMF) is the blind positive source separation method we chose. We run an original regularized NMF algorithm we developed on experimental data, and successfully obtain separated in vivo fluorescence spectra.

  4. U-SPECT-BioFluo: an integrated radionuclide, bioluminescence, and fluorescence imaging platform

    NARCIS (Netherlands)

    Van Oosterom, M.N.; Kreuger, R.; Buckle, T.; Mahn, W.A.; Bunschoten, A.; Josephson, L.; Van Leeuwen, F.W.B.; Beekman, F.J.

    2014-01-01

    Background: In vivo bioluminescence, fluorescence, and single-photon emission computed tomography (SPECT) imaging provide complementary information about biological processes. However, to date these signatures are evaluated separately on individual preclinical systems. In this paper, we introduce a

  5. Fluorescent nanoparticle probes for imaging of cancer.

    Science.gov (United States)

    Santra, Swadeshmukul; Malhotra, Astha

    2011-01-01

    Fluorescent nanoparticles (FNPs) have received immense popularity in cancer imaging in recent years because of their attractive optical properties. In comparison to traditional organic-based fluorescent dyes and fluorescent proteins, FNPs offer much improved sensitivity and photostability. FNPs in certain size range have a strong tendency to enter and retain in solid tumor tissue with abnormal (leaky) vasculature--a phenomenon known as Enhanced Permeation and Retention (EPR) effect, advancing their use for in vivo tumor imaging. Furthermore, large surface area of FNPs and their usual core-shell structure offer a platform for designing and fabricating multimodal/multifunctional nanoparticles (MMNPs). For effective cancer imaging, often the optical imaging modality is integrated with other nonoptical-based imaging modalities such as MRI, X-ray, and PET, thus creating multimodal nanoparticle (NP)-based imaging probes. Such multimodal NP probes can be further integrated with therapeutic drug as well as cancer targeting agent leading to multifunctional NPs. Biocompatibility of FNPs is an important criterion that must be seriously considered during FNP design. NP composition, size, and surface chemistry must be carefully selected to minimize potential toxicological consequences both in vitro and in vivo. In this article, we will mainly focus on three different types of FNPs: dye-loaded NPs, quantum dots (Qdots), and phosphores; briefly highlighting their potential use in translational research. PMID:21480546

  6. Fluorescence lifetime imaging in biosciences: technologies and applications

    Institute of Scientific and Technical Information of China (English)

    Raluca NIESNER; Karl-Heinz GERICKE

    2008-01-01

    The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging tech-niques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analy-sis of the total fluorescence signal originating from the sam-ple, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macro-scopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved tech-niques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosci-ences, especially for fast intravital studies are discussed in this work.

  7. Two-photon excited fluorescence from biological aerosol particles

    International Nuclear Information System (INIS)

    We used a 40 MHz mode-locked 524 nm laser source to evaluate the utility of sub-picosecond excitation of fluorescence from 2-photon absorption in biological aerosols. Individual particles of biological composition, as well as other calibration particles, suspended in an inlet air flow were illuminated and measured as they passed through an optical chamber. To our knowledge, this was the first demonstration of 2-photon excited fluorescence from micron-sized particles composed of micro-organisms. We also observed a high fluorescence signal at visible wavelengths, which was not present with single-photon excitation.

  8. Distinguishability of Biological Material Using Ultraviolet Multi-Spectral Fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P.C.; Heinen, R.J.; Rigdon, L.D.; Rosenthal, S.E.; Shokair, I.R.; Siragusa, G.R.; Tisone, G.C.; Wagner, J.S.

    1998-10-14

    Recent interest in the detection and analysis of biological samples by spectroscopic methods has led to questions concerning the degree of distinguishability and biological variability of the ultraviolet (W) fluorescent spectra from such complex samples. We show that the degree of distinguishability of such spectra is readily determined numerically.

  9. 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.

  10. Biological imaging in radiation oncology

    Energy Technology Data Exchange (ETDEWEB)

    Grosu, A.L.; Wiedenmann, N.; Molls, M. [Klinik und Poliklinik fuer Strahlentherapie und Radiologische Onkologie Klinikum rechts der Isar, Technical Univ. of Munich (Germany)

    2005-07-01

    The goal of this study was to discuss the value of integrating biological imaging (PET, SPECT, MRS etc.) in radiation treatment planning and monitoring. Studies in patients with brain tumors have shown that, compared to CT and MRI alone, the image fusion of CT/MRI and amino acid SPECT or PET allows a more correct delineation of gross tumor volume (GTV) and planning target volume (PTV). For FDG-PET, comparable results with different techniques are reported in the literature also for bronchial carcinoma, ear-nose-and-throat tumors, and cervical carcinoma, or, in the case of MRS, for prostate cancer. Imaging of hypoxia, cell proliferation, apoptosis, tumor angiogenesis, and gene expression leads to the identification of differently aggressive areas of a biologically inhomogeneous tumor mass that can be individually and more appropriately targeted using innovative IMRT. Thus, a biological, inhomogeneous dose distribution can be generated, the so-called dose painting. In addition, the biological imaging can play a significant role in the evaluation of the therapy response after radiochemotherapy. Clinical studies in ear-nose-and-throat tumors, bronchial carcinoma, esophagus carcinoma, and cervical carcinoma suggest that the sensitivity and specificity of FDG-PET for the therapy response are higher compared to anatomical imaging (CT and MRI). Clinical and experimental studies are required to define the real impact of these investigations in radiation treatment planning, and especially in the evaluation of therapy response. (orig.)

  11. LED light source for hyperspectral fluorescence imaging

    OpenAIRE

    Tendenes, Nils Ove

    2012-01-01

    This report deals with the possibility of creating a LED light source, to be used withhyperspectral fluorescence imaging. There are commercially available light sources thatcould be used, but they are expensive, they do not necessarily emit the right wavelength, the uniformity of the field is questionable and they are difficult to modify.First a batch of Light emitting diodes were acquired, these were subjected to a seriesof tests to classify their limitations and determine which diodes were ...

  12. FIZICS: fluorescent imaging zone identification system, a novel macro imaging system.

    Science.gov (United States)

    Skwish, Stephen; Asensio, Francisco; King, Greg; Clarke, Glenn; Kath, Gary; Salvatore, Michael J; Dufresne, Claude

    2004-12-01

    Constantly improving biological assay development continues to drive technological requirements. Recently, a specification was defined for capturing white light and fluorescent images of agar plates ranging in size from the NUNC Omni tray (96-well footprint, 128 x 85 mm) to the NUNC Bio Assay Dish (245 x 245 mm). An evaluation of commercially available products failed to identify any system capable of fluorescent macroimaging with discrete wavelength selection. To address the lack of a commercially available system, a custom imaging system was designed and constructed. This system provides the same capabilities of many commercially available systems with the added ability to fluorescently image up to a 245 x 245 mm area using wavelengths in the visible light spectrum.

  13. Selective Detection of Neurotransmitters by Fluorescence and Chemiluminescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ziqiang Wang; Edward S. Yeung

    2001-08-06

    In recent years, luminescence imaging has been widely employed in neurochemical analysis. It has a number of advantages for the study of neuronal and other biological cells: (1) a particular molecular species or cellular constituent can be selectively visualized in the presence of a large excess of other species in a heterogeneous environment; (2) low concentration detection limits can be achieved because of the inherent sensitivity associated with fluorescence and chemiluminescence; (3) low excitation intensities can be used so that long-term observation can be realized while the viability of the specimen is preserved; and (4) excellent spatial resolution can be obtained with the light microscope so subcellular compartments can be identified. With good sensitivity, temporal and spatial resolution, the flux of ions and molecules and the distribution and dynamics of intracellular species can be measured in real time with specific luminescence probes, substrates, or with native fluorescence. A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with CCD imaging is down to {micro}M levels of glutamate with reasonable response time. They also found that chemiluminescence associated with the ATP-dependent reaction between luciferase and luciferin can be used to image ATP at levels down to 10 nM in the millisecond time scale. Similar imaging experiments should be feasible in a broad spectrum of biological systems.

  14. Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

    Science.gov (United States)

    Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

    2015-06-23

    Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

  15. Biological oscillations: Fluorescence monitoring by confocal microscopy

    Science.gov (United States)

    Chattoraj, Shyamtanu; Bhattacharyya, Kankan

    2016-09-01

    Fluctuations play a vital role in biological systems. Single molecule spectroscopy has recently revealed many new kinds of fluctuations in biological molecules. In this account, we focus on structural fluctuations of an antigen-antibody complex, conformational dynamics of a DNA quadruplex, effects of taxol on dynamics of microtubules, intermittent red-ox oscillations at different organelles in a live cell (mitochondria, lipid droplets, endoplasmic reticulum and cell membrane) and stochastic resonance in gene silencing. We show that there are major differences in these dynamics between a cancer cell and the corresponding non-cancer cell.

  16. Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)

    Science.gov (United States)

    Belfield, Kevin D.; Yao, Sheng; Kim, Bosung; Yue, Xiling

    2016-03-01

    Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (two-photon absorption cross sections (up to 2,800 GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

  17. Porphyrin-magnetite nanoconjugates for biological imaging

    LENUS (Irish Health Repository)

    Nowostawska, Malgorzata

    2011-04-08

    Abstract Background The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, non-toxic solutions that can be manipulated via an external magnetic field. As a result, the interaction of these nanocomposites with cells is of vital importance in understanding their behaviour and biocompatibility. Here we report the preparation, characterisation and potential application of new "two-in-one" magnetic fluorescent nanocomposites composed of silica-coated magnetite nanoparticles covalently linked to a porphyrin moiety. Method The experiments were performed by administering porphyrin functionalised silica-coated magnetite nanoparticles to THP-1 cells, a human acute monocytic leukaemia cell line. Cells were cultured in RPMI 1640 medium with 25 mM HEPES supplemented with heat-inactivated foetal bovine serum (FBS). Results We have synthesised, characterised and analysed in vitro, a new multimodal (magnetic and fluorescent) porphyrin magnetic nanoparticle composite (PMNC). Initial co-incubation experiments performed with THP-1 macrophage cells were promising; however the PMNC photobleached under confocal microscopy study. β-mercaptoethanol (β-ME) was employed to counteract this problem and resulted not only in enhanced fluorescence emission, but also allowed for elongated imaging and increased exposure times of the PMNC in a cellular environment. Conclusion Our experiments have demonstrated that β-ME visibly enhances the emission intensity. No deleterious effects to the cells were witnessed upon co-incubation with β-ME alone and no increases in background fluorescence were recorded. These results should present an interest for further development of in vitro biological imaging techniques.

  18. Fluorescence lifetime imaging of oxygen in living cells

    NARCIS (Netherlands)

    Gerritsen, H.C.; Sanders, R.; Draaijer, A.; Ince, C.; Levine, Y.K.

    1997-01-01

    The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence life-time imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can

  19. Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Okabe, Kohki; Inada, Noriko; Gota, Chie; Harada, Yoshie; Funatsu, Takashi; Uchiyama, Seiichi

    2012-02-28

    Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

  20. Creating Panoramic Images for Bladder Fluorescence Endoscopy

    Directory of Open Access Journals (Sweden)

    A. Behrens

    2008-01-01

    Full Text Available The medical diagnostic analysis and therapy of urinary bladder cancer based on endoscopes are state of the art in urological medicine. Due to the limited field of view of endoscopes, the physician can examine only a small part of the whole operating field at once. This constraint makes visual control and navigation difficult, especially in hollow organs. A panoramic image, covering a larger field of view, can overcome this difficulty. Directly motivated by a physician we developed an image mosaicing algorithm for endoscopic bladder fluorescence video sequences. In this paper, we present an approach which is capable of stitching single endoscopic video images to a combined panoramic image. Based on SIFT features we estimate a 2-D homography for each image pair, using an affine model and an iterative model-fitting algorithm. We then apply the stitching process and perform a mutual linear interpolation. Our panoramic image results show a correct stitching and lead to a better overview and understanding of the operation field. 

  1. Live imaging of Tribolium castaneum embryonic development using light-sheet-based fluorescence microscopy.

    Science.gov (United States)

    Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

    2015-10-01

    Tribolium castaneum has become an important insect model organism for evolutionary developmental biology, genetics and biotechnology. However, few protocols for live fluorescence imaging of Tribolium have been reported, and little image data is available. Here we provide a protocol for recording the development of Tribolium embryos with light-sheet-based fluorescence microscopy. The protocol can be completed in 4-7 d and provides procedural details for: embryo collection, microscope configuration, embryo preparation and mounting, noninvasive live imaging for up to 120 h along multiple directions, retrieval of the live embryo once imaging is completed, and image data processing, for which exemplary data is provided. Stringent quality control criteria for developmental biology studies are also discussed. Light-sheet-based fluorescence microscopy complements existing toolkits used to study Tribolium development, can be adapted to other insect species, and requires no advanced imaging or sample preparation skills.

  2. Design Strategies of Fluorescent Biosensors Based on Biological Macromolecular Receptors

    Directory of Open Access Journals (Sweden)

    Takashi Morii

    2010-02-01

    Full Text Available Fluorescent biosensors to detect the bona fide events of biologically important molecules in living cells are increasingly demanded in the field of molecular cell biology. Recent advances in the development of fluorescent biosensors have made an outstanding contribution to elucidating not only the roles of individual biomolecules, but also the dynamic intracellular relationships between these molecules. However, rational design strategies of fluorescent biosensors are not as mature as they look. An insatiable request for the establishment of a more universal and versatile strategy continues to provide an attractive alternative, so-called modular strategy, which permits facile preparation of biosensors with tailored characteristics by a simple combination of a receptor and a signal transducer. This review describes an overview of the progress in design strategies of fluorescent biosensors, such as auto-fluorescent protein-based biosensors, protein-based biosensors covalently modified with synthetic fluorophores, and signaling aptamers, and highlights the insight into how a given receptor is converted to a fluorescent biosensor. Furthermore, we will demonstrate a significance of the modular strategy for the sensor design.

  3. A novel multiwavelength fluorescence image-guided surgery imaging system

    Science.gov (United States)

    Volpi, D.; Tullis, I. D. C.; Laios, A.; Pathiraja, P. N. J.; Haldar, K.; Ahmed, A. A.; Vojnovic, B.

    2014-02-01

    We describe the development and performance analysis of two clinical near-infrared fluorescence image-guided surgery (FIGS) devices that aim to overcome some of the limitations of current FIGS systems. The devices operate in a widefield-imaging mode and can work (1) in conjunction with a laparoscope, during minimally invasive surgery, and (2) as a hand-held, open surgery imaging system. In both cases, narrow-band excitation light, delivered at multiple wavelengths, is efficiently combined with white reflectance light. Light is delivered to ~100 cm2 surgical field at 1-2 mW/cm2 for white light and 3-7 mW/cm2 (depending on wavelength) of red - near infrared excitation, at a typical working distance of 350 mm for the hand-held device and 100 mm for the laparoscope. A single, sensitive, miniaturized color camera collects both fluorescence and white reflectance light. The use of a single imager eliminates image alignment and software overlay complexity. A novel filtering and illumination arrangement allows simultaneous detection of white reflectance and fluorescence emission from multiple dyes in real-time. We will present both fluorescence detection sensitivity modeling and practical performance data. We have demonstrated the efficiency and the advantages of the devices both pre-clinically and during live surgery on humans. Both the hand-held and the laparoscopic systems have proved to be reliable and beneficial in an ongoing clinical trial involving sentinel lymph node detection in gynecological cancers. We will show preliminary results using two clinically approved dyes, Methylene blue and indocyanine green. We anticipate that this technology can be integrated and routinely used in a larger variety of surgical procedures.

  4. Smartphone microendoscopy for high resolution fluorescence imaging

    CERN Document Server

    Hong, Xiangqian; Mugler, Dale H; Yu, Bing

    2016-01-01

    High resolution optical endoscopes are increasingly used in diagnosis of various medical conditions of internal organs, such as the gastrointestinal tracts, but they are too expensive for use in resource-poor settings. On the other hand, smartphones with high resolution cameras and Internet access have become more affordable, enabling them to diffuse into most rural areas and developing countries in the past decade. In this letter we describe a smartphone microendoscope that can take fluorescence images with a spatial resolution of 3.1 {\\mu}m. Images collected from ex vivo, in vitro and in vivo samples using the device are also presented. The compact and cost-effective smartphone microendoscope may be envisaged as a powerful tool for detecting pre-cancerous lesions of internal organs in low and middle income countries.

  5. Macro-/micro-environment-sensitive chemosensing and biological imaging.

    Science.gov (United States)

    Yang, Zhigang; Cao, Jianfang; He, Yanxia; Yang, Jung Ho; Kim, Taeyoung; Peng, Xiaojun; Kim, Jong Seung

    2014-07-01

    Environment-related parameters, including viscosity, polarity, temperature, hypoxia, and pH, play pivotal roles in controlling the physical or chemical behaviors of local molecules. In particular, in a biological environment, such factors predominantly determine the biological properties of the local environment or reflect corresponding status alterations. Abnormal changes in these factors would cause cellular malfunction or become a hallmark of the occurrence of severe diseases. Therefore, in recent years, they have increasingly attracted research interest from the fields of chemistry and biological chemistry. With the emergence of fluorescence sensing and imaging technology, several fluorescent chemosensors have been designed to respond to such parameters and to further map their distributions and variations in vitro/in vivo. In this work, we have reviewed a number of various environment-responsive chemosensors related to fluorescent recognition of viscosity, polarity, temperature, hypoxia, and pH that have been reported thus far.

  6. Tunable fluorescence from patterned silver nano-island arrays for sensitive sub-cell imaging

    International Nuclear Information System (INIS)

    Surface-enhanced fluorescence, a burgeoning technique in biological detection, provides largely enhanced fluorescence signal by exciting localized surfaces plasmons resonance with fluorescent dyes. Nanostructure and surroundings brings great impact on the emission signal, however, insufficient physics about the process limits further improvement on the nanostructure design. In this study, optical properties of Rhodamin-6G molecules on patterned silver nano-island arrays are tailored by precisely controlling the distance between the dyes and silver arrays. The fluorescence signal depends on the distance and the largest enhancement of 10 folds is achieved when the distance is 10 nm. The results are theoretically corroborated by finite difference time domain simulation and applied to cytoskeleton fluorescence imaging using phalloidin–fluorescein isothiocyanate. Our study provides insights into the physical mechanisms associated with the fluorescence enhancement and quenching, and our experiments suggest potential applications to high-sensitivity sub-cell imaging. (paper)

  7. Fluorescent resonance energy transfer based detection of biological contaminants through hybrid quantum dot-quencher interactions.

    Science.gov (United States)

    Ramadurai, D; Norton, E; Hale, J; Garland, J W; Stephenson, L D; Stroscio, M A; Sivananthan, S; Kumar, A

    2008-06-01

    A nanoscale sensor employing fluorescent resonance energy transfer interactions between fluorescent quantum dots (QDs) and organic quencher molecules can be used for the multiplexed detection of biological antigens in solution. Detection occurs when the antigens to be detected displace quencher-labelled inactivated (or dead) antigens of the same type attached to QD-antibody complexes through equilibrium reactions. This unquenches the QDs, allowing detection to take place through the observation of photoluminescence in solution or through the fluorescence imaging of unquenched QD complexes trapped on filter surfaces. Multiplexing can be accomplished by using several different sizes of QDs, with each size QD labelled with an antibody for a different antigen, providing the ability to detect several types of antigens or biological contaminants simultaneously in near real-time with high specificity and sensitivity.

  8. Quantitative fluorescence microscopy of macromolecules in gel and biological tissue

    Energy Technology Data Exchange (ETDEWEB)

    Tatarkova, Svetlana A [Department of Physics, University of Durham, Durham DH1 3LE (United Kingdom); School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom); Verma, Anita Kamra [School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom); Department of Zoology, K M College, University of Delhi, Delhi-110 007 (India); Berk, David A [School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom); Lloyd, Christopher J [School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom)

    2005-12-07

    Quantitative fluorescence microscopy provides valuable insight into drug delivery and pharmacokinetics. The technique is based on analysis of statistical fluctuations in fluorescence that arises as fluorophores pass through a small volume illuminated by a focused laser beam, and has been applied to measure particle motion and binding interactions in solutions, on surfaces and inside the cells. We examined the use of fluorescence correlation spectroscopy combined with a microscope (FCSM) to assess the transport of fluorescent beads and macromolecules in aqueous solutions, gels and living biological tissue. Obstructed diffusion of fluorescent beads in gels of various densities was tested to get a sensible estimate of diffusion in the interstitial tissue matrix consistent with previous reports. Fluorescently labelled liposomes as an artificial drug or gene carrying vehicles were used for pharmacokinetic tests of drug delivery in living tissue. The results indicate that FCS is an accurate and valuable tool for measuring the physical properties of gene vectors in vitro and for characterizing interactions with tissue in vivo.

  9. 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.

  10. Waveguide evanescent field fluorescence microscopy: Thin film fluorescence intensities and its application in cell biology

    Science.gov (United States)

    Hassanzadeh, Abdollah; Nitsche, Michael; Mittler, Silvia; Armstrong, Souzan; Dixon, Jeff; Langbein, Uwe

    2008-06-01

    We demonstrate an inexpensive alternative to total internal reflection fluorescence microscopy. A method for imaging ultrathin films and living cells located on waveguides—illuminated with their evanescent fields—is introduced. An extensive analysis of ion-exchanged waveguides focusing on their application as microscopy substrates for studying interfacial phenomena is presented. Experimental results are in excellent agreement with the simulations. As an application osteoblasts (bone matrix forming cells) and ultrathin Langmuir-Blodgett films were imaged. The fluorescence intensity has been used to determine the cell attachment.

  11. Dual-modality, fluorescent, PLGA encapsulated bismuth nanoparticles for molecular and cellular fluorescence imaging and computed tomography

    Science.gov (United States)

    Swy, Eric R.; Schwartz-Duval, Aaron S.; Shuboni, Dorela D.; Latourette, Matthew T.; Mallet, Christiane L.; Parys, Maciej; Cormode, David P.; Shapiro, Erik M.

    2014-10-01

    Reports of molecular and cellular imaging using computed tomography (CT) are rapidly increasing. Many of these reports use gold nanoparticles. Bismuth has similar CT contrast properties to gold while being approximately 1000-fold less expensive. Herein we report the design, fabrication, characterization, and CT and fluorescence imaging properties of a novel, dual modality, fluorescent, polymer encapsulated bismuth nanoparticle construct for computed tomography and fluorescence imaging. We also report on cellular internalization and preliminary in vitro and in vivo toxicity effects of these constructs. 40 nm bismuth(0) nanocrystals were synthesized and encapsulated within 120 nm Poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles by oil-in-water emulsion methodologies. Coumarin-6 was co-encapsulated to impart fluorescence. High encapsulation efficiency was achieved ~70% bismuth w/w. Particles were shown to internalize within cells following incubation in culture. Bismuth nanocrystals and PLGA encapsulated bismuth nanoparticles exhibited >90% and >70% degradation, respectively, within 24 hours in acidic, lysosomal environment mimicking media and both remained nearly 100% stable in cytosolic/extracellular fluid mimicking media. μCT and clinical CT imaging was performed at multiple X-ray tube voltages to measure concentration dependent attenuation rates as well as to establish the ability to detect the nanoparticles in an ex vivo biological sample. Dual fluorescence and CT imaging is demonstrated as well. In vivo toxicity studies in rats revealed neither clinically apparent side effects nor major alterations in serum chemistry and hematology parameters. Calculations on minimal detection requirements for in vivo targeted imaging using these nanoparticles are presented. Indeed, our results indicate that these nanoparticles may serve as a platform for sensitive and specific targeted molecular CT and fluorescence imaging.Reports of molecular and cellular imaging using

  12. In vivo cell biology of cancer cells visualized with fluorescent proteins.

    Science.gov (United States)

    Hoffman, Robert M

    2005-01-01

    This chapter describes a new cell biology where the behavior of individual cells can be visualized in the living animal. Previously it has been demonstrated that fluorescent proteins can be used for whole-body imaging of metastatic tumor growth, bacterial infection, and gene expression. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of tumor-stroma interactions and especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts, and macrophages. Another example is the color coding of cells with RFP or GFP such that both cell types can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem cell marker nestin drive GFP expression enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Highly elongated cancer cells in capillaries in living mice were observed within skin flaps. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells in the capillaries elongated to fit the width of these vessels. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

  13. Waveguide evanescent field fluorescence microscopy & its application in cell biology

    Science.gov (United States)

    Hassanzadeh, Abdollah

    There are many powerful microscopy technologies available for the investigation of bulk materials as well as for thin film samples. Nevertheless, for imaging an interface, especially live cells on a substrate and ultra thin-films, only Total Internal Reflection Fluorescence (TIRF) microscopy is available. This TIRF microscopy allows imaging without interference of the bulk. Various approaches are employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. TIRF microscopy is an evanescent field based microscopy. In this method, fluorescent dyes are only excited within an evanescent field: roughly within 100 nm above a glass coverslip. This will allow imaging surface and interfacial issues of the glass coverslip and an adjacent material. Waveguide evanescent field fluorescence (WEFF) microscopy is a new development for imaging cell-substrate interactions in real time and in vitro. It is an alternative to TIRF microscopy. In this method the light is coupled into a waveguide via an optical grating. The coupled light propagates as a waveguide mode and exhibits an evanescent field on top of the waveguide. This can be used as a surface-bound illumination source to excite fluorophores. This evanescent field serves as an extremely powerful tool for quality control of thin films, to study cell-substrate contacts, and investigating the effect of external agents and drugs on the cell-substrate interaction in real time and in vitro. This new method has been established and optimized to minimize non-uniformity, scattering and photo bleaching issues. Visualizing and quantifying of the cell-substrates and solid thin films have been carried out by WEFF microscopy. The images of the cell-substrate interface

  14. Red emitting neutral fluorescent glycoconjugates for membrane optical imaging.

    Science.gov (United States)

    Redon, Sébastien; Massin, Julien; Pouvreau, Sandrine; De Meulenaere, Evelien; Clays, Koen; Queneau, Yves; Andraud, Chantal; Girard-Egrot, Agnès; Bretonnière, Yann; Chambert, Stéphane

    2014-04-16

    A family of neutral fluorescent probes was developed, mimicking the overall structure of natural glycolipids in order to optimize their membrane affinity. Nonreducing commercially available di- or trisaccharidic structures were connected to a push-pull chromophore based on dicyanoisophorone electron-accepting group, which proved to fluoresce in the red region with a very large Stokes shift. This straightforward synthetic strategy brought structural variations to a series of probes, which were studied for their optical, biophysical, and biological properties. The insertion properties of the different probes into membranes were evaluated on a model system using the Langmuir monolayer balance technique. Confocal fluorescence microscopy performed on muscle cells showed completely different localizations and loading efficiencies depending on the structure of the probes. When compared to the commercially available ANEPPS, a family of commonly used membrane imaging dyes, the most efficient probes showed a similar brightness, but a sharper pattern was observed. According to this study, compounds bearing one chromophore, a limited size of the carbohydrate moiety, and an overall rod-like shape gave the best results.

  15. Combination of fluorescence imaging and local spectrophotometry in fluorescence diagnostics of early cancer of larynx and bronchi

    International Nuclear Information System (INIS)

    The results of comparative studies of autofluorescence and 5-ALA-induced fluorescence of protoporphyrin IX, used in the diagnostics of early cancer of larynx and bronchi, are presented. The autofluorescence and 5-ALA-induced fluorescence images of larynx and bronchial tissues are analysed during the endoscopic study. The method of local spectrophotometry is used to verify findings obtained from fluorescence images. It is shown that such a combined approach can be efficiently used to improve the diagnostics of precancer and early cancer, to detect a primary multiple tumours, as well as for the diagnostics of a residual tumour or an early recurrence after the endoscopic, surgery or X-ray treatment. The developed approach allows one to minimise the number of false-positive results and to reduce the number of biopsies, which are commonly used in the white-light bronchoscopy search for occult cancerous loci. (laser biology and medicine)

  16. Fluorescence Imaging Study of Impinging Underexpanded Jets

    Science.gov (United States)

    Inman, Jennifer A.; Danehy, Paul M.; Nowak, Robert J.; Alderfer, David W.

    2008-01-01

    An experiment was designed to create a simplified simulation of the flow through a hole in the surface of a hypersonic aerospace vehicle and the subsequent impingement of the flow on internal structures. In addition to planar laser-induced fluorescence (PLIF) flow visualization, pressure measurements were recorded on the surface of an impingement target. The PLIF images themselves provide quantitative spatial information about structure of the impinging jets. The images also help in the interpretation of impingement surface pressure profiles by highlighting the flow structures corresponding to distinctive features of these pressure profiles. The shape of the pressure distribution along the impingement surface was found to be double-peaked in cases with a sufficiently high jet-exit-to-ambient pressure ratio so as to have a Mach disk, as well as in cases where a flow feature called a recirculation bubble formed at the impingement surface. The formation of a recirculation bubble was in turn found to depend very sensitively upon the jet-exit-to-ambient pressure ratio. The pressure measured at the surface was typically less than half the nozzle plenum pressure at low jet pressure ratios and decreased with increasing jet pressure ratios. Angled impingement cases showed that impingement at a 60deg angle resulted in up to a factor of three increase in maximum pressure at the plate compared to normal incidence.

  17. Fabrication of SERS-fluorescence dual modal nanoprobes and application to multiplex cancer cell imaging

    Science.gov (United States)

    Lee, Sangyeop; Chon, Hyangah; Yoon, Soo-Young; Lee, Eun Kyu; Chang, Soo-Ik; Lim, Dong Woo; Choo, Jaebum

    2011-12-01

    We report a highly sensitive optical imaging technology using surface-enhanced Raman scattering (SERS)-fluorescence dual modal nanoprobes (DMNPs). Fluorescence microscopy is a well-known imaging technique that shows specific protein distributions within cells. However, most currently available fluorescent organic dyes have relatively weak emission intensities and are rapidly photo-bleached. Thus more sensitive and stable probes are needed. In this work we develop DMNPs, which can be used for both SERS and fluorescence detection. SERS detection is a powerful technique that allows ultrasensitive chemical or biochemical analysis through unlimited multiplexing and single molecule sensitivity. Combining advantages of fluorescence and SERS allows these dual modal nanostructures to be used as powerful probes for novel biomedical imaging. In this work, the fabrication and characterization of the SERS-fluorescence DMNPs and application to biological imaging were investigated using markers CD24 and CD44, which are co-expressed in MDA-MB-231 breast cancer cells, as a model system. SERS imaging with DMNPs was found to be a powerful tool to determine the co-localization of CD24 and CD44 in the cell.We report a highly sensitive optical imaging technology using surface-enhanced Raman scattering (SERS)-fluorescence dual modal nanoprobes (DMNPs). Fluorescence microscopy is a well-known imaging technique that shows specific protein distributions within cells. However, most currently available fluorescent organic dyes have relatively weak emission intensities and are rapidly photo-bleached. Thus more sensitive and stable probes are needed. In this work we develop DMNPs, which can be used for both SERS and fluorescence detection. SERS detection is a powerful technique that allows ultrasensitive chemical or biochemical analysis through unlimited multiplexing and single molecule sensitivity. Combining advantages of fluorescence and SERS allows these dual modal nanostructures to be used

  18. Reflectance and Fluorescence Spectral Recovery via Actively Lit RGB Images.

    Science.gov (United States)

    Fu, Ying; Lam, Antony; Sato, Imari; Okabe, Takahiro; Sato, Yoichi

    2016-07-01

    In recent years, fluorescence analysis of scenes has received attention in computer vision. Fluorescence can provide additional information about scenes, and has been used in applications such as camera spectral sensitivity estimation, 3D reconstruction, and color relighting. In particular, hyperspectral images of reflective-fluorescent scenes provide a rich amount of data. However, due to the complex nature of fluorescence, hyperspectral imaging methods rely on specialized equipment such as hyperspectral cameras and specialized illuminants. In this paper, we propose a more practical approach to hyperspectral imaging of reflective-fluorescent scenes using only a conventional RGB camera and varied colored illuminants. The key idea of our approach is to exploit a unique property of fluorescence: the chromaticity of fluorescent emissions are invariant under different illuminants. This allows us to robustly estimate spectral reflectance and fluorescent emission chromaticity. We then show that given the spectral reflectance and fluorescent chromaticity, the fluorescence absorption and emission spectra can also be estimated. We demonstrate in results that all scene spectra can be accurately estimated from RGB images. Finally, we show that our method can be used to accurately relight scenes under novel lighting. PMID:27295456

  19. Semiconductor Quantum Rods as Single Molecule FluorescentBiological Labels

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Aihua; Gu, Weiwei; Boussert, Benjamine; Koski, Kristie; Gerion, Daniele; Manna, Liberato; Le Gros, Mark; Larabell, Carolyn; Alivisatos, A. Paul

    2006-05-29

    In recent years, semiconductor quantum dots have beenapplied with great advantage in a wide range of biological imagingapplications. The continuing developments in the synthesis of nanoscalematerials and specifically in the area of colloidal semiconductornanocrystals have created an opportunity to generate a next generation ofbiological labels with complementary or in some cases enhanced propertiescompared to colloidal quantum dots. In this paper, we report thedevelopment of rod shaped semiconductor nanocrystals (quantum rods) asnew fluorescent biological labels. We have engineered biocompatiblequantum rods by surface silanization and have applied them fornon-specific cell tracking as well as specific cellular targeting. Theproperties of quantum rods as demonstrated here are enhanced sensitivityand greater resistance for degradation as compared to quantum dots.Quantum rods have many potential applications as biological labels insituations where their properties offer advantages over quantumdots.

  20. Biological detection and tagging using tailorable, reactive, highly fluorescent chemosensors.

    Energy Technology Data Exchange (ETDEWEB)

    Shepodd, Timothy J.; Zifer, Thomas; McElhanon, James Ross; Rahn, Larry A.

    2006-11-01

    This program was focused on the development of a fluorogenic chemosensor family that could tuned for reaction with electrophilic (e.g. chemical species, toxins) and nucleophilic (e.g. proteins and other biological molecules) species. Our chemosensor approach utilized the fluorescent properties of well-known berberine-type alkaloids. In situ chemosensor reaction with a target species transformed two out-of-plane, weakly conjugated, short-wavelength chromophores into one rigid, planar, conjugated, chromophore with strong long wavelength fluorescence (530-560 nm,) and large Stokes shift (100-180 nm). The chemosensor was activated with an isourea group which allowed for reaction with carboxylic acid moieties found in amino acids.

  1. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Science.gov (United States)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  2. Functionalization of hydroxyl terminated polybutadiene with biologically active fluorescent molecule

    Indian Academy of Sciences (India)

    R Murali Sankar; Subhadeep Saha; K Seeni Meera; Tushar Jana

    2009-10-01

    A biologically active molecule, 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (CBDT), has been covalently attached at the terminal carbon atoms of the hydroxyl terminated polybutadiene (HTPB) backbone. The modification of HTPB backbone by CBDT molecule does not affect the unique physico-chemical properties such as fluidity, hydroxyl value and microstructure of the parent HTPB. The formation of hydrogen bonding between the terminal hydroxyl groups and the nitrogen atoms of triazine moiety is the driving force for the terminal attachment chemistry. The functionalized HTPB (HTPB–CBDT) shows a strong fluorescence emission at 385 nm.

  3. Tip-enhanced fluorescence imaging of quantum dots

    OpenAIRE

    Huang, F.M.; Festy, F; Richards, D. R.

    2005-01-01

    We have imaged the fluorescence from a single quantum dot cluster using an apertureless scanning near-field optical microscope. When a sharp gold tip is brought within a few nanometers from the sample surface, the resulting enhancement in quantum dot fluorescence in the vicinity of the tip leads to a resolution of about 60 nm. We determine this enhancement of the fluorescence to be about fourfold in magnitude, which is consistent with the value expected as a result of competition between fluo...

  4. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    Science.gov (United States)

    Shevchuk, A. I.; Novak, P.; Velazquez, M. A.; Fleming, T. P.; Korchev, Y. E.

    2013-09-01

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development.

  5. Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

    OpenAIRE

    Guo, Hengchang; Aleyasin, Hossein; Howard, Scott S.; Dickinson, Bryan C; Lin, Vivian S.; Haskew-Layton, Renee E.; Xu, Chris; Chen, Yu; Ratan, Rajiv R.

    2013-01-01

    Abstract. We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H2O2) production in brain cells. For selective imaging of H2O2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H2O2 and mitochondria peroxy yellow 1 detects mitochondrial H2O2. Two-photon absorption cross sections for th...

  6. Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

    OpenAIRE

    Guo, Hengchang; Aleyasin, Hossein; Howard, Scott S.; Dickinson, Bryan C; Lin, Vivian S.; Haskew-Layton, Renee E.; Xu, Chris; Chen, Yu; Ratan, Rajiv R.

    2013-01-01

    Abstract. We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide ( H 2 O 2 ) production in brain cells. For selective imaging of H 2 O 2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H 2 O 2 and mitochondria peroxy yellow 1 detects mitochondrial H 2 O 2 . Two-photon absorption cross ...

  7. Analysis of human aorta using fluorescence lifetime imaging microscopy (FLIM)

    Science.gov (United States)

    Vieira-Damiani, Gislaine; Adur, J.; Ferro, D. P.; Adam, R. L.; Pelegati, V.; Thomáz, A.; Cesar, C. L.; Metze, K.

    2012-03-01

    The use of photonics has improved our understanding of biologic phenomena. For the study of the normal and pathologic architecture of the aorta the use of Two-Photon Excited Fluorescence (TPEF) and Second Harmonic Generation showed interesting details of morphologic changes of the elastin-collagen architecture during aging or development of hypertension in previous studies. In this investigation we tried to apply fluorescence lifetime imaging (FLIM) for the morphologic analysis of human aortas. The aim of our study was to use FLIM in non-stained formalin-fixed and paraffin-embedded samples of the aorta ascendants in hypertensive and normotensive patients of various ages, examining two different topographical regions. The FLIM-spectra of collagen and elastic fibers were clearly distinguishable, thus permitting an exact analysis of unstained material on the microscopic level. Moreover the FLIM spectrum of elastic fibers revealed variations between individual cases, which indicate modifications on a molecular level and might be related to FLIM age or diseases states and reflect modifications on a molecular level.

  8. Detection of rheumatoid arthritis in humans by fluorescence imaging

    Science.gov (United States)

    Ebert, Bernd; Dziekan, Thomas; Weissbach, Carmen; Mahler, Marianne; Schirner, Michael; Berliner, Birgitt; Bauer, Daniel; Voigt, Jan; Berliner, Michael; Bahner, Malte L.; Macdonald, Rainer

    2010-02-01

    The blood pool agent indo-cyanine green (ICG) has been investigated in a prospective clinical study for detection of rheumatoid arthritis using fluorescence imaging. Temporal behavior as well as spatial distribution of fluorescence intensity are suited to differentiate healthy and inflamed finger joints after i.v. injection of an ICG bolus.

  9. Cell imaging by transient fluorescence detected infrared microscopy

    Science.gov (United States)

    Ohmori, Tsutomu; Sakai, Makoto; Ishihara, Miya; Kikuchi, Makoto; Fujii, Masaaki

    2008-02-01

    Transient fluorescence detected infrared (TFD-IR) microscopy was developed to overcome the diffraction limit of infrared (IR) light without a near-field system. This microscopic technique is based on TFD-IR spectroscopy, which converts information on IR absorption to fluorescence intensity by further electronic excitation of vibrationally excited molecules by a probing UV/visible light. Roots of Arabidopsis thaliana and living A549 cells with fluorescent dyes were chosen as samples. In the measurements using the TFD-IR microscope, tunable IR picosecond laser pulses were used in the wavelength range from 2700 to 3700 nm, corresponding to CH, NH, and OH stretching modes. Fluorescence images of the root cells of A. thaliana by the TFD-IR scheme were obtained with super-resolution compared with the resolution of conventional IR microscopy. The resolution is estimated to be less than 2.6 μm by fitting of a gaussian function. However, the TFD-IR images were dominated mainly by the fluorescent dyes because they were almost the same as a conventional fluorescence image. To investigate other contributions hidden by that of fluorescent dyes, we plotted the fluorescence intensity in several 5 μm squares at various IR wavelengths, called a TFD-IR spectrum. For root cells of A. thaliana, the TFD-IR spectra show shapes similar to those of a conventional IR absorption spectrum of the fluorescent dye. Therefore, the TFD-IR images are not due to the cellular components. For an A549 cell, the TFD-IR spectra were different from a conventional IR absorption spectrum of fluorescent dyes in the wavelength region shorter than 3100 nm. We speculate that the spectral difference is due to the cellular components, possibly assigned to the combination band related to amino groups of cellular components bonded covalently to the fluorescent dyes.

  10. Easily Accessible and Highly Selective "Turn-on" Fluorescent Sensor for Imaging Cadmium in Living Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; ZHANG Ying-mu; LI Yao-xian; ZHAO Qing

    2013-01-01

    A new schiff base of phenothiazine derivative was designed for ratiometric sensing of Cd2+ selectively.Upon the addition of Cd2+ to the solution of phenothiazine derivative,the fluorescence intensity of it was enhanced in a linear fashion and the maximum fluorescence peak exhibited a blue shift from 575 nm to 525 nm.This ratiometric fluorescent sensor displays a very high sensitivity(detection limits were 0.34 and 1.0 μmol/L of Cd2+ using the visual fluorescence changes and UV-Vis changes,respectively),a rapid response time(<10 s) and high selectivity for Cd2+ over other transition metal ions.Moreover,the living cells image experiments also demonstrate the value of the sensor in fluorescent visualization of Cd2+ in the environmental and biological systems.

  11. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel;

    2013-01-01

    Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove....../reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame....... We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size asthe individual image. We have also reconstructed the image using a 3 fold larger...

  12. Fluorogen-based reporters for fluorescence imaging: a review

    Science.gov (United States)

    Jullien, Ludovic; Gautier, Arnaud

    2015-12-01

    Fluorescence bioimaging has recently jumped into a new area of spatiotemporal resolution and sensitivity thanks to synergistic advances in both optical physics and probe/biosensor design. This review focuses on the recent development of genetically encodable fluorescent reporters that bind endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activate their fluorescence. We highlight the innovative engineering and design that gave rise to these new natural and synthetic fluorescent reporters, and describe some of the emerging applications in imaging and biosensing.

  13. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    OpenAIRE

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human...

  14. 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.

  15. Shaping Small Bioactive Molecules to Untangle Their Biological Function: A Focus on Fluorescent Plant Hormones.

    Science.gov (United States)

    Lace, Beatrice; Prandi, Cristina

    2016-08-01

    Modern biology overlaps with chemistry in explaining the structure and function of all cellular processes at the molecular level. Plant hormone research is perfectly located at the interface between these two disciplines, taking advantage of synthetic and computational chemistry as a tool to decipher the complex biological mechanisms regulating the action of plant hormones. These small signaling molecules regulate a wide range of developmental processes, adapting plant growth to ever changing environmental conditions. The synthesis of small bioactive molecules mimicking the activity of endogenous hormones allows us to unveil many molecular features of their functioning, giving rise to a new field, plant chemical biology. In this framework, fluorescence labeling of plant hormones is emerging as a successful strategy to track the fate of these challenging molecules inside living organisms. Thanks to the increasing availability of new fluorescent probes as well as advanced and innovative imaging technologies, we are now in a position to investigate many of the dynamic mechanisms through which plant hormones exert their action. Such a deep and detailed comprehension is mandatory for the development of new green technologies for practical applications. In this review, we summarize the results obtained so far concerning the fluorescent labeling of plant hormones, highlighting the basic steps leading to the design and synthesis of these compelling molecular tools and their applications. PMID:27378726

  16. FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

    OpenAIRE

    Fontenete, S.; Carvalho, D; Lourenço, Anália; N.Guimarães; Madureira, P.; Figueiredo, C.; Azevedo, N. F.

    2016-01-01

    Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing sof...

  17. Imaging chemical extraction by polymer inclusion membranes using fluorescence microscopy

    International Nuclear Information System (INIS)

    Polymer inclusion membranes (PIMs) transport chemicals between bodies of liquid by simultaneously performing chemical extraction and back-extraction. The internal chemical and physical mechanisms by which this transport occurs are, however, poorly understood. Also, some PIMs, which are otherwise optimal for their task, age and lose function after only days, limiting their feasibility for industrial upscaling. Through the application of fluorescence imaging methods we are able for the first time to see where chemical extraction occurs in the membrane. Extraction of fluorescein from solution by PIMs demonstrates inhomogeneities that do not correlate to surface morphology. Fluorescence lifetime imaging demonstrates that regions of increased extraction have distinctly different fluorescence lifetimes to that of the surrounding PIM indicating localized chemical environments, and this is observed to change with membrane age. Fluorescence imaging is shown to allow probing and novel understanding of PIM internal chemical morphology. (paper)

  18. Imaging chemical extraction by polymer inclusion membranes using fluorescence microscopy

    Science.gov (United States)

    Henderson, Clare A.; Nagul, Edward A.; Cattrall, Robert W.; Kolev, Spas D.; Smith, Trevor A.

    2014-06-01

    Polymer inclusion membranes (PIMs) transport chemicals between bodies of liquid by simultaneously performing chemical extraction and back-extraction. The internal chemical and physical mechanisms by which this transport occurs are, however, poorly understood. Also, some PIMs, which are otherwise optimal for their task, age and lose function after only days, limiting their feasibility for industrial upscaling. Through the application of fluorescence imaging methods we are able for the first time to see where chemical extraction occurs in the membrane. Extraction of fluorescein from solution by PIMs demonstrates inhomogeneities that do not correlate to surface morphology. Fluorescence lifetime imaging demonstrates that regions of increased extraction have distinctly different fluorescence lifetimes to that of the surrounding PIM indicating localized chemical environments, and this is observed to change with membrane age. Fluorescence imaging is shown to allow probing and novel understanding of PIM internal chemical morphology.

  19. 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.

  20. Hyperspectral fluorescence imaging with multi wavelength LED excitation

    Science.gov (United States)

    Luthman, A. Siri; Dumitru, Sebastian; Quirós-Gonzalez, Isabel; Bohndiek, Sarah E.

    2016-04-01

    Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.

  1. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    Science.gov (United States)

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

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

    International Nuclear Information System (INIS)

    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

  3. A dual oxygenation and fluorescence imaging platform for reconstructive surgery

    Science.gov (United States)

    Ashitate, Yoshitomo; Nguyen, John N.; Venugopal, Vivek; Stockdale, Alan; Neacsu, Florin; Kettenring, Frank; Lee, Bernard T.; Frangioni, John V.; Gioux, Sylvain

    2013-03-01

    There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively, leading to a large number of failures, patient morbidity, and increased healthcare costs. Because near-infrared (NIR) optical imaging is safe, noncontact, inexpensive, and can provide relatively deep information (several mm), it offers unparalleled capabilities for providing image guidance during surgery. These capabilities are well illustrated through the clinical translation of fluorescence imaging during oncologic surgery. In this work, we introduce a novel imaging platform that combines two complementary NIR optical modalities: oxygenation imaging and fluorescence imaging. We validated this platform during facial reconstructive surgery on large animals approaching the size of humans. We demonstrate that NIR fluorescence imaging provides identification of perforator arteries, assesses arterial perfusion, and can detect thrombosis, while oxygenation imaging permits the passive monitoring of tissue vital status, as well as the detection and origin of vascular compromise simultaneously. Together, the two methods provide a comprehensive approach to identifying problems and intervening in real time during surgery before irreparable damage occurs. Taken together, this novel platform provides fully integrated and clinically friendly endogenous and exogenous NIR optical imaging for improved image-guided intervention during surgery.

  4. Fluorescence lidar multi-color imaging of vegetation

    Science.gov (United States)

    Johansson, J.; Wallinder, E.; Edner, H.; Svanberg, S.

    1992-01-01

    Multi-color imaging of vegetation fluorescence following laser excitation is reported for distances of 50 m. A mobile laser radar system equipped with a Nd:YAG laser transmitter and a 40 cm diameter telescope was used. Image processing allows extraction of information related to the physiological status of the vegetation and might prove useful in forest decline research.

  5. Fluorescence lifetime imaging of oxygen in dental biofilm

    Science.gov (United States)

    Gerritsen, Hans C.; de Grauw, Cees J.

    2000-12-01

    Dental biofilm consists of micro-colonies of bacteria embedded in a matrix of polysaccharides and salivary proteins. pH and oxygen concentration are of great importance in dental biofilm. Both can be measured using fluorescence techniques. The imaging of dental biofilm is complicated by the thickness of the biofilms that can be up to several hundred micrometers thick. Here, we employed a combination of two-photon excitation microscopy with fluorescence lifetime imaging to quantify the oxygen concentration in dental biofilm. Collisional quenching of fluorescent probes by molecular oxygen leads to a reduction of the fluorescence lifetime of the probe. We employed this mechanism to measure the oxygen concentration distribution in dental biofilm by means of fluorescence lifetime imaging. Here, TRIS Ruthenium chloride hydrate was used as an oxygen probe. A calibration procedure on buffers was use to measure the lifetime response of this Ruthenium probe. The results are in agreement with the Stern-Volmer equation. A linear relation was found between the ratio of the unquenched and the quenched lifetime and the oxygen concentration. The biofilm fluorescence lifetime imaging results show a strong oxygen gradient at the buffer - biofilm interface and the average oxygen concentration in the biofilm amounted to 50 μM.

  6. Photoswitchable Spiropyran Dyads for Biological Imaging.

    Science.gov (United States)

    Xiong, Yaoyao; Rivera-Fuentes, Pablo; Sezgin, Erdinc; Vargas Jentzsch, Andreas; Eggeling, Christian; Anderson, Harry L

    2016-08-01

    The synthesis of a small-molecule dyad consisting of a far-red-emitting silicon rhodamine dye that is covalently linked to a photochromic spironaphthothiopyran unit, which serves as a photoswitchable quencher, is reported. This system can be switched reversibly between the fluorescent and nonfluorescent states using visible light at wavelengths of 405 and 630 nm, respectively, and it works effectively in aqueous solution. Live-cell imaging demonstrates that this dyad has several desirable features, including excellent membrane permeability, fast and reversible modulation of fluorescence by visible light, and good contrast between the bright and dark states. PMID:27456166

  7. 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.

  8. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

    NARCIS (Netherlands)

    Zhao, Q.; Schelen, B.; Schouten, R., et al.

    2012-01-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device des

  9. Small BODIPY Probes for Combined Dual (19) F MRI and Fluorescence Imaging.

    Science.gov (United States)

    Huynh, Anh Minh; Müller, Andreas; Kessler, Sonja M; Henrikus, Sarah; Hoffmann, Caroline; Kiemer, Alexandra K; Bücker, Arno; Jung, Gregor

    2016-07-19

    The combination of the two complementary imaging modalities (19) F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual (19) F MRI-fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T1 and the transverse relaxation times T2 , both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual (19) F MRI-fluorescence agent was also successfully imaged in a mouse post-mortem within a 9.4 T small-animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing. PMID:27347843

  10. 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.

  11. 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.

  12. Quantitative sensing of microviscosity in protocells and amyloid materials using fluorescence lifetime imaging of molecular rotors

    Science.gov (United States)

    Thompson, Alex J.; Tang, T.-Y. Dora; Herling, Therese W.; Che Hak, C. Rohaida; Mann, Stephen; Knowles, Tuomas P. J.; Kuimova, Marina K.

    2014-03-01

    Molecular rotors are fluorophores that have a fluorescence quantum yield that depends upon intermolecular rotation. The fluorescence quantum yield, intensity and lifetime of molecular rotors all vary as functions of viscosity, as high viscosities inhibit intermolecular rotation and cause an increase in the non-radiative decay rate. As such, molecular rotors can be used to probe viscosity on microscopic scales. Here, we apply fluorescence lifetime imaging microscopy (FLIM) to measure the fluorescence lifetimes of three different molecular rotors, in order to determine the microscopic viscosity in two model systems with significant biological interest. First, the constituents of a novel protocell - a model of a prebiotic cell - were studied using the molecular rotors BODIPY C10 and kiton red. Second, amyloid formation was investigated using the molecular rotor Cy3.

  13. Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence

    Science.gov (United States)

    Su, Li-Xia; Lou, Qing; Jiao, Zhen; Shan, Chong-Xin

    2016-09-01

    Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp2-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.

  14. A portable fluorescence microscopic imaging system for cholecystectomy

    Science.gov (United States)

    Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

    2016-03-01

    In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

  15. Imaging of a targeted PDT drug with fluorescence tomography

    Science.gov (United States)

    Muffoletto, Dan; Gupta, Anurag; Xu, Zhiqiang; Mahrer, Chris; Bauer, Gretchen; Galas, Scott; Pandey, Ravindra K.; Sunar, Ulas

    2009-02-01

    We constructed a whole-body fluorescence tomography instrument to monitor novel bifunctional phototherapeutic drugs (e.g., HPPH-Cyanine dye conjugate) in small animals. The instrument allows dense source and detector sampling with a fast galvo scanner and a CCD detector for improved resolution and sensitivity (Patwardhan et al., 2005). Here we report tissue phantom measurements to evaluate the imaging performance with a newly constructed tomography instrument. Phantom measurements showed that strong fluorescence generated by HPPH-Cyanine dye (HPPH-CD), having high fluorescence quantum yield and long wavelength fluorescence emission, allowed deep tissue imaging. We also report in vivo fluorescence measurements of the conjugate in Nude mice bearing A549 human non-small cell lung carcinoma (NSCLC) tumors at 24 hr post injection to evaluate tumor detection ability of the conjugate. Our results indicate that the HPPH-CD shows preferential uptake in tumors compared to surrounding normal tissue at 24 hr post injection. This study demonstrates a potential use of HPPH-CD in detection (fluorescence imaging) and treatment (PDT) of deeply seated tumors.

  16. Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

    Directory of Open Access Journals (Sweden)

    Sean C Warren

    Full Text Available Fluorescence lifetime imaging (FLIM is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset. This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis

  17. A novel fluorescent label based on biological fluores-cent nanoparticles and its application in cell recognition

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Uniform-sized fluorescent nanoparticles have been prepared by employing silica as the shell and a highly luminescent dye complex of ruthenium ion and bipyridyl, tris(2,2′-bipyridyl) dichlororuthenium(Ⅱ) hexahydrate as the core of the nanoparticles. A novel fluorescent label method is proposed, which is based on the biological fluorescent nanoparticles on the foundation of nanotechnology, biotechnology and fluorescent label technology. In comparison with the conventional fluorophores as fluorescent labels such as fluorescein isothiocyanate (FITC) label, this new label shows more superiority in photochemical stability, detection sensitivity and application scope for the biomedicine research. SmIgG+ B lymphocytes isolated from the circulating blood of human beings can be easily recognized by using this new fluorescent label.

  18. Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Víctor M Campa

    Full Text Available The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.

  19. A Ratiometric Fluorescence Imaging System for Surgical Guidance

    Directory of Open Access Journals (Sweden)

    Brian C. Wilson

    2008-10-01

    Full Text Available A 3-chip CCD imaging system has been developed for quantitative in vivo fluorescence imaging. This incorporates a ratiometric algorithm to correct for the effects of tissue optical absorption and scattering, imaging “geometry” and tissue autofluorescence background. The performance was characterized, and the algorithm was validated in tissue-simulating optical phantoms for quantitative measurement of the fluorescent molecule protoporphyrin IX (PpIX. The technical feasibility to use this system for fluorescence-guided surgical resection of malignant brain tumor tissue was assessed in an animal model in which PpIX was induced exogenously in the tumor cells by systemic administration of aminolevulinic acid (ALA.

  20. 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.

  1. Coded Aperture Imaging for Fluorescent X-rays-Biomedical Applications

    Energy Technology Data Exchange (ETDEWEB)

    Haboub, Abdel; MacDowell, Alastair; Marchesini, Stefano; Parkinson, Dilworth

    2013-06-01

    Employing a coded aperture pattern in front of a charge couple device pixilated detector (CCD) allows for imaging of fluorescent x-rays (6-25KeV) being emitted from samples irradiated with x-rays. Coded apertures encode the angular direction of x-rays and allow for a large Numerical Aperture x- ray imaging system. The algorithm to develop the self-supported coded aperture pattern of the Non Two Holes Touching (NTHT) pattern was developed. The algorithms to reconstruct the x-ray image from the encoded pattern recorded were developed by means of modeling and confirmed by experiments. Samples were irradiated by monochromatic synchrotron x-ray radiation, and fluorescent x-rays from several different test metal samples were imaged through the newly developed coded aperture imaging system. By choice of the exciting energy the different metals were speciated.

  2. Fluorescence Lifetime Imaging of Quantum Dot Labeled DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Jonathan G. Terry

    2009-04-01

    Full Text Available Quantum dot (QD labeling combined with fluorescence lifetime imaging microscopy is proposed as a powerful transduction technique for the detection of DNA hybridization events. Fluorescence lifetime analysis of DNA microarray spots of hybridized QD labeled target indicated a characteristic lifetime value of 18.8 ns, compared to 13.3 ns obtained for spots of free QD solution, revealing that QD labels are sensitive to the spot microenvironment. Additionally, time gated detection was shown to improve the microarray image contrast ratio by 1.8, achieving femtomolar target sensitivity. Finally, lifetime multiplexing based on Qdot525 and Alexa430 was demonstrated using a single excitation-detection readout channel.

  3. Color-matched esophagus phantom for fluorescent imaging

    Science.gov (United States)

    Yang, Chenying; Hou, Vivian; Nelson, Leonard Y.; Seibel, Eric J.

    2013-02-01

    We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed "hot-spot" locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.

  4. Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish

    Science.gov (United States)

    Choi, Seon-Ae; Park, Chul Soon; Kwon, Oh Seok; Giong, Hoi-Khoanh; Lee, Jeong-Soo; Ha, Tai Hwan; Lee, Chang-Soo

    2016-05-01

    Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic ‘naphthalene’ moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

  5. FluoSTIC: miniaturized fluorescence image-guided surgery system

    Science.gov (United States)

    Gioux, Sylvain; Coutard, Jean-Guillaume; Berger, Michel; Grateau, Henri; Josserand, Véronique; Keramidas, Michelle; Righini, Christian; Coll, Jean-Luc; Dinten, Jean-Marc

    2012-10-01

    Over the last few years, near-infrared (NIR) fluorescence imaging has witnessed rapid growth and is already used in clinical trials for various procedures. However, most clinically compatible imaging systems are optimized for large, open-surgery procedures. Such systems cannot be employed during head and neck oncologic surgeries because the system is not able to image inside deep cavities or allow the surgeon access to certain tumors due to the large footprint of the system. We describe a miniaturized, low-cost, NIR fluorescence system optimized for clinical use during oral oncologic surgeries. The system, termed FluoSTIC, employs a miniature, high-quality, consumer-grade lipstick camera for collecting fluorescence light and a novel custom circular optical fiber array for illumination that combines both white light and NIR excitation. FluoSTIC maintains fluorescence imaging quality similar to that of current large-size imaging systems and is 22 mm in diameter and 200 mm in height and weighs less than 200 g.

  6. 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.

  7. Chlorophyll fluorescence analysis and imaging in plant stress and disease

    Energy Technology Data Exchange (ETDEWEB)

    Daley, P.F.

    1994-12-01

    Quantitative analysis of chlorophyll fluorescence transients and quenching has evolved rapidly in the last decade. Instrumentation capable of fluorescence detection in bright actinic light has been used in conjunction with gas exchange analysis to build an empirical foundation relating quenching parameters to photosynthetic electron transport, the state of the photoapparatus, and carbon fixation. We have developed several instruments that collect video images of chlorophyll fluorescence. Digitized versions of these images can be manipulated as numerical data arrays, supporting generation of quenching maps that represent the spatial distribution of photosynthetic activity in leaves. We have applied this technology to analysis of fluorescence quenching during application of stress hormones, herbicides, physical stresses including drought and sudden changes in humidity of the atmosphere surrounding leaves, and during stomatal oscillations in high CO{sub 2}. We describe a recently completed portable fluorescence imaging system utilizing LED illumination and a consumer-grade camcorder, that will be used in long-term, non-destructive field studies of plant virus infections.

  8. Functionalized silica nanoparticles: a platform for fluorescence imaging at the cell and small animal levels.

    Science.gov (United States)

    Wang, Kemin; He, Xiaoxiao; Yang, XiaoHai; Shi, Hui

    2013-07-16

    Going in vivo, including living cells and the whole body, is very important for gaining a better understanding of the mystery of life and requires specialized imaging techniques. The diversity, composition, and temporal-spatial variation of life activities from cells to the whole body require the analysis techniques to be fast-response, noninvasive, highly sensitive, and stable, in situ and in real-time. Functionalized nanoparticle-based fluorescence imaging techniques have the potential to meet such needs through real-time and noninvasive visualization of biological events in vivo. Functionalized silica nanoparticles (SiNPs) doped with fluorescent dyes appear to be an ideal and flexible platform for developing fluorescence imaging techniques used in living cells and the whole body. We can select and incorporate different dyes inside the silica matrix either noncovalently or covalently. These form the functionalized hybrid SiNPs, which support multiplex labeling and ratiometric sensing in living systems. Since the silica matrix protects dyes from outside quenching and degrading factors, this enhances the photostability and biocompatibility of the SiNP-based probes. This makes them ideal for real-time and long-time tracking. One nanoparticle can encapsulate large numbers of dye molecules, which amplifies their optical signal and temporal-spatial resolution response. Integrating fluorescent dye-doped SiNPs with targeting ligands using various surface modification techniques can greatly improve selective recognition. Along with the endocytosis, functionalized SiNPs can be efficiently internalized into cells for noninvasive localization, assessment, and monitoring. These unique characteristics of functionalized SiNPs substantially support their applications in fluorescence imaging in vivo. In this Account, we summarize our efforts to develop functionalized dye-doped SiNPs for fluorescence imaging at the cell and small animal levels. We first discuss how to design and

  9. Real-time absorption reduced surface fluorescence imaging.

    Science.gov (United States)

    Yang, Bin; Tunnell, James W

    2014-09-01

    We introduce a technique that limits absorption effects in fluorescence imaging and does not require extensive imaging processing, thus allowing for video rate imaging. The absorption minimization is achieved using spatial frequency domain imaging at a single high spatial frequency with standard three-phase demodulation. At a spatial frequency f ¼ 0.5 mm−1, we demonstrated in both in-vitro phantoms and ex-vivo tissue that the absorption can be significantly reduced. In the real-time implementation, we achieved a video rate of 19 frames∕s. This technique has potential in cancer visualization and tumor margin detection. PMID:25250826

  10. Fluorescence lifetime imaging of human skin and hair

    Science.gov (United States)

    Ehlers, A.; Riemann, I.; Anhut, T.; Kaatz, M.; Elsner, P.; König, K.

    2006-02-01

    Multiphoton imaging has developed into an important technique for in-vivo research in life sciences. With the laser System DermaInspect (JenLab, Germany) laser radiation from a Ti:Sapphire laser is used to generate multiphotonabsorption deep in the human skin in vivo. The resulting autofluorescence radiation arises from endogenous fluorophores such as NAD(P)H, flavines, collagen, elastin, porphyrins und melanin. Second harmonic generation (SHG) was used to detect collagen structures in the dermal layer. Femtosecond laser multiphoton imaging offers the possibility of high resolution optical tomography of human skin as well as fluorescence lifetime imaging (FLIM) with picosecond time resolution. In this work a photon detector with ultrashort rise time of less than 30ps was applied to FLIM measurements of human skin and hair with different pigmentation. Fluorescence lifetime images of different human hair types will be discussed.

  11. The Cyan Fluorescent Protein (CFP Transgenic Mouse as a Model for Imaging Pancreatic Exocrine Cells

    Directory of Open Access Journals (Sweden)

    Hop S Tran Cao

    2009-03-01

    Full Text Available The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan. Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA. Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. Main outcome measure Determination of fluorescence intensity of different organs. Results Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

  12. Polyacrylamide based ICG nanocarriers for enhanced fluorescence and photoacoustic imaging

    Science.gov (United States)

    Ray, Aniruddha; Yoon, Hyung Ki; Ryu, HeeJu; Koo Lee, Yong-Eun; Kim, Gwangseong; Wang, Xueding; Kopelman, Raoul

    2013-02-01

    Indocyanine green (ICG) is an FDA approved tricarbocyanine dye. This dye, with a strong absorbance in the near infrared (NIR) region, has been extensively used for fluorescence and photoacoustic imaging in vivo. ICG in its free form, however, has a few drawbacks that limit its in vivo applications, such as non-targetability, tendency to form aggregates which changes its optical properties, fast degradation, short plasma lifetime and reduced fluorescence at body temperature. In order to bypass these inherent drawbacks, we demonstrate a polyacrylamide based nanocarrier that was particularly designed to carry the negatively charged ICG molecules. These nanocarriers are biodegradable, biocompatible and can be specifically targeted to any cell or tissue. Using these nanocarriers we avoid all the problems associated with free ICG, such as degradation, aggregation and short plasma lifetime, and also enhance demonstrate its ability towards photoacoustics and fluorescence imaging.

  13. Fluorescence imaging of angiogenesis in green fluorescent protein-expressing tumors

    Science.gov (United States)

    Yang, Meng; Baranov, Eugene; Jiang, Ping; Li, Xiao-Ming; Wang, Jin W.; Li, Lingna; Yagi, Shigeo; Moossa, A. R.; Hoffman, Robert M.

    2002-05-01

    The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

  14. Use of fluorescence and scanning electron microscopy as tools in teaching biology

    Science.gov (United States)

    Ghosh, Nabarun; Silva, Jessica; Vazquez, Aracely; Das, A. B.; Smith, Don W.

    2011-06-01

    Recent nationwide surveys reveal significant decline in students' interest in Math and Sciences. The objective of this project was to inspire young minds in using various techniques involved in Sciences including Scanning Electron Microscopy. We used Scanning Electron Microscope in demonstrating various types of Biological samples. An SEM Tabletop model in the past decade has revolutionized the use of Scanning Electron Microscopes. Using SEM Tabletop model TM 1000 we studied biological specimens of fungal spores, pollen grains, diatoms, plant fibers, dust mites, insect parts and leaf surfaces. We also used fluorescence microscopy to view, to record and analyze various specimens with an Olympus BX40 microscope equipped with FITC and TRITC fluorescent filters, a mercury lamp source, DP-70 digital camera with Image Pro 6.0 software. Micrographs were captured using bright field microscopy, the fluoresceinisothiocyanate (FITC) filter, and the tetramethylrhodamine (TRITC) filter settings at 40X. A high pressure mercury lamp or UV source was used to excite the storage molecules or proteins which exhibited autofluorescence. We used fluorescent microscopy to confirm the localization of sugar beet viruses in plant organs by viewing the vascular bundles in the thin sections of the leaves and other tissues. We worked with the REU summer students on sample preparation and observation on various samples utilizing the SEM. Critical Point Drying (CPD) and metal coating with the sputter coater was followed before observing some cultured specimen and the samples that were soft in textures with high water content. SEM Top allowed investigating the detailed morphological features that can be used for classroom teaching. Undergraduate and graduate researchers studied biological samples of Arthropods, pollen grains and teeth collected from four species of snakes using SEM. This project inspired the research students to pursue their career in higher studies in science and 45% of the

  15. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda

    2014-08-01

    Full Text Available The development of three dimensional cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex three dimensional matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy is becoming an excellent tool for fast imaging of such three-dimensional biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

  16. 2D/3D cryo x-ray fluorescence imaging at the bionanoprobe at the advanced photon source

    Energy Technology Data Exchange (ETDEWEB)

    Chen, S., E-mail: sichen@aps.anl.gov; Vine, D. J.; Lai, B. [Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439 (United States); Paunesku, T.; Yuan, Y.; Woloschak, G. E. [Department of Radiation Oncology, Northwester University, Chicago, IL 60611 (United States); Deng, J. [Applied Physics, Northwestern University, Evanston, IL 60208 (United States); Jin, Q.; Hong, Y. P. [Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208 (United States); Flachenecker, C.; Hornberger, B. [Carl Zeiss X-ray Microscopy, Pleasanton, CA 94588 (United States); Brister, K. [Synchrotron Research Center, Northwestern University, Argonne, IL 60439 (United States); Jacobsen, C. [Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439 (United States); Applied Physics, Northwestern University, Evanston, IL 60208 (United States); Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208 (United States); Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208 (United States); Vogt, S. [Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439 (United States); Department of Radiation Oncology, Northwester University, Chicago, IL 60611 (United States)

    2016-01-28

    Trace elements, particularly metals, play very important roles in biological systems. Synchrotron-based hard X-ray fluorescence microscopy offers the most suitable capabilities to quantitatively study trace metals in thick biological samples, such as whole cells and tissues. In this manuscript, we have demonstrated X-ray fluorescence imaging of frozen-hydrated whole cells using the recent developed Bionanoprobe (BNP). The BNP provides spatial resolution down to 30 nm and cryogenic capabilities. Frozen-hydrated biological cells have been directly examined on a sub-cellular level at liquid nitrogen temperatures with minimal sample preparation.

  17. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  18. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  19. PARPi-FL - a Fluorescent PARP1 Inhibitor for Glioblastoma Imaging

    Directory of Open Access Journals (Sweden)

    Christopher P. Irwin

    2014-05-01

    Full Text Available New intravital optical imaging technologies have revolutionized our understanding of mammalian biology and continue to evolve rapidly. However, there are only a limited number of imaging probes available to date. In this study, we investigated in mouse models of glioblastoma whether a fluorescent small molecule inhibitor of the DNA repair enzyme PARP1, PARPi-FL, can be used as an imaging agent to detect glioblastomas in vivo. We demonstrated that PARPi-FL has appropriate biophysical properties, low toxicity at concentrations used for imaging, high stability in vivo, and accumulates selectively in glioblastomas due to high PARP1 expression. Importantly, subcutaneous and orthotopic glioblastoma xenografts were imaged with high contrast clearly defining tumor tissue from normal surrounding tissue. This research represents a step toward exploring and developing PARPi-FL as an optical intraoperative imaging agent for PARP1 in the clinic.

  20. Imaging cellular and molecular biological functions

    Energy Technology Data Exchange (ETDEWEB)

    Shorte, S.L. [Institut Pasteur, 75 - Paris (France). Plateforme d' Imagerie Dynamique PFID-Imagopole; Frischknecht, F. (eds.) [Heidelberg Univ. Medical School (Germany). Dept. of Parasitology

    2007-07-01

    'Imaging cellular and molecular biological function' provides a unique selection of essays by leading experts, aiming at scientist and student alike who are interested in all aspects of modern imaging, from its application and up-scaling to its development. Indeed the philosophy of this volume is to provide student, researcher, PI, professional or provost the means to enter this applications field with confidence, and to construct the means to answer their own specific questions. (orig.)

  1. microlith : Image Simulation for Biological Phase Microscopy

    CERN Document Server

    Mehta, Shalin B

    2013-01-01

    Accurate simulation of image formation remains under-exploited for biological phase microscopy methods that employ partially coherent illumination, despite being important for the design of imaging systems and the reconstruction algorithms. We present an open-source MATLAB toolbox, microlith (https://code.google.com/p/microlith), that provides accurate simulation of the 3D image of a thin specimen under any partially coherent imaging system, including coherent or incoherent systems. We demonstrate the accuracy of the microlith toolbox by comparing simulated images and experimental images of a phase-only Siemens star test target using dark field and differential interference contrast microscopes. The comparison leads to intriguing insights about the sensitivity of the dark-field microscope to sub-resolution features and effects of specimen birefringence on differential interference contrast.

  2. A novel colorimetric and off–on fluorescent sensor for Hg2+ and its application in live cell imaging

    International Nuclear Information System (INIS)

    A novel rhodamine-based fluorescent probe L2 with two “S” groups was synthesized and characterized. As expected, L2 exhibited high selectivity and sensitivity for Hg2+ over other commonly coexistent metal ions. Its selectivity is excellent, and the detection limit was measured to be 1 ppm. The significant changes in the fluorescence color could be used for naked-eye detection. Furthermore, fluorescence imaging experiments of Hg2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems. -- Highlights: • A new rhodamine-based chemodosimeter (L2) was synthesized and characterized. • L2 exhibited high selectivity and excellent sensitivity detection of Hg2+. • The significant changes in color could be used for naked-eye detection. • The fluorescence imaging experiments of Hg2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems

  3. Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens

    Science.gov (United States)

    Elson, D. S.; Jo, J. A.; Marcu, L.

    2007-05-01

    We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues.

  4. Visible to Near-Infrared Fluorescence Enhanced Cellular Imaging on Plasmonic Gold Chips.

    Science.gov (United States)

    Koh, Byumseok; Li, Xiaoyang; Zhang, Bo; Yuan, Bing; Lin, Yi; Antaris, Alexander L; Wan, Hao; Gong, Ming; Yang, Jiang; Zhang, Xiaodong; Liang, Yongye; Dai, Hongjie

    2016-01-27

    Rapid and sensitive detections of a variety of surface and intracellular proteins, nucleic acids, and other cellular biomarkers are important to elucidating biological signaling pathways and to devising disease diagnostics and therapeutics. Here, sensitive imaging and detection of cellular proteins on fluorescence-enhancing, nanostructured plasmonic gold (pGold) chips is presented. Imaging of fluorescently labeled cellular biomarkers on pGold is enhanced by 2-30-fold in the visible to near infrared (NIR) range of ≈500-900 nm. The high fluorescence enhancement of >700 nm significantly improves the dynamic range and signal/background ratios of NIR imaging, allowing high-performance multicolor imaging in the visible-NIR range using high quantum yield (QY) visible dyes and lower QY NIR fluorophores. Further, multiple cellular proteins of single cells of various cell types can be detected through microarraying of cells, useful for potentially hundreds and thousands different types of cells assayed on a single chip down to small cell numbers. This work suggests a simple, high throughput, high sensitivity, and multiplexed single-cell analysis method on fluorescence enhancing plasmonic substrates in the entire visible to NIR window. PMID:26663862

  5. Fluorescence lifetime to image epidermal ionic concentrations

    Science.gov (United States)

    Behne, Martin J.; Barry, Nicholas P.; Moll, Ingrid; Gratton, Enrico; Mauro, Theodora M.

    2004-09-01

    Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H+ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca2+, and a quantitative approach to topical drug penetration.

  6. Preparation of Fluorescent Graphene Quantum Dots as Biological Imaging Marker for Cells%荧光石墨烯量子点制备及其在细胞成像中的应用

    Institute of Scientific and Technical Information of China (English)

    谢文菁; 傅英懿; 马红; 张沫; 范楼珍

    2012-01-01

    Currently, graphene has attracted much attention in the fields of bioimaging, biolabeling and drug delivery. Theoretical and experimental studies have shown that the graphene quantum dots (GQDs) are expected to show good optical properties due to their quantum confinement and edge effect. In this report, using the electrochemical assay the fluorescent GQDs with a diameter between 5 and 10 nm could be obtained via electrolysing graphite in alkaline condition and with hydrazine hydrate as a reducing agent at room temperature. The structure of the GQDs was confimed by means of transmission electron microscope (TEM) and atomic force microscope (AFM). The finding showed that the GQDs have an uniform size, and most of them are separate graphene. The GQDs mainly consist of single layer with less than 1 nm. Their features and properties were characterized by fourier transform infrared spectroscopy (FTIR), photoluminescence spectra (PL), UV-visible spectroscopy (UV-vis) and X-ray diffraction (XRD). The results indicated that the GQDs have bright yellow luminescence with a 14 % quantum yield, which is higher than that of traditional carbon quantum dots reported previously. When they were excited by different excitation wavelengths, the intensity of photoluminescence increased to the maximum, and then decreased gradually. The fluorescent emission peak of the GQDs remained unshifted, suggesting a novel kind of quantum dots different from those of graphene oxide quantum dots depending excitation wavelengths. The luminescence of GQDs arises from the graphene modified with the phthalhydrazide-like groups and hydrazide groups at the edge. The highly fluorescent GQDs have high water solubility, good photostability and biocompatibility, indicating that the GQDs can easily enter the cells. By incorporating the GQDs with A549 (lung cancer) and MCF-7 (breast cancer) cells through MTT assay, the newly obtained GQDs exhibited low cytotoxicity with an advantage of strong photoluminescence

  7. Nonlinear spectral imaging of biological tissues

    NARCIS (Netherlands)

    Palero, J.A.

    2007-01-01

    The work presented in this thesis demonstrates live high resolution 3D imaging of tissue in its native state and environment. The nonlinear interaction between focussed femtosecond light pulses and the biological tissue results in the emission of natural autofluorescence and second-harmonic signal.

  8. Onychomycosis diagnosis using fluorescence and infrared imaging systems

    Science.gov (United States)

    da Silva, Ana Paula; Fortunato, Thereza Cury; Stringasci, Mirian D.; Kurachi, Cristina; Bagnato, Vanderlei S.; Inada, Natalia M.

    2015-06-01

    Onychomycosis is a common disease of the nail plate, constituting approximately half of all cases of nail infection. Onychomycosis diagnosis is challenging because it is hard to distinguish from other diseases of the nail lamina such as psoriasis, lichen ruber or eczematous nails. The existing methods of diagnostics so far consist of clinical and laboratory analysis, such as: Direct Mycological examination and culture, PCR and histopathology with PAS staining. However, they all share certain disadvantages in terms of sensitivity and specificity, time delay, or cost. This study aimed to evaluate the use of infrared and fluorescence imaging as new non-invasive diagnostic tools in patients with suspected onychomycosis, and compare them with established techniques. For fluorescence analysis, a Clinical Evince (MM Optics®) was used, which consists of an optical assembly with UV LED light source wavelength 400 nm +/- 10 nm and the maximum light intensity: 40 mW/cm2 +/- 20%. For infrared analysis, a Fluke® Camera FKL model Ti400 was used. Patients with onychomycosis and control group were analyzed for comparison. The fluorescence images were processed using MATLAB® routines, and infrared images were analyzed using the SmartView® 3.6 software analysis provided by the company Fluke®. The results demonstrated that both infrared and fluorescence could be complementary to diagnose different types of onychomycosis lesions. The simplicity of operation, quick response and non-invasive assessment of the nail patients in real time, are important factors to be consider for an implementation.

  9. Infrared imaging of LED lighting tubes and fluorescent tubes

    Science.gov (United States)

    Siikanen, Sami; Kivi, Sini; Kauppinen, Timo; Juuti, Mikko

    2011-05-01

    The low energy efficiency of conventional light sources is mainly caused by generation of waste heat. We used infrared (IR) imaging in order to monitor the heating of both LED tube luminaires and ordinary T8 fluorescent tubes. The IR images showed clearly how the surface temperatures of the fluorescent tube ends quickly rose up to about +50...+70°C, whereas the highest surface temperatures seen on the LED tubes were only about +30...+40°C. The IR images demonstrated how the heat produced by the individual LED chips can be efficiently guided to the supporting structure in order to keep the LED emitters cool and hence maintain efficient operation. The consumed electrical power and produced illuminance were also recorded during 24 hour measurements. In order to assess the total luminous efficacy of the luminaires, separate luminous flux measurements were made in a large integrating sphere. The currently available LED tubes showed efficacies of up to 88 lm/W, whereas a standard "cool white" T8 fluorescent tube produced ca. 75 lm/W. Both lamp types gave ca. 110 - 130 lx right below the ceiling-mounted luminaire, but the LED tubes consume only 40 - 55% of the electric power compared to fluorescent tubes.

  10. Single-atom-sensitive fluorescence imaging of ultracold quantum gases

    CERN Document Server

    Bücker, R; Manz, S; Betz, T; Koller, Ch; Plisson, T; Rottmann, J; Schumm, T; Schmiedmayer, J

    2009-01-01

    We present a novel imaging system for ultracold quantum gases in expansion. After release from a confining potential, atoms fall through a sheet of resonant excitation laser light and the emitted fluorescence photons are imaged onto an amplified CCD camera using a high numerical aperture optical system. The imaging system reaches an extraordinary dynamic range, not attainable with conventional absorption imaging. We demonstrate single-atom detection for dilute atomic clouds with high efficiency where at the same time dense Bose-Einstein condensates can be imaged without saturation or distortion. The spatial resolution can reach the sampling limit as given by the 8 microns pixel size in object space. Pulsed operation of the detector allows for slice images and hence a 3D tomography of the measured object. The scheme can easily be implemented for any atomic species and all optical components are situated outside the vacuum system. As a first application we perform thermometry on rubidium Bose-Einstein condensat...

  11. Highly stable organic fluorescent nanorods for living- cell imaging

    Institute of Scientific and Technical Information of China (English)

    Minhuan Lan[1,3; Jinfeng Zhang[1,3; Xiaoyue Zhu[1; Pengfei Wang[2; Xianfeng Chen[1; Chun-Sing Lee[1; Wenjun Zhang[1

    2015-01-01

    Metal-free, organic-dye-based fluorescent nanorods were fabricated through a simple solvent-exchange procedure. The as-prepared nanorods exhibit low toxicity to living cells and excellent photostability. Furthermore, they are stable in solutions of various pHs and high ionic strength and in solutions with interfering metal ions. Compared with the free DPP-Br molecules in THF, these nanorods exhibit larger Stokes shift, broader absorption spectra, and greatly improved photostability. We successfully demonstrated the application of the nanorods, including their aforementioned beneficial characteristics, as a good fluorescence probe for bio-imaging.

  12. Fluorescent supramolecular micelles for imaging-guided cancer therapy

    Science.gov (United States)

    Sun, Mengmeng; Yin, Wenyan; Dong, Xinghua; Yang, Wantai; Zhao, Yuliang; Yin, Meizhen

    2016-02-01

    A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSM nanomedicine platform as a nanovehicle has great potential for fluorescence imaging-guided cancer therapy.A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth

  13. Nanoscale fluorescence lifetime imaging with a single diamond NV center

    CERN Document Server

    Beams, Ryan; Johnson, Timothy W; Oh, Sang-Hyun; Novotny, Lukas; Vamivakas, Nick

    2013-01-01

    Solid-state quantum emitters, such as artificially engineered quantum dots or naturally occurring defects in solids, are being investigated for applications ranging from quantum information science and optoelectronics to biomedical imaging. Recently, these same systems have also been studied from the perspective of nanoscale metrology. In this letter we study the near-field optical properties of a diamond nanocrystal hosting a single nitrogen vacancy center. We find that the nitrogen vacancy center is a sensitive probe of the surrounding electromagnetic mode structure. We exploit this sensitivity to demonstrate nanoscale fluorescence lifetime imaging microscopy (FLIM) with a single nitrogen vacancy center by imaging the local density of states of an optical antenna.

  14. Sulfonate-based fluorescent probes for imaging hydrogen peroxide in living cells

    Institute of Scientific and Technical Information of China (English)

    XU KeHua; LIU Fen; WANG HuiXia; WANG ShanShan; WANG LuLu; TANG Bo

    2009-01-01

    Based on the mechanism of H2O2-mediated hydrolysis of sulfonates, two fluorescein disulfonates compounds (FS-1 and FS-2) were designed and synthesized as the highly selective and sensitive fluo-rescent probes for imaging H2O2 in living cells. The probes were detected with elemental analysis, IR, 1H NMR and 13C NMR. Upon reaction with H2O2, the probes exhibit strong fluorescence responses and high selectivity for H202 over other reactive oxygen species and some biological compounds. Fur-thermore, the sulfonate-based probes, as novel fluorescent reagents, are cell-permeable and can detect micromolar changes in H202 concentrations in living cells by using confocal microscopy.

  15. Sulfonate-based fluorescent probes for imaging hydrogen peroxide in living cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Based on the mechanism of H2O2-mediated hydrolysis of sulfonates, two fluorescein disulfonates compounds (FS-1 and FS-2) were designed and synthesized as the highly selective and sensitive fluorescent probes for imaging H2O2 in living cells. The probes were detected with elemental analysis, IR, 1H NMR and 13C NMR. Upon reaction with H2O2, the probes exhibit strong fluorescence responses and high selectivity for H2O2 over other reactive oxygen species and some biological compounds. Furthermore, the sulfonate-based probes, as novel fluorescent reagents, are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells by using confocal microscopy.

  16. Submicron hard X-ray fluorescence imaging of synthetic elements

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, Mark P., E-mail: mjensen@anl.gov [Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Aryal, Baikuntha P. [Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Department of Chemistry, University of Chicago, Chicago, IL 60637 (United States); Gorman-Lewis, Drew [Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Paunesku, Tatjana [Department of Radiation Oncology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Department of Radiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Lai, Barry; Vogt, Stefan [X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Woloschak, Gayle E. [Department of Radiation Oncology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Department of Radiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Actinide elements are mapped with L-edge X-rays and better than 400 nm resolution. Black-Right-Pointing-Pointer A typical detection limit was 2.9 Multiplication-Sign 10{sup -20} moles Pu {mu}m{sup -2}. Black-Right-Pointing-Pointer XANES measurements provide chemical information in 0.1 {mu}m{sup 2} spots. Black-Right-Pointing-Pointer Selection of materials for encapsulation is important for avoiding interferences. - Abstract: Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as {mu}-XANES (X-ray absorption near edge structure). We have used XFM to image and simultaneously quantify the transuranic element plutonium at the L{sub 3} or L{sub 2}-edge as well as Th and lighter biologically essential elements in individual rat pheochromocytoma (PC12) cells after exposure to the long-lived plutonium isotope {sup 242}Pu. Elemental maps demonstrate that plutonium localizes principally in the cytoplasm of the cells and avoids the cell nucleus, which is marked by the highest concentrations of phosphorus and zinc, under the conditions of our experiments. The minimum detection limit under typical acquisition conditions with an incident X-ray energy of 18 keV for an average 202 {mu}m{sup 2} cell is 1.4 fg Pu or 2.9 Multiplication-Sign 10{sup -20} moles Pu {mu}m{sup -2}, which is similar to the detection limit of K-edge XFM of transition metals at 10 keV. Copper electron microscopy grids were used to avoid interference from gold X-ray emissions, but traces of strontium present in naturally occurring calcium can still interfere with plutonium detection using its L{sub {alpha}} X-ray emission.

  17. Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry.

    Science.gov (United States)

    Maguire, Orla; Wallace, Paul K; Minderman, Hans

    2016-01-01

    The emergence of imaging flow cytometry (IFC) has brought novel applications exploiting its advantages over conventional flow cytometry and microscopy. One of the new applications is fluorescence in situ hybridization in suspension (FISH-IS). Conventional FISH is a slide-based approach in which the spotlike imagery resulting from hybridization with fluorescently tagged probes is evaluated by fluorescence microscopy. The FISH-IS approach evaluated by IFC enables the evaluation of tens to hundreds of thousands of cells in suspension and the analysis can be automated and standardized diminishing operator bias from the analysis. The high cell number throughput of FISH-IS improves the detection of rare events compared to conventional FISH. The applicability of FISH-IS is currently limited to detection of abnormal quantitative differences of hybridization targets such as occur in numerical chromosome abnormalities, deletions and amplifications.Here, we describe a protocol for FISH-IS using chromosome enumeration probes as an example. PMID:27460240

  18. Reconstruction of Elemental Distribution Images from Synchrotron Radiation X-Ray Fluorescence Spectra

    Science.gov (United States)

    Toque, Jay Arre; Ide-Ektessabi, Ari

    Synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) is a powerful technique for studying trace elements in biological samples and other materials in general. Its features including capability to perform measurements in air and water, noncontact and nondestructive assay are superior to other elemental analysis techniques. In this study, a technique for reconstructing elemental distribution mapping of trace elements from spectral data was developed. The reconstruction was made possible by using the measured fluorescent signals to obtain local differences in elemental concentrations. The proposed technique features interpolation and background subtraction using matrix transformations of the spectral data to produce an enhanced distribution images. It is achieved by employing polychromatic or monochromatic color assignments proportional to the fluorescence intensities for displaying single-element or multiple-element distributions respectively. Some typical applications (i.e., macrophage and tissue surrounding an implant) were presented and the samples were imaged using the proposed method. The distribution images of the trace elements of the selected samples were used in conjunction with other analytical techniques to draw relevant observations, which cannot be achieved using conventional techniques such as metallic uptake and corresponding cellular response. The elemental distribution images produced from this study were found to have better quality compared to images produced using other analytical techniques (e.g., SIMS, PIXE, XPS, etc).

  19. 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.

  20. 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.

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

    International Nuclear Information System (INIS)

    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

  2. Fluorescence imaging to study cancer burden on lymph nodes

    Science.gov (United States)

    D'Souza, Alisha V.; Elliott, Jonathan T.; Gunn, Jason R.; Samkoe, Kimberley S.; Tichauer, Kenneth M.; Pogue, Brian W.

    2015-03-01

    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 deploy and test an intra-nodal cancer-cell injection model to enable planar fluorescence imaging of a clinically relevant blue dye, specifically methylene blue along with a cancer targeting tracer, Affibody labeled with IRDYE800CW and subsequently segregate tumor-bearing from normal lymph nodes. This direct-injection based tumor model was employed in athymic rats (6 normal, 4 controls, 6 cancer-bearing), where luciferase-expressing breast cancer cells were injected into axillary lymph nodes. Tumor presence in nodes was confirmed by bioluminescence imaging before and after fluorescence imaging. Lymphatic uptake from the injection site (intradermal on forepaw) to lymph node was imaged at approximately 2 frames/minute. Large variability was observed within each cohort.

  3. Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

    Directory of Open Access Journals (Sweden)

    D. A. Healy

    2014-02-01

    particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAP observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

  4. Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

    Science.gov (United States)

    Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

    2014-08-01

    particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are relatively insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse-mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAPs observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

  5. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    International Nuclear Information System (INIS)

    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

  6. Correlative imaging of fluorescent proteins in resin-embedded plant material.

    Science.gov (United States)

    Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

    2013-04-01

    Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology.

  7. Proton-induced x-ray fluorescence CT imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bazalova-Carter, Magdalena, E-mail: bazalova@stanford.edu; Xing, Lei [Department of Radiation Oncology, Stanford University, Stanford, California 94305-5847 and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo 060-8648 (Japan); Ahmad, Moiz [Department of Radiation Oncology, Stanford University, Stanford, California 94305-5847 (United States); Matsuura, Taeko; Takao, Seishin; Shirato, Hiroki; Umegaki, Kikuo [Department of Medical Physics, Proton Beam Therapy Center, Hokkaido University Hospital, Sapporo 060-8648, Japan and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo 060-8648 (Japan); Matsuo, Yuto [Department of Medical Physics, Proton Beam Therapy Center, Hokkaido University Hospital, Sapporo 060-8648 (Japan); Fahrig, Rebecca [Department of Radiology, Stanford University, Stanford, California 94305 (United States)

    2015-02-15

    Purpose: To demonstrate the feasibility of proton-induced x-ray fluorescence CT (pXFCT) imaging of gold in a small animal sized object by means of experiments and Monte Carlo (MC) simulations. Methods: First, proton-induced gold x-ray fluorescence (pXRF) was measured as a function of gold concentration. Vials of 2.2 cm in diameter filled with 0%–5% Au solutions were irradiated with a 220 MeV proton beam and x-ray fluorescence induced by the interaction of protons, and Au was detected with a 3 × 3 mm{sup 2} CdTe detector placed at 90° with respect to the incident proton beam at a distance of 45 cm from the vials. Second, a 7-cm diameter water phantom containing three 2.2-diameter vials with 3%–5% Au solutions was imaged with a 7-mm FWHM 220 MeV proton beam in a first generation CT scanning geometry. X-rays scattered perpendicular to the incident proton beam were acquired with the CdTe detector placed at 45 cm from the phantom positioned on a translation/rotation stage. Twenty one translational steps spaced by 3 mm at each of 36 projection angles spaced by 10° were acquired, and pXFCT images of the phantom were reconstructed with filtered back projection. A simplified geometry of the experimental data acquisition setup was modeled with the MC TOPAS code, and simulation results were compared to the experimental data. Results: A linear relationship between gold pXRF and gold concentration was observed in both experimental and MC simulation data (R{sup 2} > 0.99). All Au vials were apparent in the experimental and simulated pXFCT images. Specifically, the 3% Au vial was detectable in the experimental [contrast-to-noise ratio (CNR) = 5.8] and simulated (CNR = 11.5) pXFCT image. Due to fluorescence x-ray attenuation in the higher concentration vials, the 4% and 5% Au contrast were underestimated by 10% and 15%, respectively, in both the experimental and simulated pXFCT images. Conclusions: Proton-induced x-ray fluorescence CT imaging of 3%–5% gold solutions in a

  8. Proton-induced x-ray fluorescence CT imaging

    International Nuclear Information System (INIS)

    Purpose: To demonstrate the feasibility of proton-induced x-ray fluorescence CT (pXFCT) imaging of gold in a small animal sized object by means of experiments and Monte Carlo (MC) simulations. Methods: First, proton-induced gold x-ray fluorescence (pXRF) was measured as a function of gold concentration. Vials of 2.2 cm in diameter filled with 0%–5% Au solutions were irradiated with a 220 MeV proton beam and x-ray fluorescence induced by the interaction of protons, and Au was detected with a 3 × 3 mm2 CdTe detector placed at 90° with respect to the incident proton beam at a distance of 45 cm from the vials. Second, a 7-cm diameter water phantom containing three 2.2-diameter vials with 3%–5% Au solutions was imaged with a 7-mm FWHM 220 MeV proton beam in a first generation CT scanning geometry. X-rays scattered perpendicular to the incident proton beam were acquired with the CdTe detector placed at 45 cm from the phantom positioned on a translation/rotation stage. Twenty one translational steps spaced by 3 mm at each of 36 projection angles spaced by 10° were acquired, and pXFCT images of the phantom were reconstructed with filtered back projection. A simplified geometry of the experimental data acquisition setup was modeled with the MC TOPAS code, and simulation results were compared to the experimental data. Results: A linear relationship between gold pXRF and gold concentration was observed in both experimental and MC simulation data (R2 > 0.99). All Au vials were apparent in the experimental and simulated pXFCT images. Specifically, the 3% Au vial was detectable in the experimental [contrast-to-noise ratio (CNR) = 5.8] and simulated (CNR = 11.5) pXFCT image. Due to fluorescence x-ray attenuation in the higher concentration vials, the 4% and 5% Au contrast were underestimated by 10% and 15%, respectively, in both the experimental and simulated pXFCT images. Conclusions: Proton-induced x-ray fluorescence CT imaging of 3%–5% gold solutions in a small animal

  9. Combined phase and X-Ray fluorescence imaging at the sub-cellular level

    International Nuclear Information System (INIS)

    This work presents some recent developments in the field of hard X-ray imaging applied to biomedical research. As the discipline is evolving quickly, new questions appear and the list of needs becomes bigger. Some of them are dealt with in this manuscript. It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimental setup to investigate diverse aspects of cellular research. Together with its high spatial resolution, high flux and high energy range the experimental setup provides bigger field of view, is less sensitive to radiation damages (while taking phase contrast images) and suits well chemical analysis with emphasis on endogenous metals (Zn, Fe, Mn) but also with a possibility for exogenous one's like these found in nanoparticles (Au, Pt, Ag) study. Two synchrotron-based imaging techniques, fluorescence and phase contrast imaging were used in this research project. They were correlated with each other on a number of biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA, red blood cells). The explorations made in the chapter 5 allowed preparation of more established and detailed analysis, described in the next chapter where both techniques, X-ray fluorescence and phase contrast imaging, were exploited in order to access absolute metal projected mass fraction in a whole cell. The final image presents for the first time true quantitative information at the sub-cellular level, not biased by the cell thickness. Thus for the first time a fluorescence map serves as a complete quantitative image of a cell without any risk of misinterpretation. Once both maps are divided by each other pixel by pixel (fluorescence map divided by the phase map) they present a complete and final result of the metal (Zn in this work) projected mass fraction in ppm of dry weight. For the purpose of this calculation the analysis was extended to calibration (non-biological) samples. Polystyrene spheres of a known diameter and known

  10. Deep vascular imaging in wounds by two-photon fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Ciceron O Yanez

    Full Text Available Deep imaging within tissue (over 300 μm at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM. The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.

  11. Reviewing the relevance of fluorescence in biological systems.

    Science.gov (United States)

    Lagorio, M Gabriela; Cordon, Gabriela B; Iriel, Analia

    2015-09-26

    Fluorescence is emitted by diverse living organisms. The analysis and interpretation of these signals may give information about their physiological state, ways of communication among species and the presence of specific chemicals. In this manuscript we review the state of the art in the research on the fluorescence emitted by plant leaves, fruits, flowers, avians, butterflies, beetles, dragonflies, millipedes, cockroaches, bees, spiders, scorpions and sea organisms and discuss its relevance in nature. PMID:26103563

  12. Imaging Atherosclerotic Plaque Calcification: Translating Biology.

    Science.gov (United States)

    Bailey, Grant; Meadows, Judith; Morrison, Alan R

    2016-08-01

    Calcification of atherosclerotic lesions was long thought to be an age - related, passive process, but increasingly data has revealed that atherosclerotic calcification is a more active process, involving complex signaling pathways and bone-like genetic programs. Initially, imaging of atherosclerotic calcification was limited to gross assessment of calcium burden, which is associated with total atherosclerotic burden and risk of cardiovascular mortality and of all cause mortality. More recently, sophisticated molecular imaging studies of the various processes involved in calcification have begun to elucidate information about plaque calcium composition and consequent vulnerability to rupture, leading to hard cardiovascular events like myocardial infarction. As such, there has been renewed interest in imaging calcification to advance risk assessment accuracy in an evolving era of precision medicine. Here we summarize recent advances in our understanding of the biologic process of atherosclerotic calcification as well as some of the molecular imaging tools used to assess it. PMID:27339750

  13. Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

    Science.gov (United States)

    Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

    2016-02-01

    Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

  14. 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.

  15. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

    Science.gov (United States)

    Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

    2012-12-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

  16. Neural imaging in songbirds using fiber optic fluorescence microscopy

    Science.gov (United States)

    Nooshabadi, Fatemeh; Hearn, Gentry; Lints, Thierry; Maitland, Kristen C.

    2012-02-01

    The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.

  17. Picosecond fluorescence lifetime imaging microscope for imaging of living glioma cells

    Science.gov (United States)

    Fang, Qiyin; Wang, Jingjing; Sun, Yinghua; Vernier, Thomas; Papaioannou, Thanassis; Jo, Javier; Thu, Mya M.; Gundersen, Martin A.; Marcu, Laura

    2005-03-01

    In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.

  18. Scintillating Optical Fiber Imagers for biology

    International Nuclear Information System (INIS)

    S.O.F.I (Scintillating Optical Fiber Imager) is a detector developed to replace the autoradiographic films used in molecular biology for the location of radiolabelled (32P) DNA molecules in blotting experiments. It analyses samples on a 25 x 25 cm2 square area still 25 times faster than autoradiographic films, with a 1.75 and 3 mm resolution for two orthogonal directions. This device performs numerised images with a dynamic upper than 100 which allows the direct quantitation of the analysed samples. First, this thesis describes the S.O.F.I. development (Scintillating Optical Fibers, coding of these fibers and specific electronic for the treatment of the Multi-Anode Photo-Multiplier signals) and experiments made in collaboration with molecular biology laboratories. In a second place, we prove the feasibility of an automatic DNA sequencer issued from S.O.F.I

  19. Elastic registration for auto-fluorescence image averaging.

    Science.gov (United States)

    Kubecka, Libor; Jan, Jiri; Kolar, Radim; Jirik, Radovan

    2006-01-01

    The paper describes restitution of geometrical distortions and improvement of signal-to-noise ratio of auto-fluorescence retinal images, finally aimed at segmentation and area estimation of the lipofuscin spots as one of the features to be included in glaucoma diagnosis. The main problems - geometrical and illumination incompatibility of frames in the image sequence and a non-negligible "shear" distortion in the individual frames - have been solved by the presented registration procedure. The concept and some details of the MI-based regularized registration, together with evaluation of test results form the core of the contribution. PMID:17945684

  20. Compact instrument for fluorescence image-guided surgery

    Science.gov (United States)

    Wang, Xinghua; Bhaumik, Srabani; Li, Qing; Staudinger, V. Paul; Yazdanfar, Siavash

    2010-03-01

    Fluorescence image-guided surgery (FIGS) is an emerging technique in oncology, neurology, and cardiology. To adapt intraoperative imaging for various surgical applications, increasingly flexible and compact FIGS instruments are necessary. We present a compact, portable FIGS system and demonstrate its use in cardiovascular mapping in a preclinical model of myocardial ischemia. Our system uses fiber optic delivery of laser diode excitation, custom optics with high collection efficiency, and compact consumer-grade cameras as a low-cost and compact alternative to open surgical FIGS systems. Dramatic size and weight reduction increases flexibility and access, and allows for handheld use or unobtrusive positioning over the surgical field.

  1. A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues.

    Science.gov (United States)

    Jin, Qiang; Feng, Lei; Wang, Dan-Dan; Dai, Zi-Ru; Wang, Ping; Zou, Li-Wei; Liu, Zhi-Hong; Wang, Jia-Yue; Yu, Yang; Ge, Guang-Bo; Cui, Jing-Nan; Yang, Ling

    2015-12-30

    In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

  2. Potential Application of Fluorescence Imaging for Assessing Fecal Contamination of Soil and Compost Maturity

    Directory of Open Access Journals (Sweden)

    Hyunjeong Cho

    2016-08-01

    Full Text Available Pathogenic microorganisms can lead to serious outbreaks of foodborne illnesses, particularly if fresh produce becomes contaminated and then happens to be inappropriately handled in a manner that can incubate pathogens. Pathogenic microbial contamination of produce can occur through a variety of pathways, such as from the excrement of domesticated and wild animals, biological soil amendment, agricultural water, worker health and hygiene, and field tools used during growth and harvest. The use of mature manure compost and preventative control of fecal contamination from wildlife and livestock are subject to safety standards to minimize the risk of foodborne illness associated with produce. However, in a field production environment, neither traces of animal feces nor the degree of maturity of manure compost can be identified by the naked eye. In this study, we investigated hyperspectral fluorescence imaging techniques to characterize fecal samples from bovine, swine, poultry, and sheep species, and to determine feasibilities for both detecting the presence of animal feces as well as identifying the species origin of the feces in mixtures of soil and feces. In addition, the imaging techniques were evaluated for assessing the maturity of manure compost. The animal feces exhibited dynamic and unique fluorescence emission features that allowed for the detection of the presence of feces and showed that identification of the species origin of fecal matter present in soil-feces mixtures is feasible. Furthermore, the results indicate that using simple single-band fluorescence imaging at the fluorescence emission maximum for animal feces, simpler than full-spectrum hyperspectral fluorescence imaging, can be used to assess the maturity of manure compost.

  3. Development of Photoactivated Fluorescent N-Hydroxyoxindoles and Their Application for Cell-Selective Imaging.

    Science.gov (United States)

    Lai, Jinping; Yu, An; Yang, Letao; Zhang, Yixiao; Shah, Birju P; Lee, Ki Bum

    2016-04-25

    Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N-hydroxyoxindoles formed through the intramolecular photocyclization of substituted o-nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra-high fluorescence enhancement (up to 800-fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure-activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE-based fluorescent probes with desired photophysical and biological properties. As a proof-of-concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365-405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs). PMID:27004772

  4. Volumetric retinal fluorescence microscopic imaging with extended depth of field

    Science.gov (United States)

    Li, Zengzhuo; Fischer, Andrew; Li, Wei; Li, Guoqiang

    2016-03-01

    Wavefront-engineered microscope with greatly extended depth of field (EDoF) is designed and demonstrated for volumetric imaging with near-diffraction limited optical performance. A bright field infinity-corrected transmissive/reflective light microscope is built with Kohler illumination. A home-made phase mask is placed in between the objective lens and the tube lens for ease of use. General polynomial function is adopted in the design of the phase plate for robustness and custom merit function is used in Zemax for optimization. The resulting EDoF system achieves an engineered point spread function (PSF) that is much less sensitive to object depth variation than conventional systems and therefore 3D volumetric information can be acquired in a single frame with expanded tolerance of defocus. In Zemax simulation for a setup using 32X objective (NA = 0.6), the EDoF is 20μm whereas a conventional one has a DoF of 1.5μm, indicating a 13 times increase. In experiment, a 20X objective lens with NA = 0.4 was used and the corresponding phase plate was designed and fabricated. Retinal fluorescence images of the EDoF microscope using passive adaptive optical phase element illustrate a DoF around 100μm and it is able to recover the volumetric fluorescence images that are almost identical to in-focus images after post processing. The image obtained from the EDoF microscope is also better in resolution and contrast, and the retinal structure is better defined. Hence, due to its high tolerance of defocus and fine restored image quality, EDoF optical systems have promising potential in consumer portable medical imaging devices where user's ability to achieve focus is not optimal, and other medical imaging equipment where achieving best focus is not a necessary.

  5. Synthesis, Characterization and Biological Activities of a New Fluorescent Indicator for the Intracellular Calcium Ions

    Institute of Scientific and Technical Information of China (English)

    HE Huaizhen; LEI Lei; LI Jianli; SHI Zhen

    2009-01-01

    A novel calcium-selective fluorescent indicator Fluo-3M AM was synthesized by introduction of a methyl group into the Ca2+-chelating moiety and adequately characterized by spectral methods (1H NMR, GC-MS, IR and MALDI-TOF MS). Meanwhile, its fluorescence spectra and some biological activities have been also studied. The results indicate that the new fluorescent indicator has relatively high affinity to calcium and a strong fluorescence signal, which should be useful for biomedical researchers to investigate the effects of calcium ions in biosystems.

  6. New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging.

    Science.gov (United States)

    Oheim, Martin; van 't Hoff, Marcel; Feltz, Anne; Zamaleeva, Alsu; Mallet, Jean-Maurice; Collot, Mayeul

    2014-10-01

    Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca(2+) probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-sensitive indicators have been developed since 2009 than in the thirty years before. In this topical review, we survey the physicochemical properties of these red-emitting Ca(2+) probes and discuss their utility for biological Ca(2+) imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3-16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca(2+) indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck

  7. Development of Fluorescence Imaging Lidar for Boat-Based Coral Observation

    Science.gov (United States)

    Sasano, Masahiko; Imasato, Motonobu; Yamano, Hiroya; Oguma, Hiroyuki

    2016-06-01

    A fluorescence imaging lidar system installed in a boat-towable buoy has been developed for the observation of reef-building corals. Long-range fluorescent images of the sea bed can be recorded in the daytime with this system. The viability of corals is clear in these fluorescent images because of the innate fluorescent proteins. In this study, the specifications and performance of the system are shown.

  8. Development of Fluorescence Imaging Lidar for Boat-Based Coral Observation

    Directory of Open Access Journals (Sweden)

    Sasano Masahiko

    2016-01-01

    Full Text Available A fluorescence imaging lidar system installed in a boat-towable buoy has been developed for the observation of reef-building corals. Long-range fluorescent images of the sea bed can be recorded in the daytime with this system. The viability of corals is clear in these fluorescent images because of the innate fluorescent proteins. In this study, the specifications and performance of the system are shown.

  9. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging.

    Science.gov (United States)

    Yankelevich, Diego R; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Elson, Daniel S; Marcu, Laura

    2014-03-01

    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 time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

  10. Imaging morphogenesis: technological advances and biological insights.

    Science.gov (United States)

    Keller, Philipp J

    2013-06-01

    Morphogenesis, the development of the shape of an organism, is a dynamic process on a multitude of scales, from fast subcellular rearrangements and cell movements to slow structural changes at the whole-organism level. Live-imaging approaches based on light microscopy reveal the intricate dynamics of this process and are thus indispensable for investigating the underlying mechanisms. This Review discusses emerging imaging techniques that can record morphogenesis at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert highly complex image data sets into biological insights.

  11. Femtosecond diffractive imaging of biological cells

    Science.gov (United States)

    Marvin Seibert, M.; Boutet, Sébastien; Svenda, Martin; Ekeberg, Tomas; Maia, Filipe R. N. C.; Bogan, Michael J.; Tîmneanu, Nicusor; Barty, Anton; Hau-Riege, Stefan; Caleman, Carl; Frank, Matthias; Benner, Henry; Y Lee, Joanna; Marchesini, Stefano; Shaevitz, Joshua W.; Fletcher, Daniel A.; Bajt, Sasa; Andersson, Inger; Chapman, Henry N.; Hajdu, Janos

    2010-10-01

    In a flash diffraction experiment, a short and extremely intense x-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval by iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al 2006 Nat. Phys. 2 839-43). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 fs soft x-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show that the pulse heated the sample to about 160 000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest that the average displacement of ions and atoms in the hottest surface layers remained below 3 Å during the pulse.

  12. Femtosecond diffractive imaging of biological cells

    Energy Technology Data Exchange (ETDEWEB)

    Marvin Seibert, M; Boutet, Sebastien; Svenda, Martin; Ekeberg, Tomas; Maia, Filipe R N C; TImneanu, Nicusor; Caleman, Carl; Hajdu, Janos [Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3, Box 596, SE-75124 Uppsala (Sweden); Bogan, Michael J [SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025 (United States); Barty, Anton; Hau-Riege, Stefan; Frank, Matthias; Benner, Henry [Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550 (United States); Lee, Joanna Y [Department of Biology, Stanford University, Stanford, CA 94305 (United States); Marchesini, Stefano [Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Shaevitz, Joshua W [150 Carl Icahn Laboratory, Princeton University, Princeton, NJ 08544 (United States); Fletcher, Daniel A [Bioengineering and Biophysics, University of California, Berkeley, CA 94720 (United States); Bajt, Sasa [Photon Science, DESY, Notkestrasse 85, 22607 Hamburg (Germany); Andersson, Inger [Department of Molecular Biology, Swedish University of Agricultural Sciences, Husargatan 3, Box 590, SE-751 24 Uppsala (Sweden); Chapman, Henry N, E-mail: marvin@xray.bmc.uu.s, E-mail: janos@xray.bmc.uu.s [Center for Free-Electron Laser Science, University of Hamburg and DESY, Notkestrasse 85, Hamburg (Germany)

    2010-10-14

    In a flash diffraction experiment, a short and extremely intense x-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval by iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al 2006 Nat. Phys. 2 839-43). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 fs soft x-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show that the pulse heated the sample to about 160 000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest that the average displacement of ions and atoms in the hottest surface layers remained below 3 A during the pulse.

  13. 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

    2013-05-01

    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.

  14. A microcomputed tomography guided fluorescence tomography system for small animal molecular imaging

    International Nuclear Information System (INIS)

    A prototype small animal imaging system was created for coupling fluorescence tomography (FT) with x-ray microcomputed tomography (microCT). The FT system has the potential to provide synergistic information content resultant from using microCT images as prior spatial information and then allows overlay of the FT image onto the original microCT image. The FT system was designed to use single photon counting to provide maximal sensitivity measurements in a noncontact geometry. Five parallel detector locations are used, each allowing simultaneous sampling of the fluorescence and transmitted excitation signals through the tissue. The calibration and linearity range performance of the system are outlined in a series of basic performance tests and phantom studies. The ability to image protoporphyrin IX in mouse phantoms was assessed and the system is ready for in vivo use to study biological production of this endogenous marker of tumors. This multimodality imaging system will have a wide range of applications in preclinical cancer research ranging from studies of the tumor microenvironment and treatment efficacy for emerging cancer therapeutics.

  15. Molecular Imaging Approaches to Understanding the Roles of Hydrogen Peroxide Biology in Stress and Development

    OpenAIRE

    Dickinson, Bryan Craig

    2010-01-01

    The production of hydrogen peroxide (H2O2) in biological systems is associated with a variety of pathologies including neurodegenerative diseases, cancer, and the general process of aging. However, a growing body of evidence suggests that the reactivity of this particular reactive oxygen species (ROS) is also harnessed for physiological processes. Molecular imaging using fluorescence microscopy offers a valuable approach for deciphering the multifaceted roles of H2O2 in biological processes. ...

  16. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-22

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  17. A novel, cell-permeable, fluorescent probe for ratiometric imaging of zinc ion.

    Science.gov (United States)

    Maruyama, Satoko; Kikuchi, Kazuya; Hirano, Tomoya; Urano, Yasuteru; Nagano, Tetsuo

    2002-09-11

    Zn(2+) plays important roles in various biological systems; as a result, the development of tools that can visualize chelatable Zn(2+) has attracted much attention recently. We report here newly synthesized fluorescent sensors for Zn(2+), ZnAF-Rs, whose excitation maximum is shifted by Zn(2+) under physiological conditions. Thus, these sensors enable ratiometric imaging, which is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a probe. Ratiometric measurement can provide precise data, and some probes allow quantitative detection. ZnAF-Rs are the first ratiometric fluorescent sensors for Zn(2+) that enable quantitative analysis under physiological conditions. ZnAF-Rs also possess suitable K(d) for applications, and high selectivity against other biologically relevant cations, especially Ca(2+). Using these probes, changes of intracellular Zn(2+) concentration in cultured cells were monitored successfully. We believe that these probes will be extremely useful in studies on the biological functions of Zn(2+).

  18. Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.

    Science.gov (United States)

    Alexander, Nathan S; Palczewska, Grazyna; Stremplewski, Patrycjusz; Wojtkowski, Maciej; Kern, Timothy S; Palczewski, Krzysztof

    2016-07-01

    Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)]. PMID:27446697

  19. Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.

    Science.gov (United States)

    Alexander, Nathan S; Palczewska, Grazyna; Stremplewski, Patrycjusz; Wojtkowski, Maciej; Kern, Timothy S; Palczewski, Krzysztof

    2016-07-01

    Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)].

  20. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    Science.gov (United States)

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  1. Fluorescence and image guided resection in high grade glioma.

    Science.gov (United States)

    Panciani, Pier Paolo; Fontanella, Marco; Schatlo, Bawarjan; Garbossa, Diego; Agnoletti, Alessandro; Ducati, Alessandro; Lanotte, Michele

    2012-01-01

    The extent of resection in high grade glioma is increasingly been shown to positively effect survival. Nevertheless, heterogeneity and migratory behavior of glioma cells make gross total resection very challenging. Several techniques were used in order to improve the detection of residual tumor. Aim of this study was to analyze advantages and limitations of fluorescence and image guided resection. A multicentric prospective study was designed to evaluate the accuracy of each method. Furthermore, the role of 5-aminolevulinc acid and neuronavigation were reviewed. Twenty-three patients harboring suspected high grade glioma, amenable to complete resection, were enrolled. Fluorescence and image guides were used to perform surgery. Multiple samples were obtained from the resection cavity of each lesion according to 5-ALA staining positivity and boundaries as delineated by neuronavigation. All samples were analyzed by a pathologist blinded to the intra-operative labeling. Decision-making based on fluorescence showed a sensitivity of 91.1% and a specificity of 89.4% (pimage-guided resection accuracy was low (sensitivity: 57.8%; specificity: 57.4%; p=0.346). We observed that the sensitivity of 5-ALA can be improved by the combined use of neuronavigation, but this leads to a significant reduction in specificity. Thus, the use of auxiliary techniques should always be subject to critical skills of the surgeon. We advocate a large-scale study to further improve the assessment of multimodal approaches.

  2. 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.

  3. Measurement of Nanoparticle Magnetic Hyperthermia Using Fluorescent Microthermal Imaging

    Science.gov (United States)

    Zheng, Xiaowan; van Keuren, Edward

    Nanoparticle magnetic hyperthermia uses the application of an AC magnetic field to ferromagnetic nanoparticles to elevate the temperature of cancer cells. The principle of hyperthermia as a true cell-specific therapy is that tumor cells are more sensitive to high temperature, so it is of great importance to control the locality and magnitude of the temperature differences. One technique to measure temperature variations on microscopic length scales is fluorescent microthermal imaging (FMI). Since it is the local temperature that is measured in FMI, effects such as heating due to nearby field coils can be accounted for. A dye, the rare earth chelate europium thenoyltrifluoroacetonate (Eu:TTA), with a strong temperature-dependent fluorescence emission has been incorporated into magnetic nanoparticles dispersed in a polymer films. FMI experiments were carried out on these samples under an applied high frequency magnetic field. Preliminary results show that FMI is a promising technique for characterizing the local generation of heat in nanoparticle magnetic hyperthermia.

  4. Compact wearable dual-mode imaging system for real-time fluorescence image-guided surgery

    Science.gov (United States)

    Zhu, Nan; Huang, Chih-Yu; Mondal, Suman; Gao, Shengkui; Huang, Chongyuan; Gruev, Viktor; Achilefu, Samuel; Liang, Rongguang

    2015-09-01

    A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance.

  5. Probabilistic mapping and image clustering for quantitative assessment of fluorescent protein localizations in Arabidopsis guard cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Takumi Higaki, Natsumaro Kutsuna & Seiichiro Hasezawa ### Abstract The protocol reported here describes a method to quantitatively evaluate fluorescently-tagged protein localizations from fluorescent microscopic images with a combination of probabilistic mapping and image clustering. We demonstrate the use of this protocol using kidney-shaped guard cells of plants. ### Introduction Microscopic assessment of protein localizations with fluorescent protein taggin...

  6. Detection of biological warfare agents using ultra violet-laser induced fluorescence LIDAR

    Science.gov (United States)

    Joshi, Deepti; Kumar, Deepak; Maini, Anil K.; Sharma, Ramesh C.

    This review has been written to highlight the threat of biological warfare agents, their types and detection. Bacterial biological agent Bacillus anthracis (bacteria causing the disease anthrax) which is most likely to be employed in biological warfare is being discussed in detail. Standoff detection of biological warfare agents in aerosol form using Ultra violet-Laser Induced Fluorescence (UV-LIF) spectroscopy method has been studied. Range-resolved detection and identification of biological aerosols by both nano-second and non-linear femto-second LIDAR is also discussed. Calculated received fluorescence signal for a cloud of typical biological agent Bacillus globigii (Simulants of B. anthracis) at a location of ˜5.0 km at different concentrations in presence of solar background radiation has been described. Overview of current research efforts in internationally available working UV-LIF LIDAR systems are also mentioned briefly.

  7. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil

    Directory of Open Access Journals (Sweden)

    F. Boschi

    2011-06-01

    Full Text Available Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils.

  8. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil.

    Science.gov (United States)

    Boschi, F; Fontanella, M; Calderan, L; Sbarbati, A

    2011-01-01

    Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils. PMID:22193298

  9. SIMA: Python software for analysis of dynamic fluorescence imaging data

    Directory of Open Access Journals (Sweden)

    Patrick eKaifosh

    2014-09-01

    Full Text Available Fluorescence imaging is a powerful method for monitoring dynamic signals in the nervous system. However, analysis of dynamic fluorescence imaging data remains burdensome, in part due to the shortage of available software tools. To address this need, we have developed SIMA, an open source Python package that facilitates common analysis tasks related to fluorescence imaging. Functionality of this package includes correction of motion artifacts occurring during in vivo imaging with laser-scanning microscopy, segmentation of imaged fields into regions of interest (ROIs, and extraction of signals from the segmented ROIs. We have also developed a graphical user interface (GUI for manual editing of the automatically segmented ROIs and automated registration of ROIs across multiple imaging datasets. This software has been designed with flexibility in mind to allow for future extension with different analysis methods and potential integration with other packages. Software, documentation, and source code for the SIMA package and ROI Buddy GUI are freely available at http://www.losonczylab.org/sima/.

  10. Green fluorescent protein is lighting up fungal biology

    Science.gov (United States)

    Lorang, J.M.; Tuori, R.P; Martinez, J.P; Sawyer, T.L.; Redman, R.S.; Rollins, J. A.; Wolpert, T.J.; Johnson, K.B.; Rodriguez, R.J.; Dickman, M. B.; Ciuffetti, L.M.

    2001-01-01

    Prasher (42) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfishAequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria andCaenorhabditis elegans cells in culture (10,31), followed by yeast (24, 39), mammals (40), Drosophila (66),Dictyostelium(23, 30), plants (28,49), and filamentous fungi (54). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (β-glucuronidase, β-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation ofgfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17and 67), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 (67).

  11. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  12. Synthesis and evaluation of [{sup 14}C]-Labelled and fluorescent-Tagged paclitaxel derivatives as new biological probes

    Energy Technology Data Exchange (ETDEWEB)

    Rao, C.S.; Chu, J.-J.; Lai, Y.-K. [Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, R.-S. [Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan (China)

    1998-11-01

    Our present report deals with the preparation of hitherto unreported 7-([carbonyl-{sup 14}C]-acetyl)paclitaxel 4 and two new bioactive 7-substituted fluorescent taxoids (FITC 9 and rhodamine 11), as well as evaluation towards their applications as biological probes. The results in this report demonstrate that (a) the new paclitaxel derivatives 4, 9, 11 could be prepared with good yields starting from paclitaxel; (b) the [{sup 14}C]acetylation step was found to be better by using [{sup 14}C]acetic anhydride rather than [{sup 14}C]sodium acetate; (c) the radiochemical purity of 4 was 96% and its specific activity was 48 mCi/mmol; (d) the cytotoxicity of 4 was close to that of paclitaxel whereas 9, 11 were far less active than paclitaxel, but these cytotoxic levels were good enough for their biological applications; (e) the drug-quantitation by flow cytometric analysis using 9 and 11 was proved to be equally efficient with respect to the radioactivity-based determination employing 4; (f) the intracellular fluorescence mapping by 9 and 11 was found to be effective and the microtubule network pattern was visible in both the cases; (g) the overall fluorescence imaging efficiency was better with 11 while the intensity of fluorescence was higher with 9; (h) staining of nucleolus was observed in fluorescence studies of both 9 and 11. Based on these results, the newly prepared paclitaxel derivatives can be considered as efficient biological probes and should find further use in relevant applications. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  13. Two-photon fluorescence imaging through multicore fiber with digital phase conjugation

    Science.gov (United States)

    Stasio, Nicolino; Conkey, Donald B.; Moser, Christophe; Psaltis, Demetri

    2016-03-01

    We present near diffraction limited two photon fluorescence (TPF) imaging through a lensless, multi-core fiber (MCF) endoscope utilizing digital phase conjugation. The ultra-small size of MCFs make them desirable tools for imaging deep into the body. TPF imaging enables optical sectioning and is widely used in brain and biological imaging and is a desired modality for fiber endoscopes. Previous implementations of TPF imaging through MCFs focus and scan the light from individual cores for image formation. In such systems the resolution is limited by the MCF core spacing, although a lens may be used to improve the resolution at the expense of the field of view. Other, more recent work has improved the resolution limitation using custom built MCFs for focusing and scanning of ultrafast pulses using wavefront shaping. Here we present digital phase conjugation for ultrafast pulse focusing through a MCF for an imaging resolution independent of the MCF core spacing. Furthermore, the phase conjugation technique does not require the use of a lens at the fiber end for focus formation and is compatible with commercially available MCFs with a large number of cores. Here, we present a 3000 core MCF endoscope and demonstrate ultrafast pulse focusing with sufficient focus spot contrast and power for TPF endoscope imaging. We construct TPF images by digital scanning of the phase conjugated focus on the target object and collection of the emitted fluorescence through the MCF. This work demonstrates the viability of digital conjugation combined with commercially available MCFs for higher resolution lensless, two photon endoscopy.

  14. 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.

  15. Fluorescent probes in biology and medicine: measurement of intracellular pH values in individual cells

    Science.gov (United States)

    Slavik, Jan; Cimprich, Petr; Gregor, Martin; Smetana, Karel, Jr.

    1997-12-01

    The application possibilities of fluorescent probes have increased dramatically in the last few years. The main areas are as follows (Slavik, 1994, 1996, 1998). Intracellular ionic cell composition: There are selective ion-sensitive dyes for H+, Ca2+, Mg2+, K+, Na+, Fe3+, Cl-, Zn2+, Cd2+, Hg2+, Pb2+, Ba2+, La3+. Membrane potential: Using the so-called slow (Nernstian dyes) or electrochromic dyes one can assess the value of the transmembrane potential. Membrane fluidity: Fluorescent probes inform about the freedom of rotational and translational movement of membrane proteins and lipids. Selective labeling: Almost any object of interest inside the cell or on its surface can be selectively fluorescently labeled. There are dyes specific for DNA, RNA, oligonucleotides (FISH), Golgi, endoplasmic reticulum, mitochondria, vacuoles, cytoskeleton, etc. Using fluorescent dyes specific receptors may be localized, their conformational changes followed and the polarity of corresponding binding sites accessed. The endocytic pathway may be followed, enzymes and their local enzymatic activity localized. For really selective labeling fluorescent labeled antibodies exist. Imaging: One of the main advantages of fluorescence imaging is its versatility. It allow choice among ratio imaging in excitation, ratio imaging in emission and lifetime imaging. These approaches can be applied to both the classical wide-field fluorescence microscopy and to the laser confocal fluorescence microscopy, one day possibly to the scanning near field optical microscopy. Simultaneous application of several fluorescent dyes: The technical progress in both excitation sources and in detectors allows to extend the excitation deeper in the blue and ultraviolet side and the detection further in the NIR and IR. Consequently, up to 6 peaks in excitation and up to 6 peaks in emission can be followed without any substantial difficulties. Application of dyes such with longer fluorescence lifetimes such as rare earth

  16. 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.

  17. 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

  18. Dynamic fluorescence imaging for multiparametric measurement of tumor vasculature

    Science.gov (United States)

    Choi, Myunghwan; Choi, Kyungsun; Ryu, Seung-Wook; Lee, Jungwhoi; Choi, Chulhee

    2011-04-01

    Angiogenesis is essential for tumor growth and a promising target for cancer therapy. Blood vessel monitoring is an indispensable tool for evaluation and development of anti-angiogenic drugs. Here, we report a new noninvasive in vivo imaging tool, named dynamic fluorescence imaging (DyFI), for the simultaneous measurement of multiple vascular parameters including vascular density, perfusion rate, and permeability using spatiotemporal profiles of indocyanine green. Using DyFI in a tumor xenograft model, we quantitatively measured multiple vascular parameters in tumors and normal tissues with high spatial resolution. The multimodality of this method allowed us to find negative spatial correlations between perfusion and permeability. Moreover, DyFI was effective for revealing the early effects of an anti-angiogenic drug. We suggest that DyFI could be a useful tool for the preclinical development of anti-angiogenic drugs.

  19. Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron.

    Science.gov (United States)

    Kim, Kyoung-Ran; Lee, Yong-Deok; Lee, Taemin; Kim, Byeong-Su; Kim, Sehoon; Ahn, Dae-Ro

    2013-07-01

    Sentinel lymph nodes (SLNs) are the first lymph nodes which cancer cells reach after traveling through lymphatic vessels from the primary tumor. Evaluating the nodal status is crucial in accurate staging of human cancers and accordingly determines prognosis and the most appropriate treatment. The commonly used methods for SLN identification in clinics are based on employment of a colloid of radionuclide or injection of a small dye. Although these methods have certainly contributed to improve surgical practice, new imaging materials are still required to overcome drawbacks of the techniques such as inconvenience of handling radioactive materials and short retention time of small dyes in SLNs. Here, we prepare a fluorescence-labeled DNA tetrahedron and perform SLN imaging by using the DNA nanoconstruct. With a successful identification of SLNs by the DNA nanoconstruct, we suggest that DNA tetrahedron hold great promises for clinical applications.

  20. Inorganic phosphate nanorods are a novel fluorescent label in cell biology

    Directory of Open Access Journals (Sweden)

    Mukherjee Priyabrata

    2006-10-01

    Full Text Available Abstract We report the first use of inorganic fluorescent lanthanide (europium and terbium ortho phosphate [LnPO4·H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC, 786-O cells, or renal carcinoma cells (RCC. The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS, differential interference contrast (DIC microscopy, confocal microscopy, and transmission electron microscopy (TEM. At concentrations up to 50 μg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL, and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes

  1. A galvanic replacement route to prepare strongly fluorescent and highly stable gold nanodots for cellular imaging.

    Science.gov (United States)

    Wang, Chuanxi; Wang, Yu; Xu, Lin; Shi, Xiaodong; Li, Xiangwei; Xu, Xiaowei; Sun, Hongchen; Yang, Bai; Lin, Quan

    2013-02-11

    Fluorescent gold nanodots (GNDs) are an important kind of nanoprobes. Herein, the application of galvanic replacement for the preparation of fluorescent GNDs is reported. Using presynthesized and size-controlled Ag nanodots (Ag NDs) as templates, the as-prepared GNDs have strong fluorescence (quantum yields ~10%) with high stability and surface bioactivity. The resultant GNDs show excellent photoluminescence properties with high photo-, time-, metal-, and pH-stability, which are attributed to the protective surface layer of glutathione (GSH) and the presence of Au(I)-S complexes on the surface of the gold core. GSH, a naturally occurring and readily available tripeptide with carboxyl and amino functional groups, allows good dispersion of the as-prepared GNDs in aqueous solution and favorable biocompatibility. These advantages, combined with their small size, mean that the as-prepared GNDs have potential application in biological labeling, especially as a DNA probe for the specific detection of nucleic acids. In this study, the CAL-27 cells are used as a model to evaluate the fluorescence imaging of GNDs.

  2. Light-induced fluorescence endoscopy (LIFE) imaging system for early cancer detection

    Science.gov (United States)

    Zeng, Haishan; MacAulay, Calum E.; Lam, Stephen; Palcic, Branko

    1999-09-01

    This paper summarizes our experiences on the development of a Light Induced Fluorescence Endoscopy (LIFE) imaging system for early cancer detection in the respiratory and gastrointestinal tract. The system utilizes tissue autofluorescence to provide real time video imaging of the examined organ. No exogenous fluorescent tumor markers are needed. It is used by a physician in adjunct to conventional white-light endoscopy. Suspicious areas are identified in pseudo color to guide biopsy. A multi- center clinical trial has demonstrated that in the lung, the relative sensitivity of white-light imaging + LIFE imaging vs. white-light imaging alone was 6.3 for intraepithelial neoplastic lesion detection and 2.71 when invasive carcinomas were also included. The following issues will be discussed: (1) spectroscopy study design for imaging system development; (2) architecture of the imaging systems; (3) different imaging modalities (white-light imaging, dual channel fluorescence imaging, and combined fluorescence/reflectance imaging); and (4) clinical applications.

  3. In vivo optical imaging of acute cell death using a near-infrared fluorescent zinc-dipicolylamine probe.

    Science.gov (United States)

    Smith, Bryan A; Gammon, Seth T; Xiao, Shuzhang; Wang, Wei; Chapman, Sarah; McDermott, Ryan; Suckow, Mark A; Johnson, James R; Piwnica-Worms, David; Gokel, George W; Smith, Bradley D; Leevy, W Matthew

    2011-04-01

    Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near-infrared (NIR) conjugate of zinc(II)-dipicolylamine (Zn²+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as cytotoxins. The Zn²+-DPA fluorescent probe or corresponding control was subsequently injected, and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a NIR fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn²+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates.

  4. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    Science.gov (United States)

    Veetil, Jithesh V.; Jin, Sha; Ye, Kaiming

    2012-01-01

    Background One of the major hurdles in studying diabetes pathophysiology is the lack of adequate methodology that allows for direct and real-time determination of glucose transport and metabolism in cells and tissues. In this article, we present a new methodology that adopts frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) to visualize and quantify the dynamics of intracellular glucose within living cells using a biosensor protein based on fluorescence resonance energy transfer (FRET). Method The biosensor protein was developed by fusing a FRET pair, an AcGFP1 donor and a mCherry acceptor to N- and C- termini of a mutant glucose-binding protein (GBP), respectively. The probe was expressed and biosynthesized inside the cells, offering continuous monitoring of glucose dynamics in real time through fluorescence lifetime imaging microscopy (FLIM) measurement. Results We transfected the deoxyribonucleic acid of the AcGFP1-GBP-mCherry sensor into murine myoblast cells, C2C12, and continuously monitored the changes in intracellular glucose concentrations in response to the variation in extracellular glucose, from which we determined glucose uptake and clearance rates. The distribution of intracellular glucose concentration was also characterized. We detected a high glucose concentration in a region close to the cell membrane and a low glucose concentration in a region close to the nucleus. The monoexponential decay of AcGFP1 was distinguished using FD-FLIM. Conclusions This work enables continuous glucose monitoring (CGM) within living cells using FD-FLIM and a biosensor protein. The sensor protein developed offers a new means for quantitatively analyzing glucose homeostasis at the cellular level. Data accumulated from these studies will help increase our understanding of the pathology of diabetes. PMID:23294772

  5. Frequency Domain Fluorescent Molecular Tomography and Molecular Probes for Small Animal Imaging

    Science.gov (United States)

    Kujala, Naresh Gandhi

    Fluorescent molecular tomography (FMT) is a noninvasive biomedical optical imaging that enables 3-dimensional quantitative determination of fluorochromes distributed in biological tissues. There are three methods for imaging large volume tissues based on different light sources: (a) using a light source of constant intensity, through a continuous or constant wave, (b) using a light source that is intensity modulated with a radio frequency (RF), and (c) using ultrafast pulses in the femtosecond range. In this study, we have developed a frequency domain fluorescent molecular tomographic system based on the heterodyne technique, using a single source and detector pair that can be used for small animal imaging. In our system, the intensity of the laser source is modulated with a RF frequency to produce a diffuse photon density wave in the tissue. The phase of the diffuse photon density wave is measured by comparing the reference signal with the signal from the tissue using a phasemeter. The data acquisition was performed by using a Labview program. The results suggest that we can measure the phase change from the heterogeneous inside tissue. Combined with fiber optics and filter sets, the system can be used to sensitively image the targeted fluorescent molecular probes, allowing the detection of cancer at an early stage. We used the system to detect the tumor-targeting molecular probe Alexa Fluor 680 and Alexa Fluor 750 bombesin peptide conjugates in phantoms as well as mouse tissues. We also developed and evaluated fluorescent Bombesin (BBN) probes to target gastrin-releasing peptide (GRP) receptors for optical molecular imaging. GRP receptors are over-expressed in several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. BBN is a 14 amino acid peptide that is an analogue to human gastrin-releasing peptide that binds specifically to GRPr receptors. BBN conjugates are significant in cancer detection and therapy. The

  6. High Resolution Fluorescence Imaging of Cancers Using Lanthanide Ion-Doped Upconverting Nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Naccache, Rafik; Rodríguez, Emma Martín; Bogdan, Nicoleta [Department of Chemistry and Biochemistry, Concordia University, Montreal H4B 1R6 (Canada); Sanz-Rodríguez, Francisco [Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 28049 (Spain); Cruz, Maria del Carmen Iglesias de la [Departamento de Fisiología. Facultad de Medicina, Universidad Autónoma de Madrid, Madrid 28029 (Spain); Fuente, Ángeles Juarranz de la [Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 28049 (Spain); Vetrone, Fiorenzo [Institut National de la Recherche Scientifique-Énergie, Matériaux et Télécommunications, Université du Québec, Varennes J3X 1S2 (Canada); Jaque, Daniel; Solé, José García, E-mail: jose.garcia_sole@uam.es [Departamento de Física de Materiales, Universidad Autónoma de Madrid, Madrid 28049 (Spain); Capobianco, John A., E-mail: jose.garcia_sole@uam.es [Department of Chemistry and Biochemistry, Concordia University, Montreal H4B 1R6 (Canada)

    2012-10-22

    During the last decade inorganic luminescent nanoparticles that emit visible light under near infrared (NIR) excitation (in the biological window) have played a relevant role for high resolution imaging of cancer. Indeed, semiconductor quantum dots (QDs) and metal nanoparticles, mostly gold nanorods (GNRs), are already commercially available for this purpose. In this work we review the role which is being played by a relatively new class of nanoparticles, based on lanthanide ion doped nanocrystals, to target and image cancer cells using upconversion fluorescence microscopy. These nanoparticles are insulating nanocrystals that are usually doped with small percentages of two different rare earth (lanthanide) ions: The excited donor ions (usually Yb{sup 3+} ion) that absorb the NIR excitation and the acceptor ions (usually Er{sup 3+}, Ho{sup 3+} or Tm{sup 3+}), that are responsible for the emitted visible (or also near infrared) radiation. The higher conversion efficiency of these nanoparticles in respect to those based on QDs and GNRs, as well as the almost independent excitation/emission properties from the particle size, make them particularly promising for fluorescence imaging. The different approaches of these novel nanoparticles devoted to “in vitro” and “in vivo” cancer imaging, selective targeting and treatment are examined in this review.

  7. High Resolution Fluorescence Imaging of Cancers Using Lanthanide Ion-Doped Upconverting Nanocrystals

    International Nuclear Information System (INIS)

    During the last decade inorganic luminescent nanoparticles that emit visible light under near infrared (NIR) excitation (in the biological window) have played a relevant role for high resolution imaging of cancer. Indeed, semiconductor quantum dots (QDs) and metal nanoparticles, mostly gold nanorods (GNRs), are already commercially available for this purpose. In this work we review the role which is being played by a relatively new class of nanoparticles, based on lanthanide ion doped nanocrystals, to target and image cancer cells using upconversion fluorescence microscopy. These nanoparticles are insulating nanocrystals that are usually doped with small percentages of two different rare earth (lanthanide) ions: The excited donor ions (usually Yb3+ ion) that absorb the NIR excitation and the acceptor ions (usually Er3+, Ho3+ or Tm3+), that are responsible for the emitted visible (or also near infrared) radiation. The higher conversion efficiency of these nanoparticles in respect to those based on QDs and GNRs, as well as the almost independent excitation/emission properties from the particle size, make them particularly promising for fluorescence imaging. The different approaches of these novel nanoparticles devoted to “in vitro” and “in vivo” cancer imaging, selective targeting and treatment are examined in this review

  8. Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging.

    Science.gov (United States)

    Shakiba, Mojdeh; Ng, Kenneth K; Huynh, Elizabeth; Chan, Harley; Charron, Danielle M; Chen, Juan; Muhanna, Nidal; Foster, F Stuart; Wilson, Brian C; Zheng, Gang

    2016-07-01

    J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J-aggregates for in vivo bioimaging applications. We find that 15 mol% Bchl-lipid embedded within a saturated phospholipid bilayer vesicle was optimal in terms of maximizing Bchl-lipid dye loading, while maintaining a spherical nanoparticle morphology and retaining spectral properties characteristic of J-aggregates. The addition of cholesterol maintains the stability of the J-aggregate absorption band for up to 6 hours in the presence of 90% FBS. In a proof-of-concept experiment, we successfully applied JNPs as a fluorescence contrast agent for real-time intraoperative detection of metastatic lymph nodes in a rabbit head-and-neck cancer model. Lymph node metastasis delineation was further verified by visualizing the JNP within the excised lymph node using photoacoustic imaging. Using JNPs, we demonstrate the possibility of using J-aggregates as fluorescence and photoacoustic contrast agents and may potentially spur the development of other nanomaterials that can stably induce J-aggregation for in vivo cancer bioimaging applications. PMID:26731304

  9. Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging.

    Science.gov (United States)

    Shakiba, Mojdeh; Ng, Kenneth K; Huynh, Elizabeth; Chan, Harley; Charron, Danielle M; Chen, Juan; Muhanna, Nidal; Foster, F Stuart; Wilson, Brian C; Zheng, Gang

    2016-07-01

    J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J-aggregates for in vivo bioimaging applications. We find that 15 mol% Bchl-lipid embedded within a saturated phospholipid bilayer vesicle was optimal in terms of maximizing Bchl-lipid dye loading, while maintaining a spherical nanoparticle morphology and retaining spectral properties characteristic of J-aggregates. The addition of cholesterol maintains the stability of the J-aggregate absorption band for up to 6 hours in the presence of 90% FBS. In a proof-of-concept experiment, we successfully applied JNPs as a fluorescence contrast agent for real-time intraoperative detection of metastatic lymph nodes in a rabbit head-and-neck cancer model. Lymph node metastasis delineation was further verified by visualizing the JNP within the excised lymph node using photoacoustic imaging. Using JNPs, we demonstrate the possibility of using J-aggregates as fluorescence and photoacoustic contrast agents and may potentially spur the development of other nanomaterials that can stably induce J-aggregation for in vivo cancer bioimaging applications.

  10. An (125)I-labeled octavalent peptide fluorescent nanoprobe for tumor-homing imaging in vivo.

    Science.gov (United States)

    Luo, Haiming; Shi, Jiyun; Jin, Honglin; Fan, Di; Lu, Lisen; Wang, Fan; Zhang, Zhihong

    2012-06-01

    Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, (125)I-Octa-FNP, (125)I-tfRFP and (125)I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that (125)I-Octa-FNP had a significantly higher cellular binding capability than (125)I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, (125)I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of (125)I-tfRFP and (125)I-peptide, respectively. Moreover, γ-imaging at 24 h post-injection revealed a remarkable accumulation of (125)I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics.

  11. Combining OCT and a fluorescence intensity imaging method for atherosclerosis detection

    Science.gov (United States)

    Liang, Shanshan; Saidi, Arya; Jing, Joe; Liu, Gangjun; Yin, Jiechen; Narula, Jagat; Chen, Zhongping

    2012-02-01

    Coronary heart disease (like myocardial infarction) is caused by atherosclerosis. It cause over 30% of all deaths in North America and are the most common cause of death in European men under 65 years of age and the second most common cause in women. To diagnose this atherosclerosis before it gets rupture is the most effect way to increase the chance of survival for patients who suffer from this disease. The crucial tusk is how to find out vulnerable plaques. In resent years optical coherence tomography (OCT) has become a very useful tool for intravascular imaging, since it has high axial and transverse resolution. OCT can tell the detail structure inside the plaque like the thickness of plaque cap which is an important factor to identify vulnerable plaques. But we still need to find out the biochemical characteristics that is unique for vulnerable plaques (like inflammation). Fluorescence molecular imaging is a standard way to exam the biochemical property of biological samples. So we integrate these two techniques together into one probe. Our probe is comprised of a double-clad fiber (DCF) and a grin lens, and rotates with a micro mirror in front. The single-mode inner core of the DCF transmits both OCT and fluorescence excitation light, and the multimode inner cladding is used to detect fluorescence signal. In vitro result shows that this is a possible way for more accurate diagnose of vulnerable plaques.

  12. Improved detection of fluorescently labeled microspheres and vessel architecture with an imaging cryomicrotome

    NARCIS (Netherlands)

    P. van Horssen; M. Siebes; I. Hoefer; J.A.E. Spaan; J.P.H.M. van den Wijngaard

    2010-01-01

    Due to spectral overlap, the number of fluorescent labels for imaging cryomicrotome detection was limited to 4. The aim of this study was to increase the separation of fluorescent labels. In the new imaging cryomicrotome, the sample is cut in slices of 40 mu m. Six images are taken for each cutting

  13. Determination of uranium in seawater, biological samples and sediments using laser induced fluorescence spectrometry

    International Nuclear Information System (INIS)

    Uranium has been determined in seawater, biological samples and sediments using laser induced fluorescence spectrometry (LIFS). The biological samples and sediments are digested with a mixture of HNO3, HClO4 and HF. The conductivity of the seawater should be below 5.0 mS and the pH of the sample should be in the range 6.5-9.0. The volume of the reagent used to enhance the fluorescence intensity was 0.5 ml. Comparison with other methods was favorable, LIFS being rapid, simple and sensitive, and well suited to environmental monitoring. (author)

  14. Analyzing free zinc(II) ion concentrations in cell biology with fluorescent chelating molecules.

    Science.gov (United States)

    Maret, Wolfgang

    2015-02-01

    Essential metal ions are tightly controlled in biological systems. An understanding of metal metabolism and homeostasis is being developed from quantitative information of the sizes, concentrations, and dynamics of cellular and subcellular metal ion pools. In the case of human zinc metabolism, minimally 24 proteins of two zinc transporter families and a dozen metallothioneins participate in cellular uptake, extrusion, and re-distribution among cellular compartments. Significantly, zinc(ii) ions are now considered signaling ions in intra- and intercellular communication. Such functions require transients of free zinc ions. It is experimentally quite challenging to distinguish zinc that is protein-bound from zinc that is not bound to proteins. Measurement of total zinc is relatively straightforward with analytical techniques such as atomic absorption/emission spectroscopy or inductively coupled plasma mass spectrometry. Total zinc concentrations of human cells are 200-300 μM. In contrast, the pool of non-protein bound zinc is mostly examined with fluorescence microscopy/spectroscopy. There are two widely applied fluorescence approaches, one employing low molecular weight chelating agents ("probes") and the other metal-binding proteins ("sensors"). The protein sensors, such as the CALWY, Zap/ZifCY, and carbonic anhydrase-based sensors, can be genetically encoded and have certain advantages in terms of controlling intracellular concentration, localization, and calibration. When employed correctly, both probes and sensors can establish qualitative differences in free zinc ion concentrations. However, when quantitative information is sought, the assumptions underlying the applications of probes and sensors must be carefully examined and even then measured pools of free zinc ions remain methodologically defined. A consensus is building that the steady-state free zinc ion concentrations in the cytosol are in the picomolar range but there is no consensus on their

  15. Motion corrected photoacoustic difference imaging of fluorescent contrast agents

    Science.gov (United States)

    Märk, Julia; Wagener, Asja; Pönick, Sarah; Grötzinger, Carsten; Zhang, Edward; Laufer, Jan

    2016-03-01

    In fluorophores, such as exogenous dyes and genetically expressed proteins, the excited state lifetime can be modulated using pump-probe excitation at wavelengths corresponding to the absorption and fluorescence spectra. Simultaneous pump-probe pulses induce stimulated emission (SE) which, in turn, modulates the thermalized energy, and hence the photoacoustic (PA) signal amplitude. For time-delayed pulses, by contrast, SE is suppressed. Since this is not observed in endogenous chromophores, the location of the fluorophore can be determined by subtracting images acquired using simultaneous and time-delayed pump-probe excitation. This simple experimental approach exploits a fluorophorespecific contrast mechanism, and has the potential to enable deep-tissue molecular imaging at fluences below the MPE. In this study, some of the challenges to its in vivo implementation are addressed. First, the PA signal amplitude generated in fluorophores in vivo is often much smaller than that in blood. Second, tissue motion can give rise to artifacts that correspond to endogenous chromophores in the difference image. This would not allow the unambiguous detection of fluorophores. A method to suppress motion artifacts based on fast switching between simultaneous and time-delayed pump-probe excitation was developed. This enables the acquisition of PA signals using the two excitation modes with minimal time delay (20 ms), thus minimizing the effects of tissue motion. The feasibility of this method is demonstrated by visualizing a fluorophore (Atto680) in tissue phantoms, which were moved during the image acquisition to mimic tissue motion.

  16. Fluorescence Imaging of the Cytoskeleton in Plant Roots.

    Science.gov (United States)

    Dyachok, Julia; Paez-Garcia, Ana; Yoo, Cheol-Min; Palanichelvam, Karuppaiah; Blancaflor, Elison B

    2016-01-01

    During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.

  17. Solid-State Camera System for Fluorescence Lifetime Microscopy

    NARCIS (Netherlands)

    Zhao, Q.

    2014-01-01

    Fluorescence microscopy is a well-established platform for biology and biomedical research (Chapter 2). Based on this platform, fluorescence lifetime imaging microscopy (FLIM) has been developed to measure fluorescence lifetimes, which are independent of fluorophore concentration and excitation inte

  18. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.

    Science.gov (United States)

    Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

    2016-01-01

    Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series. PMID:26967231

  19. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    Science.gov (United States)

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  20. Method for single illumination source combined optical coherence tomography and fluorescence imaging of fluorescently labeled ocular structures in transgenic mice.

    Science.gov (United States)

    McNabb, Ryan P; Blanco, Tomas; Bomze, Howard M; Tseng, Henry C; Saban, Daniel R; Izatt, Joseph A; Kuo, Anthony N

    2016-10-01

    In vivo imaging permits longitudinal study of ocular disease processes in the same animal over time. Two different in vivo optical imaging modalities - optical coherence tomography (OCT) and fluorescence - provide important structural and cellular data respectively about disease processes. In this Methods in Eye Research article, we describe and demonstrate the combination of these two modalities producing a truly simultaneous OCT and fluorescence imaging system for imaging of fluorescently labeled animal models. This system uses only a single light source to illuminate both modalities, and both share the same field of view. This allows simultaneous acquisition of OCT and fluorescence images, and the benefits of both techniques are realized without incurring increased costs in variability, light exposure, time, and post-processing effort as would occur when the modalities are used separately. We then utilized this system to demonstrate multi-modal imaging in a progression of samples exhibiting both fluorescence and OCT scattering beginning with resolution targets, ex vivo thy1-YFP labeled neurons in mouse eyes, and finally an in vivo longitudinal time course of GFP labeled myeloid cells in a mouse model of ocular allergy. PMID:27519152

  1. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    Science.gov (United States)

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

    2015-09-01

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min-1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels-1.

  2. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    Energy Technology Data Exchange (ETDEWEB)

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J., E-mail: tmuldoon@uark.edu [Department of Biomedical Engineering, University of Arkansas, 120 Engineering Hall, Fayetteville, Arkansas 72701 (United States)

    2015-09-15

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.

  3. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    International Nuclear Information System (INIS)

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min−1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels−1

  4. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization.

    Science.gov (United States)

    Hutcheson, Joshua A; Majid, Aneeka A; Powless, Amy J; Muldoon, Timothy J

    2015-09-01

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min(-1) with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels(-1). PMID:26429450

  5. Full Field X-Ray Fluorescence Imaging Using Micro Pore Optics for Planetary Surface Exploration

    Science.gov (United States)

    Sarrazin, P.; Blake, D. F.; Gailhanou, M.; Walter, P.; Schyns, E.; Marchis, F.; Thompson, K.; Bristow, T.

    2016-01-01

    Many planetary surface processes leave evidence as small features in the sub-millimetre scale. Current planetary X-ray fluorescence spectrometers lack the spatial resolution to analyse such small features as they only provide global analyses of areas greater than 100 mm(exp 2). A micro-XRF spectrometer will be deployed on the NASA Mars 2020 rover to analyse spots as small as 120m. When using its line-scanning capacity combined to perpendicular scanning by the rover arm, elemental maps can be generated. We present a new instrument that provides full-field XRF imaging, alleviating the need for precise positioning and scanning mechanisms. The Mapping X-ray Fluorescence Spectrometer - "Map-X" - will allow elemental imaging with approximately 100µm spatial resolution and simultaneously provide elemental chemistry at the scale where many relict physical, chemical and biological features can be imaged in ancient rocks. The arm-mounted Map-X instrument is placed directly on the surface of an object and held in a fixed position during measurements. A 25x25 mm(exp 2) surface area is uniformly illuminated with X-rays or alpha-particles and gamma-rays. A novel Micro Pore Optic focusses a fraction of the emitted X-ray fluorescence onto a CCD operated at a few frames per second. On board processing allows measuring the energy and coordinates of each X-ray photon collected. Large sets of frames are reduced into 2d histograms used to compute higher level data products such as elemental maps and XRF spectra from selected regions of interest. XRF spectra are processed on the ground to further determine quantitative elemental compositions. The instrument development will be presented with an emphasis on the characterization and modelling of the X-ray focussing Micro Pore Optic. An outlook on possible alternative XRF imaging applications will be discussed.

  6. Fluorescent Cy5 silica nanoparticles for cancer cell imaging

    Science.gov (United States)

    O'Connell, Claire; Nooney, Robert I.; Glynn, MacDara; Ducree, Jens; McDonagh, Colette

    2015-08-01

    Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter ~50 nm which contain a large concentration of Cy5 dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free dye. They do not photo bleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other dyes will be doped into the silica NPs and their use in multiplexing will be investigated.

  7. Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays

    Directory of Open Access Journals (Sweden)

    Robert K. Henderson

    2012-05-01

    Full Text Available We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD-based cameras for fluorescence lifetime imaging microscopy (FLIM by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast.

  8. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    International Nuclear Information System (INIS)

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  9. Monomeric red fluorescent protein variants used for imaging studies in different species

    NARCIS (Netherlands)

    Mueller-Taubenberger, Annette; Vos, Michel J.; Boettger, Angelika; Lasi, Margherita; Lai, Frank P. L.; Fischer, Markus; Rottner, Klemens

    2006-01-01

    Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation

  10. Quantitative imaging of cell-permeable magnetic resonance contrast agents using x-ray fluorescence.

    Science.gov (United States)

    Endres, Paul J; Macrenaris, Keith W; Vogt, Stefan; Allen, Matthew J; Meade, Thomas J

    2006-01-01

    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-dimethylamino)stilbene. 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 10(3) better than (153)Gd gamma counting, without altering the agent by organic fluorophore conjugation. The transduction efficiency of the intracellular agents was evaluated by T(1) analysis and inductively coupled plasma mass spectrometry to determine the efficacy of each chelate-transporter combination. PMID:17150161

  11. Single aflatoxin contaminated corn kernel analysis with fluorescence hyperspectral image

    Science.gov (United States)

    Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Ononye, Ambrose; Brown, Robert L.; Cleveland, Thomas E.

    2010-04-01

    Aflatoxins are toxic secondary metabolites of the fungi Aspergillus flavus and Aspergillus parasiticus, among others. Aflatoxin contaminated corn is toxic to domestic animals when ingested in feed and is a known carcinogen associated with liver and lung cancer in humans. Consequently, aflatoxin levels in food and feed are regulated by the Food and Drug Administration (FDA) in the US, allowing 20 ppb (parts per billion) limits in food and 100 ppb in feed for interstate commerce. Currently, aflatoxin detection and quantification methods are based on analytical tests including thin-layer chromatography (TCL) and high performance liquid chromatography (HPLC). These analytical tests require the destruction of samples, and are costly and time consuming. Thus, the ability to detect aflatoxin in a rapid, nondestructive way is crucial to the grain industry, particularly to corn industry. Hyperspectral imaging technology offers a non-invasive approach toward screening for food safety inspection and quality control based on its spectral signature. The focus of this paper is to classify aflatoxin contaminated single corn kernels using fluorescence hyperspectral imagery. Field inoculated corn kernels were used in the study. Contaminated and control kernels under long wavelength ultraviolet excitation were imaged using a visible near-infrared (VNIR) hyperspectral camera. The imaged kernels were chemically analyzed to provide reference information for image analysis. This paper describes a procedure to process corn kernels located in different images for statistical training and classification. Two classification algorithms, Maximum Likelihood and Binary Encoding, were used to classify each corn kernel into "control" or "contaminated" through pixel classification. The Binary Encoding approach had a slightly better performance with accuracy equals to 87% or 88% when 20 ppb or 100 ppb was used as classification threshold, respectively.

  12. Near-infrared (NIR) fluorescence imaging of head and neck squamous cell carcinoma for fluorescence-guided surgery (Conference Presentation)

    Science.gov (United States)

    Moore, Lindsay; Warram, Jason M.; de Boer, Esther; Carroll, William R.; Morlandt, Anthony; Withrow, Kirk P.; Rosenthal, Eben L.

    2016-03-01

    During fluorescence-guided surgery, a cancer-specific optical probe is injected and visualized using a compatible device intraoperatively to provide visual contrast between diseased and normal tissues to maximize resection of cancer and minimize the resection of precious adjacent normal tissues. Six patients with squamous cell carcinomas of the head and neck region (oral cavity (n=4) or cutaneous (n=2)) were injected with an EGFR-targeting antibody (Cetuximab) conjugated to a near-infrared (NIR) fluorescent dye (IRDye800) 3, 4, or 7 days prior to surgical resection of the cancer. Each patient's tumor was then imaged using a commercially available, open-field NIR fluorescence imaging device each day prior to surgery, intraoperatively, and post-operatively. The mean fluorescence intensity (MFI) of the tumor was calculated for each specimen at each imaging time point. Adjacent normal tissue served as an internal anatomic control for each patient to establish a patient-matched "background" fluorescence. Resected tissues were also imaged using a closed-field NIR imaging device. Tumor to background ratios (TBRs) were calculated for each patient using both devices. Fluorescence histology was correlated with traditional pathology assessment to verify the specificity of antibody-dye conjugate binding. Peak TBRs using the open-field device ranged from 2.2 to 11.3, with an average TBR of 4.9. Peak TBRs were achieved between days 1 and 4. This study demonstrated that a commercially available NIR imaging device suited for intraoperative and clinical use can successfully be used with a fluorescently-labeled dye to delineate between diseased and normal tissue in this single cohort human study, illuminated the potential for its use in fluoresence-guided surgery.

  13. FEM-based simulation of a fluorescence tomography experiment using anatomical MR images

    Science.gov (United States)

    Ren, Wuwei; Elmer, Andreas; Augath, Mark-Aurel; Rudin, Markus

    2016-03-01

    A hybrid system combining fluorescence molecular tomography (FMT) and magnetic resonance imaging (MRI) is attractive for preclinical imaging as it allows fusion of molecular information derived from FMT and anatomical reference data derived from MRI. We have previously developed such a system and demonstrated its performance in biological applications. For reconstruction slab geometry with homogeneous optical parameters was assumed, which led to undesirable artifacts. In order to exploit the power of the hybrid system, the use of MRI derived anatomical information, as a constraint for FMT reconstruction, appears logical. Heterogeneity of tissues and irregular surface derived from MRI can be accounted for by generating a mesh using the finite element method (FEM), and attributing optical parameters to individual mesh points. We have established a forward simulation tool based on TOAST++ to mimic an FMT experiment. MRI images were recorded on a 9.4T MR scanner using a T1-weighted pulse sequence. The voxelized dataset was processed by iso2mesh to yield a 3D-mesh. Four steps of FMT simulation were included: 1) Assignment of optical properties, 2) Specification of boundary conditions and generation of 3) excitation and 4) emission maps. FEM-derived results were compared with those obtained using the analytical solution of Green's function and with experimental data with a single fluorescent inclusion in a silicon phantom. Once, the forward modeling method is properly validated it will be used as a central element of a reconstruction algorithm for analyzing data derived from a hybrid FMT/MRI setup.

  14. Lateral width of shower image in the Auger fluorescence detector

    CERN Document Server

    Barbosa, H; Dobrigkeit, C; Engel, R; Gora, D; Heck, D; Homola, P; Klages, H; Medina-Tanco, G; Ortiz, J A; Pekala, J; Risse, M; Wilczynska, B; Wilczynski, H

    2005-01-01

    The impact of the lateral distribution of light in extensive air showers on the detection and reconstruction of shower profiles is investigated for the Auger fluorescence telescopes. Based on three-dimensional simulations, the capability of the Auger telescopes to measure the lateral distribution of light is evaluated. The ability to infer the actual lateral distribution is confirmed by the comparison of detailed simulations with real data. The contribution of pixels located far from the axis of the shower image is calculated and the accepted signal is rescaled in order to reconstruct a correct shower profile. The analysis presented here shows that: (a) the Auger telescopes are able to observe the lateral distribution of showers and (b) the energy corrections to account for the signal in outlying pixels can exceed 10%, depending on shower geometry.

  15. A review of imaging techniques for systems biology

    Directory of Open Access Journals (Sweden)

    Po Ming J

    2008-08-01

    Full Text Available Abstract This paper presents a review of imaging techniques and of their utility in system biology. During the last decade systems biology has matured into a distinct field and imaging has been increasingly used to enable the interplay of experimental and theoretical biology. In this review, we describe and compare the roles of microscopy, ultrasound, CT (Computed Tomography, MRI (Magnetic Resonance Imaging, PET (Positron Emission Tomography, and molecular probes such as quantum dots and nanoshells in systems biology. As a unified application area among these different imaging techniques, examples in cancer targeting are highlighted.

  16. Design, synthesis, and biological evaluation of potent discodermolide fluorescent and photoaffinity molecular probes.

    Science.gov (United States)

    Smith, Amos B; Rucker, Paul V; Brouard, Ignacio; Freeze, B Scott; Xia, Shujun; Horwitz, Susan Band

    2005-11-10

    [structure: see text] The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent appendages has been achieved. Stereoselective olefin cross-metathesis comprised a key tactic for construction of two of the molecular probes. Three photoaffinity probes were radiolabeled with tritium.

  17. Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function

    Science.gov (United States)

    Moerner, W. E.

    2011-03-01

    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. 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. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS {103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature {388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB (Nat. Meth. {5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. {12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS {132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on

  18. Facile and green approach to prepare fluorescent carbon dots: Emergent nanomaterial for cell imaging and detection of vitamin B2.

    Science.gov (United States)

    Kundu, Aniruddha; Nandi, Sudipta; Das, Pradip; Nandi, Arun K

    2016-04-15

    Carbon dots (CDs) are a new representative in carbonaceous family and have initiated remarkable research interests over the past one decade in a large variety of fields. Herein, we have utilized a facile, one-step carbonization method to prepare fluorescent carbon dots using poly(vinyl alcohol) (PVA) both as a carbon source and as a surface passivating agent. The as prepared CDs emit bright blue fluorescence under ultraviolet illumination. The structure and optical properties of the CDs are thoroughly investigated by several methods such as high-resolution transmission electron microscopy; dynamic light scattering; UV-vis, fluorescence and Fourier transform infrared spectroscopy. The CDs exhibit excellent water solubility and demonstrate average hydrodynamic diameter of 11.3 nm, holding great promise for biological applications. The biocompatibility evaluation and in vitro imaging study reveals that the synthesized CDs can be used as effective fluorescent probes in bio-imaging without noticeable cytotoxicity. In addition, a unique sensor for the detection of vitamin B2 in aqueous solution is proposed on the basis of spontaneous fluorescence resonance energy transfer from CD to vitamin B2. These findings therefore suggest that the CDs can find potential applications in cellular imaging along with sensing of vitamin B2.

  19. Facile and green approach to prepare fluorescent carbon dots: Emergent nanomaterial for cell imaging and detection of vitamin B2.

    Science.gov (United States)

    Kundu, Aniruddha; Nandi, Sudipta; Das, Pradip; Nandi, Arun K

    2016-04-15

    Carbon dots (CDs) are a new representative in carbonaceous family and have initiated remarkable research interests over the past one decade in a large variety of fields. Herein, we have utilized a facile, one-step carbonization method to prepare fluorescent carbon dots using poly(vinyl alcohol) (PVA) both as a carbon source and as a surface passivating agent. The as prepared CDs emit bright blue fluorescence under ultraviolet illumination. The structure and optical properties of the CDs are thoroughly investigated by several methods such as high-resolution transmission electron microscopy; dynamic light scattering; UV-vis, fluorescence and Fourier transform infrared spectroscopy. The CDs exhibit excellent water solubility and demonstrate average hydrodynamic diameter of 11.3 nm, holding great promise for biological applications. The biocompatibility evaluation and in vitro imaging study reveals that the synthesized CDs can be used as effective fluorescent probes in bio-imaging without noticeable cytotoxicity. In addition, a unique sensor for the detection of vitamin B2 in aqueous solution is proposed on the basis of spontaneous fluorescence resonance energy transfer from CD to vitamin B2. These findings therefore suggest that the CDs can find potential applications in cellular imaging along with sensing of vitamin B2. PMID:26852351

  20. Two-photon fluorescent sensor for K+ imaging in live cells (Conference Presentation)

    Science.gov (United States)

    Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D.

    2016-03-01

    It is difficult to overstate the physiological importance of potassium for life as its indispensable roles in a variety of biological processes are widely known. As a result, efficient methods for determining physiological levels of potassium are of paramount importance. Despite this, relatively few K+ fluorescence sensors have been reported, with only one being commercially available. A new two-photon excited fluorescent K+ sensor is reported. The sensor is comprised of three moieties, a highly selective K+ chelator as the K+ recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K+ sensing in living cells.

  1. Attenuation-corrected fluorescence extraction for image-guided surgery in spatial frequency domain.

    Science.gov (United States)

    Yang, Bin; Sharma, Manu; Tunnell, James W

    2013-08-01

    A new approach to retrieve the attenuation-corrected fluorescence using spatial frequency-domain imaging is demonstrated. Both in vitro and ex vivo experiments showed the technique can compensate for the fluorescence attenuation from tissue absorption and scattering. This approach has potential in molecular image-guided surgery. PMID:23955392

  2. Attenuation-corrected fluorescence extraction for image-guided surgery in spatial frequency domain

    OpenAIRE

    Yang, Bin; Sharma, Manu; Tunnell, James W.

    2013-01-01

    Abstract. A new approach to retrieve the attenuation-corrected fluorescence using spatial frequency-domain imaging is demonstrated. Both in vitro and ex vivo experiments showed the technique can compensate for the fluorescence attenuation from tissue absorption and scattering. This approach has potential in molecular image-guided surgery.

  3. Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

    NARCIS (Netherlands)

    Uzunbajakava, Natalia; Otto, Cees

    2003-01-01

    We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Ram

  4. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    Science.gov (United States)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

  5. Wavelength dispersive X-ray fluorescence imaging using a high-sensitivity imaging sensor

    Science.gov (United States)

    Ohmori, Takashi; Kato, Shuichi; Doi, Makoto; Shoji, Takashi; Tsuji, Kouichi

    2013-05-01

    A new wavelength-dispersive X-ray fluorescence (WD-XRF) imaging spectrometer equipped with a high-sensitivity imaging sensor was developed in our laboratory. In this instrument, a straight polycapillary optic was applied instead of a Soller slit as well as a 2D imaging X-ray detector instead of X-ray counters, which are used in conventional WD-XRF spectrometers. Therefore, images of elemental distribution were available after a short exposure time. Ni Kα images and Cu Kα images were clearly obtained at corresponding diffraction angles for a short exposure time of 10 s. By optimizing the spectrometer, the time required for imaging is reduced, leading to XRF image movies. It is difficult to distinguish two peaks (Ti Kα (4.508 keV) and Ba Lα (4.465 keV)) due to the poor energy resolution of EDXRS. However, Ti and Ba images could be successfully observed by the WD-XRF imaging spectrometer. The energy resolution of the developed spectrometer was 25 eV at the Ti Kα peak.

  6. A Biologically Inspired CMOS Image Sensor

    CERN Document Server

    Sarkar, Mukul

    2013-01-01

    Biological systems are a source of inspiration in the development of small autonomous sensor nodes. The two major types of optical vision systems found in nature are the single aperture human eye and the compound eye of insects. The latter are among the most compact and smallest vision sensors. The eye is a compound of individual lenses with their own photoreceptor arrays.  The visual system of insects allows them to fly with a limited intelligence and brain processing power. A CMOS image sensor replicating the perception of vision in insects is discussed and designed in this book for industrial (machine vision) and medical applications. The CMOS metal layer is used to create an embedded micro-polarizer able to sense polarization information. This polarization information is shown to be useful in applications like real time material classification and autonomous agent navigation. Further the sensor is equipped with in pixel analog and digital memories which allow variation of the dynamic range and in-pixel b...

  7. Accurate study of FosPeg® distribution in a mouse model using fluorescence imaging technique and fluorescence white monte carlo simulations

    DEFF Research Database (Denmark)

    Xie, Haiyan; Liu, Haichun; Svenmarker, Pontus;

    2010-01-01

    Fluorescence imaging is used for quantitative in vivo assessment of drug concentration. Light attenuation in tissue is compensated for through Monte-Carlo simulations. The intrinsic fluorescence intensity, directly proportional to the drug concentration, could be obtained....

  8. Fluorescence guided lymph node biopsy in large animals using direct image projection device

    Science.gov (United States)

    Ringhausen, Elizabeth; Wang, Tylon; Pitts, Jonathan; Akers, Walter J.

    2016-03-01

    The use of fluorescence imaging for aiding oncologic surgery is a fast growing field in biomedical imaging, revolutionizing open and minimally invasive surgery practices. We have designed, constructed, and tested a system for fluorescence image acquisition and direct display on the surgical field for fluorescence guided surgery. The system uses a near-infrared sensitive CMOS camera for image acquisition, a near-infra LED light source for excitation, and DLP digital projector for projection of fluorescence image data onto the operating field in real time. Instrument control was implemented in Matlab for image capture, processing of acquired data and alignment of image parameters with the projected pattern. Accuracy of alignment was evaluated statistically to demonstrate sensitivity to small objects and alignment throughout the imaging field. After verification of accurate alignment, feasibility for clinical application was demonstrated in large animal models of sentinel lymph node biopsy. Indocyanine green was injected subcutaneously in Yorkshire pigs at various locations to model sentinel lymph node biopsy in gynecologic cancers, head and neck cancer, and melanoma. Fluorescence was detected by the camera system during operations and projected onto the imaging field, accurately identifying tissues containing the fluorescent tracer at up to 15 frames per second. Fluorescence information was projected as binary green regions after thresholding and denoising raw intensity data. Promising results with this initial clinical scale prototype provided encouraging results for the feasibility of optical projection of acquired luminescence during open oncologic surgeries.

  9. Enhancing contrast and quantitation by spatial frequency domain fluorescence molecular imaging

    Science.gov (United States)

    Sun, Jessica; Hathi, Deep; Zhou, Haiying; Shokeen, Monica; Akers, Walter J.

    2016-03-01

    Optical imaging with fluorescent contrast agents is highly sensitive for molecular imaging but is limited in depth to a few centimeters below the skin. Planar fluorescence imaging with full-field, uniform illumination and scientific camera image capture provides a portable and robust configuration for real-time, sensitive fluorescence detection with scalable resolution, but is inherently surface weighted and therefore limited in depth to a few millimeters. At the NIR region (700-1000 nm), tissue absorption and autofluorescence are relatively reduced, increasing depth penetration and reducing background signal, respectively. Optical imaging resolution scales with depth, limiting microscopic resolution with multiphoton microscopy and optical coherence tomography to skin and peri-tumoral tissues are not uniform, varying in thickness and color, complicating subsurface fluorescence measurements. Diffuse optical imaging methods have been developed that better quantify optical signals relative to faster full-field planar reflectance imaging, but require long scan times, complex instrumentation, and reconstruction algorithms. Here we report a novel strategy for rapid measurement of subsurface fluorescence using structured light illumination to improve quantitation of deep-seated fluorescence molecular probe accumulation. This technique, in combination with highly specific, tumor-avid fluorescent molecular probes, will easily integrate noninvasive diagnostics for superficial cancers and fluorescence guided surgery.

  10. Lifetime estimation of moving subcellular objects in frequency-domain fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Roudot, Philippe; Kervrann, Charles; Blouin, Cedric M; Waharte, Francois

    2015-10-01

    Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells. The aim is then to measure the fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation for both background and small moving structures with a focus on intracellular vesicle trafficking. PMID:26479936

  11. Diversity oriented fluorescence library approach (DOFLA) for live cell imaging probe development.

    Science.gov (United States)

    Yun, Seong-Wook; Kang, Nam-Young; Park, Sung-Jin; Ha, Hyung-Ho; Kim, Yun Kyung; Lee, Jun-Seok; Chang, Young-Tae

    2014-04-15

    A cell is the smallest functional unit of life. All forms of life rely on cellular processes to maintain normal functions, and changes in cell function induced by metabolic disturbances, physicochemical damage, infection, or abnormal gene expression may cause disease. To understand basic biology and to develop therapeutics for diseases, researchers need to study live cells. Along with advances in fluorescence microscopy and in vitro cell culture, live-cell imaging has become an essential tool in modern biology for the study of molecular and cellular events. Although researchers have often used fluorescent proteins to visualize cell-type-specific markers, this method requires genetic manipulations, which may not be appropriate in nontransgenic cells. Immunodetection of cellular markers requires the use of xenogenic antibodies, which may not detect intracellular markers in live cells. One option for overcoming these problems is the use of fluorescent small molecules targeted to specific cell types, which can enter live cells and interact with molecules of interest. We have used combinatorial chemistry to develop a large number of fluorescent small molecules as new imaging probes even without prior information about the probes' binding targets and mechanism, a strategy that we call the diversity oriented fluorescence library approach (DOFLA). We have used DOFLA to produce novel sensors and probes that detect a variety of biological and chemical molecules in vivo as well as in vitro. In this Account, we describe a series of fluorescent small molecules developed using DOFLA that bind specifically to particular cell types. These molecules provide new ways to detect and isolate these cells. The fluorescent probes CDy1, CDg4, and CDb8 tag embryonic stem cells and induced pluripotent stem cells but not fibroblasts or germ-line cells. CDr3 binds to an intracellular neural stem cell marker, fatty acid binding protein 7, which allows researchers to separate neural stem cells

  12. Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

    Science.gov (United States)

    Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

    2008-09-15

    A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously.

  13. Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

    Science.gov (United States)

    Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

    2008-09-15

    A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously. PMID:18794943

  14. Optical gating in fluorescence imaging through a tissue-like medium

    International Nuclear Information System (INIS)

    Full text: In this paper, Monte Carlo simulation of light propagation through turbid media is used to study fluorescence imaging under a confocal microscope. Optical gating methods, such as a pinhole gating method, have been investigated. The results reveal that the pinhole gating method is efficient in 1-photon fluorescence imaging, but is limited in 2-photon fluorescence imaging. The effect of other system parameters in a microscope such as the numerical aperture of an objective is also studied. The results show that an image with a higher numerical aperture objective gives a stronger signal while retains equal or higher resolution. Copyright (1999) Australian Optical Society

  15. Fluorescence imaging of experimental rheumatoid arthritis in vivo using a fast flying-spot scanner

    Science.gov (United States)

    Berger, J.; Voigt, J.; Seifert, F.; Ebert, B.; Macdonald, R.; Gemeinhardt, I.; Gemeinhardt, O.; Schnorr, J.; Taupitz, M.; Vater, A.; Vollmer, S.; Licha, K.; Schirner, M.

    2007-07-01

    We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR) spectral range following intravenous administration of contrast agents. The new imaging system has been characterized with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint inflammation have been compared with findings from contrast enhanced MR imaging and histology.

  16. 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

  17. A Combined Light Sheet Fluorescence and Differential Interference Contrast Microscope for Live Imaging of Multicellular Specimens

    Science.gov (United States)

    Baker, Ryan; Taormina, Michael; Jemielita, Matthew; Parthasarathy, Raghuveer

    2015-03-01

    We present a microscope capable of both light sheet fluorescence microscopy (LSFM) and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: LSFM provides high speed three-dimensional imaging of fluorescently labeled components of multicellular systems, large fields of view, and low phototoxicity, while DICM reveals the unlabeled neighborhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a shared detection path for both imaging modes enables simple integration of the two techniques in one microscope. To demonstrate the instrument's utility, we provide several examples which focus on the digestive tract of the larval zebrafish. We show that DICM can sometimes circumvent the need for fluorescent based techniques, augmenting the number of parameters obtainable per experiment when used alongside LSFM, and that DICM can be used to augment each experiment by imaging complementary features, such as non-fluorescent local environments near fluorescent samples (e.g. fluorescent enteric neurons imaged alongside the non-fluorescent gut wall), interactions between fluorescent and non-fluorescent samples (e.g. bacteria), and more. NSF Award 0922951, NIH Award 1P50 GM098911

  18. Iodinated oil-loaded, fluorescent mesoporous silica-coated iron oxide nanoparticles for magnetic resonance imaging/computed tomography/fluorescence trimodal imaging.

    Science.gov (United States)

    Xue, Sihan; Wang, Yao; Wang, Mengxing; Zhang, Lu; Du, Xiaoxia; Gu, Hongchen; Zhang, Chunfu

    2014-01-01

    In this study, a novel magnetic resonance imaging (MRI)/computed tomography (CT)/fluorescence trifunctional probe was prepared by loading iodinated oil into fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (i-fmSiO4@SPIONs). Fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs) were prepared by growing fluorescent dye-doped silica onto superparamagnetic iron oxide nanoparticles (SPIONs) directed by a cetyltrimethylammonium bromide template. As prepared, fmSiO4@SPIONs had a uniform size, a large surface area, and a large pore volume, which demonstrated high efficiency for iodinated oil loading. Iodinated oil loading did not change the sizes of fmSiO4@SPIONs, but they reduced the MRI T2 relaxivity (r2) markedly. I-fmSiO4@SPIONs were stable in their physical condition and did not demonstrate cytotoxic effects under the conditions investigated. In vitro studies indicated that the contrast enhancement of MRI and CT, and the fluorescence signal intensity of i-fmSiO4@SPION aqueous suspensions and macrophages, were intensified with increased i-fmSiO4@SPION concentrations in suspension and cell culture media. Moreover, for the in vivo study, the accumulation of i-fmSiO4@SPIONs in the liver could also be detected by MRI, CT, and fluorescence imaging. Our study demonstrated that i-fmSiO4@SPIONs had great potential for MRI/CT/fluorescence trimodal imaging. PMID:24904212

  19. High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.

    Directory of Open Access Journals (Sweden)

    Liangxing Wu

    Full Text Available We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID imaging microscopy. A triazole-substituted BIPS (TzBIPS is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP and a far-red absorbing merocyanine (MC state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C₁₂-TzBIPS is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.

  20. Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging

    Science.gov (United States)

    Shakiba, Mojdeh; Ng, Kenneth K.; Huynh, Elizabeth; Chan, Harley; Charron, Danielle M.; Chen, Juan; Muhanna, Nidal; Foster, F. Stuart; Wilson, Brian C.; Zheng, Gang

    2016-06-01

    J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J-aggregates for in vivo bioimaging applications. We find that 15 mol% Bchl-lipid embedded within a saturated phospholipid bilayer vesicle was optimal in terms of maximizing Bchl-lipid dye loading, while maintaining a spherical nanoparticle morphology and retaining spectral properties characteristic of J-aggregates. The addition of cholesterol maintains the stability of the J-aggregate absorption band for up to 6 hours in the presence of 90% FBS. In a proof-of-concept experiment, we successfully applied JNPs as a fluorescence contrast agent for real-time intraoperative detection of metastatic lymph nodes in a rabbit head-and-neck cancer model. Lymph node metastasis delineation was further verified by visualizing the JNP within the excised lymph node using photoacoustic imaging. Using JNPs, we demonstrate the possibility of using J-aggregates as fluorescence and photoacoustic contrast agents and may potentially spur the development of other nanomaterials that can stably induce J-aggregation for in vivo cancer bioimaging applications.J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J

  1. Segmenting Intracellular Distribution Images Derived by Fluorescent Dyes Using a Potts Model Hamiltonian

    CERN Document Server

    Hu, Dandan; Ronhovde, Peter; Bloch, Sharon; Achilefu, Samuel; Nussinov, Zohar

    2012-01-01

    We apply a multiresolution community detection algorithm to perform unsupervised segmentation of complex intracellular signals derived using fluorescent dyes. In our earlier work, when applying our method to benchmarks, our algorithm was shown to be one of the best and to be especially suited to the detection of camouflage images. In the current manuscript, we have explored this algorithm in a more complex scenario. The current image processing problem is framed as identifying clusters with respective average fluorescent lifetimes (FLTs) against a background or "solvent" in fluorescence lifetime imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the proposed algorithm from different starting points. Our method is more efficient than a well-known image segmentation...

  2. 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.

  3. Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

    Directory of Open Access Journals (Sweden)

    Jacob Beal

    Full Text Available We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.

  4. Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging

    International Nuclear Information System (INIS)

    Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters and the tissue is relatively homogeneous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins.

  5. Autofluorescence of atmospheric bioaerosols - fluorescent biomolecules, biological standard particles and potential interferences

    Science.gov (United States)

    Pöhlker, C.; Huffmann, J. A.; Pöschl, U.

    2012-04-01

    Primary biological aerosol particles (PBAP) such as pollen, fungal spores, bacteria, biogenic polymers and debris from larger organisms are known to influence atmospheric chemistry and physics, the biosphere and public health. PBAP account for up to ~30% of fine and up to ~70% of coarse particulate matter in urban, rural and pristine environment and are released with estimated emission rates of up to ~1000 Tg/a [1]. Continuous measurements of the abundance, variability and diversity of PBAP have been difficult until recently, however. The application of on-line instruments able to detect autofluorescence from biological particles in real-time has been a promising development for the measurement of PBAP concentrations and fluxes in different environments [2,3]. The detected fluorescent biological aerosol particles (FBAP) can be regarded as a subset of PBAP, although the exact relationship between PBAP and FBAP is still being investigated. Autofluorescence of FBAP is usually a superposition of fluorescence from a mixture of individual fluorescent molecules (fluorophores). Numerous biogenic fluorophores such as amino acids (e.g., tryptophan, tyrosine), coenzymes (e.g., NAD(P)H, riboflavin) and biopolymers (e.g., cellulose) emit fluorescent light due to heterocyclic aromatic rings or conjugated double bonds within their molecular structures. The tryptophan emission peak is a common feature of most bioparticles because the amino acid is a constituent of many proteins and peptides. The influence of the coenzymes NAD(P)H and riboflavin on the autofluorescence of bacteria can be regarded as an indicator for bacterial metabolism and has been utilized to discriminate between viable and non-viable organisms [4]. However, very little information is available about other essential biofluorophores in fungal spores and pollen. In order to better understand the autofluorescence behavior of FBAP, we have used fluorescence spectroscopy and fluorescence microscopy to analyze standard

  6. Electron beam dispersion measurements in nitrogen using two-dimensional imaging of N2(+) fluorescence

    Science.gov (United States)

    Clapp, L. H.; Twiss, R. G.; Cattolica, R. J.

    Experimental results are presented related to the radial spread of fluorescence excited by 10 and 20 KeV electron beams passing through nonflowing rarefied nitrogen at 293 K. An imaging technique for obtaining species distributions from measured beam-excited fluorescence is described, based on a signal inversion scheme mathematically equivalent to the inversion of the Abel integral equation. From fluorescence image data, measurements of beam radius, integrated signal intensity, and spatially resolved distributions of N2(+) first-negative-band fluorescence-emitting species have been made. Data are compared with earlier measurements and with an heuristic beam spread model.

  7. Preparation and Characterization of Highly Fluorescent, Glutathione-coated Near Infrared Quantum Dots for in Vivo Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Yoshichika Yoshioka

    2008-10-01

    Full Text Available Fluorescent probes that emit in the near-infrared (NIR, 700-1,300 nm region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4. The GSH-QDs (800 nm emission were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer, and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM, the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIRfluorescence imaging of a lymph node in a mouse is presented.

  8. Low Light CMOS Contact Imager with an Integrated Poly-Acrylic Emission Filter for Fluorescence Detection

    Directory of Open Access Journals (Sweden)

    Yonathan Dattner

    2010-05-01

    Full Text Available This study presents the fabrication of a low cost poly-acrylic acid (PAA based emission filter integrated with a low light CMOS contact imager for fluorescence detection. The process involves the use of PAA as an adhesive for the emission filter. The poly-acrylic solution was chosen due its optical transparent properties, adhesive properties, miscibility with polar protic solvents and most importantly its bio-compatibility with a biological environment. The emission filter, also known as an absorption filter, involves dissolving an absorbing specimen in a polar protic solvent and mixing it with the PAA to uniformly bond the absorbing specimen and harden the filter. The PAA is optically transparent in solid form and therefore does not contribute to the absorbance of light in the visible spectrum. Many combinations of absorbing specimen and polar protic solvents can be derived, yielding different filter characteristics in different parts of the spectrum. We report a specific combination as a first example of implementation of our technology. The filter reported has excitation in the green spectrum and emission in the red spectrum, utilizing the increased quantum efficiency of the photo sensitive sensor array. The thickness of the filter (20 μm was chosen by calculating the desired SNR using Beer-Lambert’s law for liquids, Quantum Yield of the fluorophore and the Quantum Efficiency of the sensor array. The filters promising characteristics make it suitable for low light fluorescence detection. The filter was integrated with a fully functional low noise, low light CMOS contact imager and experimental results using fluorescence polystyrene micro-spheres are presented.

  9. Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia

    Science.gov (United States)

    Ishikawa, Hiroaki; Marshall, Wallace F.

    2016-01-01

    Intraflagellar transport (IFT) is a motile process critical for building most cilia, including those of mammalian cells. Defects in IFT lead to short or missing cilia, and in animals can cause defects in development, for example in hedgehog mediated signaling, as well as disease symptoms such as polycystic kidney disease or retinal degeneration. Understanding how IFT works is thus a high priority in ciliary biology. Imaging of living cells has played a key role in understanding the mechanism of IFT, and this is particularly the case in mammalian cells where biochemical analysis of IFT is extremely difficult, due to the difficulty of isolating cilia away from the rest of the cell. Imaging IFT in living mammalian cells requires solution to several problems: constructing cell lines that express fluorescent protein tagged IFT proteins, obtaining cell populations with a high degree of ciliation, confocal or TIRF imaging with sufficient time resolution and signal to noise ratio to observe the majority of IFT particles as they travel back and forth inside the cilium, and analyzing the image data to extract quantitative measurements of IFT. We describe optimized solutions to each of these technical challenges. Using the approaches described here, mammalian cultured cells become powerful platforms for quantitative analysis of IFT dynamics. PMID:25837392

  10. Fluorescence imaging under background light with a self-reset complementary metal–oxide–semiconductor image sensor

    Directory of Open Access Journals (Sweden)

    Takahiro Yamaguchi

    2015-11-01

    Full Text Available The authors propose and demonstrate the fluorescence imaging of green fluorescence protein (GFP expressed in a brain slice with a self-reset complementary metal–oxide–semiconductor image sensor under background light. By using a self-reset function to avoid pixel saturation, the weak fluorescence of GFP was successfully observed, even under background light. The result suggests that the sensor can be applied to in vivo imaging of laboratory animals during light–dark cycles, so that they can observe the different responses to bright and dark environments.

  11. Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

    Science.gov (United States)

    Mondal, Samsuzzoha; Rakshit, Ananya; Pal, Suranjana; Datta, Ankona

    2016-07-15

    Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.

  12. Deployment of a Fully-Automated Green Fluorescent Protein Imaging System in a High Arctic Autonomous Greenhouse

    Directory of Open Access Journals (Sweden)

    Alain Berinstain

    2013-03-01

    Full Text Available Higher plants are an integral part of strategies for sustained human presence in space. Space-based greenhouses have the potential to provide closed-loop recycling of oxygen, water and food. Plant monitoring systems with the capacity to remotely observe the condition of crops in real-time within these systems would permit operators to take immediate action to ensure optimum system yield and reliability. One such plant health monitoring technique involves the use of reporter genes driving fluorescent proteins as biological sensors of plant stress. In 2006 an initial prototype green fluorescent protein imager system was deployed at the Arthur Clarke Mars Greenhouse located in the Canadian High Arctic. This prototype demonstrated the advantageous of this biosensor technology and underscored the challenges in collecting and managing telemetric data from exigent environments. We present here the design and deployment of a second prototype imaging system deployed within and connected to the infrastructure of the Arthur Clarke Mars Greenhouse. This is the first imager to run autonomously for one year in the un-crewed greenhouse with command and control conducted through the greenhouse satellite control system. Images were saved locally in high resolution and sent telemetrically in low resolution. Imager hardware is described, including the custom designed LED growth light and fluorescent excitation light boards, filters, data acquisition and control system, and basic sensing and environmental control. Several critical lessons learned related to the hardware of small plant growth payloads are also elaborated.

  13. Phenotypic charactheristics of fluorescent pseudomonss, biological control agent of lincat disease of temanggung tobacco

    Directory of Open Access Journals (Sweden)

    NINING NURUL AZIZAH

    2007-04-01

    Full Text Available Fluorescent pseudomonass isolated from local plants-rishosphere in temanggung controlled lincat disease of tobacco. This report describe phenotypic charactheristics of the bacteria in order to be used as a base for the development of the bacteria as a biological control agent of lincat disease. Phenotypic charactheristics of six isolates of fluorescent Pseudomonass which controlled lincat disease in the field were determined in the laboratory of Plant Bacteriology, Faculty of Agriculture, Gadjah Mada University. Plant pathogenicity tests were conducted by hypersensitive reaction into tobacco leaf and inoculation to tobacco plants. Antagonism test between fluorescent Pseudomonass and other candidate of biological control agents were also conducted. The results indicated that the bacteria were rod shape, Gram negative, positive reaction in catalase and oxidase tests. Nitrate reduce to nitrite, arginine was hydrolysed, fluorescent pigment were produced on King’s B medium, levan formation positive and all bacteria denitrifiy. The bacteria used urea, tween 80 and amylum were not hydrolised, poly--hydroxybutyrate was not accumulated in the cells. Negative reactions were observed for lysine decarboxylation, indol production, VP/MR reaction, and gelatn liquefation. Some compounds could be used as solely carbon sources. All isolates grew on the medium containing 2% NaCl. The best pH for growth was 6-7 and all isolates grew at 20-41C. Negative result were obtained for hypersensitive reaction and pathogenicity tests.

  14. Using genetically encoded fluorescent reporters to image lipid signalling in living plants

    NARCIS (Netherlands)

    J.E.M. Vermeer; T. Munnik

    2013-01-01

    The discovery of the green fluorescent protein has revolutionized cell biology as it allowed researchers to visualize dynamic processes in living cells. The fusion of fluorescent protein variants with lipid binding domains that bind to specific phospholipids have been very instrumental in investigat

  15. Aqueous synthesis of PbS quantum dots for noninvasive near-infrared fluorescence imaging in a mouse model

    Science.gov (United States)

    Deng, Dawei; Chen, Xinyang; Zhang, Jian; Liu, Fei; Cao, Jie; Gu, Yueqing

    2010-02-01

    In this paper, we present a new facile and environmental friendly method to prepare water-soluble near-infrared (NIR)-emitting PbS quantum dots (QDs) at room temperature under ambient conditions, using dihydrolipoic acid (DHLA) as a stabilizer. The photoluminescence (PL) emissions of the prepared DHLA-capped PbS QDs are tunable between 870 and 1010 nm. A PL quantum yield (QY) of ~10% can be achieved under optimized conditions without any post-preparative treatment. Here, we further use the produced DHLA-capped PbS QDs for NIR fluorescence imaging in a mouse model. The obtained experimental results showed that the NIR fluorescence of the PbS QDs in living tissues generated from the excitation with semiconductor laser (λmax=765.9 nm) could penetrate living tissues and be detected easily by the noninvasive in vivo NIR fluorescence imaging system. In addition, the preliminary studies on the cytotoxicity and in vivo toxicity of the QDs also indicates fully that these water-soluble DHLA-capped PbS QDs are very lowly toxic, and as such they should have greater potential in biological and medical applications especially in noninvasive in vivo fluorescence imaging of mice, compared to other existing highly toxic aqueous NIR-emitting quantum dots (CdTe, HgTe, etc).

  16. Application of X-ray fluorescence analytical techniques in phytoremediation and plant biology studies

    Science.gov (United States)

    Nečemer, Marijan; Kump, Peter; Ščančar, Janez; Jaćimović, Radojko; Simčič, Jurij; Pelicon, Primož; Budnar, Miloš; Jeran, Zvonka; Pongrac, Paula; Regvar, Marjana; Vogel-Mikuš, Katarina

    2008-11-01

    Phytoremediation is an emerging technology that employs the use of higher plants for the clean-up of contaminated environments. Progress in the field is however handicapped by limited knowledge of the biological processes involved in plant metal uptake, translocation, tolerance and plant-microbe-soil interactions; therefore a better understanding of the basic biological mechanisms involved in plant/microbe/soil/contaminant interactions would allow further optimization of phytoremediation technologies. In view of the needs of global environmental protection, it is important that in phytoremediation and plant biology studies the analytical procedures for elemental determination in plant tissues and soil should be fast and cheap, with simple sample preparation, and of adequate accuracy and reproducibility. The aim of this study was therefore to present the main characteristics, sample preparation protocols and applications of X-ray fluorescence-based analytical techniques (energy dispersive X-ray fluorescence spectrometry—EDXRF, total reflection X-ray fluorescence spectrometry—TXRF and micro-proton induced X-ray emission—micro-PIXE). Element concentrations in plant leaves from metal polluted and non-polluted sites, as well as standard reference materials, were analyzed by the mentioned techniques, and additionally by instrumental neutron activation analysis (INAA) and atomic absorption spectrometry (AAS). The results were compared and critically evaluated in order to assess the performance and capability of X-ray fluorescence-based techniques in phytoremediation and plant biology studies. It is the EDXRF, which is recommended as suitable to be used in the analyses of a large number of samples, because it is multi-elemental, requires only simple preparation of sample material, and it is analytically comparable to the most frequently used instrumental chemical techniques. The TXRF is compatible to FAAS in sample preparation, but relative to AAS it is fast

  17. Biological synthesis of fluorescent nanoparticles by cadmium and tellurite resistant Antarctic bacteria: exploring novel natural nanofactories

    OpenAIRE

    Plaza, D. O.; Gallardo, C.; Straub, Y. D.; Bravo, D.; Pérez-Donoso, J. M.

    2016-01-01

    Background Fluorescent nanoparticles or quantum dots (QDs) have been intensely studied for basic and applied research due to their unique size-dependent properties. There is an increasing interest in developing ecofriendly methods to synthesize these nanoparticles since they improve biocompatibility and avoid the generation of toxic byproducts. The use of biological systems, particularly prokaryotes, has emerged as a promising alternative. Recent studies indicate that QDs biosynthesis is rela...

  18. New developments of X-ray fluorescence imaging techniques in laboratory

    Science.gov (United States)

    Tsuji, Kouichi; Matsuno, Tsuyoshi; Takimoto, Yuki; Yamanashi, Masaki; Kometani, Noritsugu; Sasaki, Yuji C.; Hasegawa, Takeshi; Kato, Shuichi; Yamada, Takashi; Shoji, Takashi; Kawahara, Naoki

    2015-11-01

    X-ray fluorescence (XRF) analysis is a well-established analytical technique with a long research history. Many applications have been reported in various fields, such as in the environmental, archeological, biological, and forensic sciences as well as in industry. This is because XRF has a unique advantage of being a nondestructive analytical tool with good precision for quantitative analysis. Recent advances in XRF analysis have been realized by the development of new x-ray optics and x-ray detectors. Advanced x-ray focusing optics enables the making of a micro x-ray beam, leading to micro-XRF analysis and XRF imaging. A confocal micro-XRF technique has been applied for the visualization of elemental distributions inside the samples. This technique was applied for liquid samples and for monitoring chemical reactions such as the metal corrosion of steel samples in the NaCl solutions. In addition, a principal component analysis was applied for reducing the background intensity in XRF spectra obtained during XRF mapping, leading to improved spatial resolution of confocal micro-XRF images. In parallel, the authors have proposed a wavelength dispersive XRF (WD-XRF) imaging spectrometer for a fast elemental imaging. A new two dimensional x-ray detector, the Pilatus detector was applied for WD-XRF imaging. Fast XRF imaging in 1 s or even less was demonstrated for Euro coins and industrial samples. In this review paper, these recent advances in laboratory-based XRF imaging, especially in a laboratory setting, will be introduced.

  19. Imaging of Flow Patterns with Fluorescent Molecular Rotors

    OpenAIRE

    Mustafic, Adnan; Huang, Hsuan-Ming; Theodorakis, Emmanuel A.; Haidekker, Mark A

    2010-01-01

    Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer states (TICT) upon photoexcitation. Some classes of molecular rotors, among them those that are built on the benzylidene malononitrile motif, return to the ground state either by nonradiative intramolecular rotation or by fluorescence emission. In low-viscosity solvents, intramolecular rotation dominates, and the fluorescence quantum yield is low. Higher solvent viscosities reduce the intram...

  20. Photoactivation and imaging of optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, George H

    2011-07-01

    A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins, which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be "turned on" by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks.

  1. Determination of selenium in biological samples with an energy-dispersive X-ray fluorescence spectrometer.

    Science.gov (United States)

    Li, Xiaoli; Yu, Zhaoshui

    2016-05-01

    Selenium is both a nutrient and a toxin. Selenium-especially organic selenium-is a core component of human nutrition. Thus, it is very important to measure selenium in biological samples. The limited sensitivity of conventional XRF hampers its widespread use in biological samples. Here, we describe the use of high-energy (100kV, 600W) linearly polarized beam energy-dispersive X-Ray fluorescence spectroscopy (EDXRF) in tandem with a three-dimensional optics design to determine 0.1-5.1μgg(-1) levels of selenium in biological samples. The effects of various experimental parameters such as applied voltage, acquisition time, secondary target and various filters were thoroughly investigated. The detection limit of selenium in biological samples via high-energy (100kV, 600W) linearly polarized beam energy-dispersive X-ray fluorescence spectroscopy was decreased by one order of magnitude versus conventional XRF (Paltridge et al., 2012) and found to be 0.1μg/g. To the best of our knowledge, this is the first report to describe EDXRF measurements of Se in biological samples with important implications for the nutrition and analytical chemistry communities. PMID:26922394

  2. Influence of angle's ranges for recording an X-ray fluorescence hologram on reconstructed atomic images

    Institute of Scientific and Technical Information of China (English)

    XIE Hong-Lan; CHEN Jian-Wen; GAO Hong-Yi; ZHU Hua-Feng; LI Ru-Xin; XU Zhi-Zhan

    2004-01-01

    X-ray fluorescence holography (XFH) is a novel method for three-dimensional (3D) imaging of atomic structure. Theoretically, in an XFH experiment, one has to measure the fluorescence energy on a spherical surface to get well-resolved 3D images of atoms. But in practice, the experimental system arrangement does not allow the measurement of the fluorescent intensity oscillations in the full sphere. The holographic information losses because of the limited sampling range (less than 4π) will directly result in defective reconstructed atomic images. In this work, the atomic image of a Fe single crystal (001) was reconstructed by numerically simulating X-ray fluorescence holograms of the crystal at different recording angle's ranges and step lengths. Influences of the ranges of azimuth angles and polar angles and the step length of polar angles on the reconstructed atomic images were discussed.

  3. Performance evaluation of CCD- and mobile-phone-based near-infrared fluorescence imaging systems with molded and 3D-printed phantoms

    Science.gov (United States)

    Wang, Bohan; Ghassemi, Pejhman; Wang, Jianting; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

    2016-03-01

    Increasing numbers of devices are emerging which involve biophotonic imaging on a mobile platform. Therefore, effective test methods are needed to ensure that these devices provide a high level of image quality. We have developed novel phantoms for performance assessment of near infrared fluorescence (NIRF) imaging devices. Resin molding and 3D printing techniques were applied for phantom fabrication. Comparisons between two imaging approaches - a CCD-based scientific camera and an NIR-enabled mobile phone - were made based on evaluation of the contrast transfer function and penetration depth. Optical properties of the phantoms were evaluated, including absorption and scattering spectra and fluorescence excitation-emission matrices. The potential viability of contrastenhanced biological NIRF imaging with a mobile phone is demonstrated, and color-channel-specific variations in image quality are documented. Our results provide evidence of the utility of novel phantom-based test methods for quantifying image quality in emerging NIRF devices.

  4. Dual-Modal Nanoprobes for Imaging of Mesenchymal Stem Cell Transplant by MRI and Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Chang Kyu; Hong, Kyung Ah; Lin, Shun Mei [Seoul Metropolitan Boramae Medical Center, Seoul (Korea, Republic of)] (and others)

    2009-12-15

    To determine the feasibility of labeling human mesenchymal stem cells (hMSCs) with bifunctional nanoparticles and assessing their potential as imaging probes in the monitoring of hMSC transplantation. The T1 and T2 relaxivities of the nanoparticles (MNP SiO{sub 2}[RITC]-PEG) were measured at 1.5T and 3T magnetic resonance scanner. Using hMSCs and the nanoparticles, labeling efficiency, toxicity, and proliferation were assessed. Confocal laser scanning microscopy and transmission electron microscopy were used to specify the intracellular localization of the endocytosed iron nanoparticles. We also observed in vitro and in vivo visualization of the labeled hMSCs with a 3T MR scanner and optical imaging. MNP SiO{sub 2}(RITC)-PEG showed both superparamagnetic and fluorescent properties. The r{sub 1} and r{sub 2} relaxivity values of the MNP SiO{sub 2}(RITC)-PEG were 0.33 and 398 mM{sup -1} s{sup -1} at 1.5T, respectively, and 0.29 and 453 mM{sup -1} s{sup -1} at 3T, respectively. The effective internalization of MNP SiO{sub 2}(RITC)-PEG into hMSCs was observed by confocal laser scanning fluorescence microscopy. The transmission electron microscopy images showed that MNP SiO{sub 2}(RITC)-PEG was internalized into the cells and mainly resided in the cytoplasm. The viability and proliferation of MNP SiO{sub 2}(RITC)-PEG-labeled hMSCs were not significantly different from the control cells. MNP SiO{sub 2}(RITC)-PEG-labeled hMSCs were observed in vitro and in vivo with optical and MR imaging. MNP SiO{sub 2}(RITC)-PEG can be a useful contrast agent for stem cell imaging, which is suitable for a bimodal detection by MRI and optical imaging.

  5. Enhanced fluorescence imaging performance of hydrophobic colloidal ZnO nanoparticles by a facile method

    International Nuclear Information System (INIS)

    Highlights: • A dual phase hydrothermal method was developed to synthesize ZnO nanoparticles. • ZnO nanoparticles show a stability and solubility in the aqueous environment. • ZnO nanoparticles with a blue emission wavelength at around 420 nm and small size (30 nm). • ZnO nanoparticles as biological labeling agent was also shown. - Abstract: A facile synthesis method for the formation of ZnO nanoparticles by using a double-phase reaction was demonstrated in this paper. The morphology of the synthesized ZnO nanoparticles shows a flower-shape. Hydrogen peroxide was used as a unique oxygenic source to promote the formation of ZnO in the presence of organic zinc precursor. The as-synthesized ZnO nanoparticles also show a stability and solubility in the aqueous environment. The structure and properties of ZnO nanoparticles were investigated by the transmission electron microscopy (TEM) and X-ray diffraction (XRD) as well as UV–vis and photoluminescence spectroscopy. The as-prepared hydrophobic colloidal ZnO nanoparticles could be modified to become water-soluble via ligand exchange with amineothanethiol⋅HCl while retaining the photoluminescence properties. In addition, the potential application for biological label of water-soluble ZnO nanoparticles were also demonstrated. These results not only have applications towards using colloidal ZnO nanoparticles effectively in biological fluorescence imaging, but also promote its application in the field of targeted drug delivery

  6. Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging

    Science.gov (United States)

    Husvogt, Lennart; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Hornegger, Joachim; Connolly, James L.; Fujimoto, James G.

    2016-01-01

    We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems. PMID:27500636

  7. First X-ray fluorescence CT experimental results at the SSRF X-ray imaging beamline

    Institute of Scientific and Technical Information of China (English)

    DENG Biao; YANG Qun; XIE Hong-Lan; DU Guo-Hao; XIAO Wi-Qiao

    2011-01-01

    X-ray fluorescence CT is a non-destructive technique for detecting elemental composition and distribution inside a specimen. In this paper, the first experimental results of X-ray fluorescence CT obtained at the SSRF X-ray imaging beamline (BL13W1) are described. The test samples were investigated and the 2D elemental image was reconstructed using a filtered back-projection algorithm. In the sample the element Cd was observed. Up to now, the X-ray fluorescence CT could be carried out at the SSRF X-ray imaging beamline.

  8. A two-photon fluorescent probe with a large turn-on signal for imaging hydrogen sulfide in living tissues

    International Nuclear Information System (INIS)

    Highlights: • A two-photon fluorescent probe for sensing H2S was developed. • The probe shows a large turn on signal (120-fold enhancement). • The probe is suitable for fluorescence imaging of H2S in living cells and tissues. • The probe was capable of detecting H2S up to 170 μm depth in live tissues. - Abstract: A two-photon fluorescence turn-on H2S probe GCTPOC–H2S based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive H2S recognition site, dinitrophenyl ether was constructed. The probe GCTPOC–H2S exhibits desirable properties such as high sensitivity, high selectivity, functioning well at physiological pH and low cytotoxicity. In particular, the probe shows a 120-fold enhancement in the presence of Na2S (500 μM), which is larger than the reported two-photon fluorescent H2S probes. The large fluorescence enhancement of the two-photon probe GCTPOC–H2S renders it attractive for imaging H2S in living tissues with deep tissue penetration. Significantly, we have demonstrated that the probe GCTPOC–H2S is suitable for fluorescence imaging of H2S in living tissues with deep penetration by using two-photon microscopy. The further application of the two-photon probe for the investigation of biological functions and pathological roles of H2S in living systems is under progress

  9. Simplified and optimized multispectral imaging for 5-ALA-based fluorescence diagnosis of malignant lesions.

    Science.gov (United States)

    Minamikawa, Takeo; Matsuo, Hisataka; Kato, Yoshiyuki; Harada, Yoshinori; Otsuji, Eigo; Yanagisawa, Akio; Tanaka, Hideo; Takamatsu, Tetsuro

    2016-01-01

    5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is now clinically applied for accurate and ultrarapid diagnosis of malignant lesions such as lymph node metastasis during surgery. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA, protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue chromophores, such as collagen and flavins. In this study, we demonstrated PPIX fluorescence estimation with autofluorescence elimination for 5-ALA-based fluorescence diagnosis of malignant lesions by simplified and optimized multispectral imaging. We computationally optimized observation wavelength regions for the estimation of PPIX fluorescence in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen and flavins. By using the fluorescence intensities of the optimized wavelength regions, we verified quantitative detection of PPIX fluorescence by using chemical mixtures of PPIX, flavins, and collagen. Furthermore, we demonstrated detection capability by using metastatic and non-metastatic lymph nodes of colorectal cancer patients. These results suggest the potential and usefulness of the background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions, and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis. PMID:27149301

  10. 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.

  11. 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. PMID:27406825

  12. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  13. Wavefront sensorless approaches to adaptive optics for in vivo fluorescence imaging of mouse retina

    Science.gov (United States)

    Wahl, Daniel J.; Bonora, Stefano; Mata, Oscar S.; Haunerland, Bengt K.; Zawadzki, Robert J.; Sarunic, Marinko V.; Jian, Yifan

    2016-03-01

    Adaptive optics (AO) is necessary to correct aberrations when imaging the mouse eye with high numerical aperture. In order to obtain cellular resolution, we have implemented wavefront sensorless adaptive optics for in vivo fluorescence imaging of mouse retina. Our approach includes a lens-based system and MEMS deformable mirror for aberration correction. The AO system was constructed with a reflectance channel for structural images and fluorescence channel for functional images. The structural imaging was used in real-time for navigation on the retina using landmarks such as blood vessels. We have also implemented a tunable liquid lens to select the retinal layer of interest at which to perform the optimization. At the desired location on the mouse retina, the optimization algorithm used the fluorescence image data to drive a modal hill-climbing algorithm using an intensity or sharpness image quality metric. The optimization requires ~30 seconds to complete a search up to the 20th Zernike mode. In this report, we have demonstrated the AO performance for high-resolution images of the capillaries in a fluorescence angiography. We have also made progress on an approach to AO with pupil segmentation as a possible sensorless technique suitable for small animal retinal imaging. Pupil segmentation AO was implemented on the same ophthalmic system and imaging performance was demonstrated on fluorescent beads with induced aberrations.

  14. Photoacoustic tomography imaging of biological tissues

    Science.gov (United States)

    Su, Yixiong; Wang, Ruikang K.; Xu, Kexin; Zhang, Fan; Yao, Jianquan

    2005-01-01

    Non-invasive laser-induced photoacoustic tomography is attracting more and more attentions in the biomedical optical imaging field. This imaging modality takes the advantages in that the tomography image has the optical contrast similar to the optical techniques while enjoying the high spatial resolution comparable to the ultrasound. Currently, its biomedical applications are mainly focused on breast cancer diagnosis and small animal imaging. In this paper, we report in detail a photoacoustic tomography experiment system constructed in our laboratory. In our system, a Q-switched ND:YAG pulse laser operated at 532nm with a 10ns pulse width is employed to generate photoacoustic signal. A tissue-mimicking phantom was built to test the system. When imaged, the phantom and detectors were immersed in a water tank to facilitate the acoustic detection. Based on filtered back-projection process of photoacoustic imaging, the two-dimension distribution of optical absorption in tissue phantom was reconstructed.

  15. Image navigation as a means to expand the boundaries of fluorescence-guided surgery

    Science.gov (United States)

    Brouwer, Oscar R.; Buckle, Tessa; Bunschoten, Anton; Kuil, Joeri; Vahrmeijer, Alexander L.; Wendler, Thomas; Valdés-Olmos, Renato A.; van der Poel, Henk G.; van Leeuwen, Fijs W. B.

    2012-05-01

    Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy.

  16. In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

    Science.gov (United States)

    Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

    2014-03-01

    Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

  17. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    International Nuclear Information System (INIS)

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

  18. Multispectral fluorescence imaging for detection of bovine feces on Romaine lettuce and baby spinach leaves

    Science.gov (United States)

    Hyperspectral fluorescence imaging with ultraviolet-A excitation was used to evaluate the feasibility of two-waveband fluorescence algorithms for the detection of bovine fecal contaminants on the abaxial and adaxial surfaces of Romaine lettuce and baby spinach leaves. Correlation analysis was used t...

  19. Investigation of formalin influence over hard and soft biological tissues fluorescent spectra in vitro

    Science.gov (United States)

    Borisova, E.; Uzunov, Tz.; Vladimirov, B.; Avramov, L.

    2007-05-01

    In order to investigate the formalin influence over fluorescence properties of hard and soft biological tissues during conservation, emission spectra have been registered. Nitrogen laser at 337 nm and light-emitting diode with maximum at 405 nm have been used as excitation sources. For investigation of formalin influence over hard tissues, an experiment was made on teeth samples. Sound teeth were demineralized with a phosphoric acid for 10 seconds to obtain enamel structure near to the tooth lesion, and were fixed in formalin. Before and after teeth treatment spectra from the areas of interest were detected. There were not observed changes in the shape of the teeth spectra, related to the introduction of formalin fluorescence. Samples from mucosa of esophagus and stomach, where initially an ALA/Protoporphyrin IX diagnosis was applied, were used as soft tissue specimens. After fluorescent diagnosis in vivo biopsy samples were obtained from normal and cancerous areas and were conserved in formalin. Initially, spectrum observed has one autofluorescence maximum from the mucous tissue at 500-600 nm and secondary maxima from the protoporphyrin fluorescence at 635 nm and 720 nm, as well as pronounced minima at 540 and 575 nm related to hemoglobin absorption. After formalin conservation hemoglobin absorption was strongly reduced that increases mucous emission signal in green-yellow spectral region. Simultaneously the maxima at 635 nm and 720 nm were reduced. As conclusion we could say that formalin has negligible influence over fluorescence spectra of conserved hard tissues and has more pronounced influence over fluorescence spectra obtained in the case of soft tissue conservation, which has to be taking into account in measurements in vitro.

  20. Targeted Thiazole Orange Derivative with Folate: Synthesis, Fluorescence and in Vivo Fluorescence Imaging

    OpenAIRE

    Qingyang Meng; Baolian Zhang; Yiqi Wang; Xuening Fei; Yingchun Gu

    2010-01-01

    A Thiazole Orange conjugated with folate derivative was synthesized in two steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to obtain the title folate-conjugated compound. The compound was evaluated by 1H-NMR MS, TG/DTA and fluorescence spectroscopic methods. The title compound could selectively target folate receptor expressing tumors according to the in vivo fluorescence ...

  1. Fluorescence imaging of single Kinesin motors on immobilized microtubules.

    Science.gov (United States)

    Korten, Till; Nitzsche, Bert; Gell, Chris; Ruhnow, Felix; Leduc, Cécile; Diez, Stefan

    2011-01-01

    Recent developments in optical microscopy and nanometer tracking have greatly improved our understanding of cytoskeletal motor proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules mainly using a geometry, where fluorescently labeled motors move on surface-immobilized filaments. In this chapter, we review recent methods related to single-molecule kinesin motility assays. In particular, we aim to provide practical advice on: how to set up the assays, how to acquire high-precision data from fluorescently labeled kinesin motors and attached quantum dots, and how to analyze data by nanometer tracking.

  2. Early detection of tumor masses by in vivo hematoporphyrin-mediated fluorescence imaging

    Science.gov (United States)

    Autiero, Maddalena; Celentano, Luigi; Cozzolino, Rosanna; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Cristina Montesi, Maria; Quarto, Maria; Riccio, Patrizia; Roberti, Giuseppe; Russo, Paolo

    2007-02-01

    We investigated the capability of fluorescence reflectance imaging (FRI) for the early detection of surface tumors in mice. We used a hematoporphyrin (HP) compound (HP dichlorohydrate) as a red fluorescent marker and a low noise, high sensitivity, digital CCD camera for fluorescence imaging. In this preliminary study, highly malignant anaplastic human thyroid carcinoma cells were implanted subcutaneously in one mouse and their growth was monitored daily for 5 days by FRI. The selective HP uptake by the tumor tissues was successfully observed: we observed the fluorescence of tumor only 3 days after cancer cells injection, i.e. when the tumor mass was neither visible (to the naked eye) or palpable. These measurements indicate that FRI is a suitable technique to detect minute subcutaneous tumor masses. This FRI system will be coupled to a radionuclide imaging system based on a CdTe detector for in vivo multimodal imaging in mice.

  3. Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Davidson Michael W

    2008-03-01

    Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

  4. 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.

  5. Cell depth imaging by point laser scanning fluorescence microscopy with an optical disk pickup head

    Science.gov (United States)

    Tsai, Rung-Ywan; Chen, Jung-Po; Lee, Yuan-Chin; Chiang, Hung-Chih; Cheng, Chih-Ming; Huang, Chun-Chieh; Huang, Tai-Ting; Cheng, Chung-Ta; Tiao, Golden

    2015-09-01

    A compact, cost-effective, and position-addressable digital laser scanning microscopy (DLSM) instrument is made using a commercially available Blu-ray disc read-only memory (BD-ROM) pickup head. Fluorescent cell images captured by DLSM have resolutions of 0.38 µm. Because of the position-addressable function, multispectral fluorescence cell images are captured using the same sample slide with different excitation laser sources. Specially designed objective lenses with the same working distance as the image-capturing beam are used for the different excitation laser sources. By accurately controlling the tilting angles of the sample slide or by moving the collimator lens of the image-capturing beam, the fluorescence cell images along different depth positions of the sample are obtained. Thus, z-section images with micrometer-depth resolutions are achievable.

  6. Strategies for Biologic Image-Guided Dose Escalation: A Review

    International Nuclear Information System (INIS)

    There is increasing interest in how to incorporate functional and molecular information obtained by noninvasive, three-dimensional tumor imaging into radiotherapy. The key issues are to identify radioresistant regions that can be targeted for dose escalation, and to develop radiation dose prescription and delivery strategies providing optimal treatment for the individual patient. In the present work, we review the proposed strategies for biologic image-guided dose escalation with intensity-modulated radiation therapy. Biologic imaging modalities and the derived images are discussed, as are methods for target volume delineation. Different dose escalation strategies and techniques for treatment delivery and treatment plan evaluation are also addressed. Furthermore, we consider the need for response monitoring during treatment. We conclude with a summary of the current status of biologic image-based dose escalation and of areas where further work is needed for this strategy to become incorporated into clinical practice

  7. Observations of fluorescent and biological aerosol at a high-altitude site in central France

    Science.gov (United States)

    Gabey, A. M.; Vaitilingom, M.; Freney, E.; Boulon, J.; Sellegri, K.; Gallagher, M. W.; Crawford, I. P.; Robinson, N. H.; Stanley, W. R.; Kaye, P. H.

    2013-08-01

    Total bacteria, fungal spore and yeast counts were compared with ultraviolet-light-induced fluorescence (UV-LIF) measurements of ambient aerosol at the summit of the Puy de Dôme (PdD) mountain in central France (1465 m a.s.l), which represents a background elevated site. Bacteria, fungal spores and yeast were enumerated by epifluorescence microscopy (EFM) and found to number 2.2 to 23 L-1 and 0.8 to 2 L-1, respectively. Bacteria counts on two successive nights were an order of magnitude larger than in the intervening day. A wide issue bioaerosol spectrometer, version 3 (WIBS-3) was used to perform UV-LIF measurements on ambient aerosol sized 0.8 to 20 μm. Mean total number concentration was 270 L-1 (σ = 66 L-1), found predominantly in a size mode at 2 μm for most of the campaign. Total concentration (fluorescent + non-fluorescent aerosol) peaked at 500 L-1 with a size mode at 1 μm because of a change in air mass origin lasting around 48 h. The WIBS-3 features two excitation and fluorescence detection wavelengths corresponding to different biological molecules, although non-biological interferents also contribute. The mean fluorescent particle concentration after short-wave (280 nm; associated with tryptophan) excitation was 12 L-1 (σ = 6 L-1), and did not vary much throughout the campaign. In contrast, the mean concentration of particles fluorescent after long-wave (370 nm; associated with NADH) excitation was 95 L-1 (σ = 25 L-1), and a nightly rise and subsequent fall of up to 100 L-1 formed a strong diurnal cycle in the latter. The two fluorescent populations exhibited size modes at 3 μm and 2 to 3 μm, respectively. A hierarchical agglomerative cluster analysis algorithm was applied to the data and used to extract different particle factors. A cluster concentration time series representative of bacteria was identified. This was found to exhibit a diurnal cycle with a maximum peak appearing during the day. Analysis of organic mass spectra recorded using

  8. Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

    Science.gov (United States)

    Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

    2015-10-01

    Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

  9. Time efficient methods for scanning a fluorescent membrane with a fluorescent microscopic imager for the quality assurance of food

    Science.gov (United States)

    Lerm, Steffen; Holder, Silvio; Schellhorn, Mathias; Brückner, Peter; Linß, Gerhard

    2013-05-01

    An important part of the quality assurance of meat is the estimation of germs in the meat exudes. The kind and the number of the germs in the meat affect the medical risk for the consumer of the meat. State-of-the-art analyses of meat are incubator test procedures. The main disadvantages of such incubator tests are the time consumption, the necessary equipment and the need of special skilled employees. These facts cause in high inspection cost. For this reason a new method for the quality assurance is necessary which combines low detection limits and less time consumption. One approach for such a new method is fluorescence microscopic imaging. The germs in the meat exude are caught in special membranes by body-antibody reactions. The germ typical signature could be enhanced with fluorescent chemical markers instead of reproduction of the germs. Each fluorescent marker connects with a free germ or run off the membrane. An image processing system is used to detect the number of fluorescent particles. Each fluorescent spot should be a marker which is connected with a germ. Caused by the small object sizes of germs, the image processing system needs a high optical magnification of the camera. However, this leads to a small field of view and a small depth of focus. For this reasons the whole area of the membrane has to be scanned in three dimensions. To minimize the time consumption, the optimal path has to be found. This optimization problem is influenced by features of the hardware and is presented in this paper. The traversing range in each direction, the step width, the velocity, the shape of the inspection volume and the field of view have influence on the optimal path to scan the membrane.

  10. Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

    OpenAIRE

    Putman, Constant A.J.; Hansma, Helen G.; Gaub, Hermann E.; Hansma, Paul K.

    1992-01-01

    The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microscope enables a direct comparison between features observed in the fluorescence microscope and those observed in the images obtained with the AFM, in air or under liquid. The cracks in polymerized La...

  11. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

    Full Text Available Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

  12. Evaluation of chicory seeds maturity by chlorophyll fluorescence imaging

    OpenAIRE

    Ooms, David; Destain, Marie-France

    2011-01-01

    Chicory (Cichorium intybus L.) seed production includes sorting to remove foreign materials and non-viable seeds. A machine vision system was developed to monitor the fluorescence in order to detect the immature chicory seeds. It comprised a monochromatic light source, a highpass filter and a monochromatic CCD camera sensitive to red and infrared. With this device, blue light reflected by the seeds was blocked whilst red fluorescence was measured by the camera. A segmentation a...

  13. Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer

    International Nuclear Information System (INIS)

    Pancreatic cancer is a devastating disease characterized by dismal 5-year survival rates and limited treatment options. In an effort to provide useful models for preclinical evaluation of new experimental therapeutics, we and others have developed orthotopic mouse models of pancreatic cancer. The utility of these models for pre-clinical testing is dependent upon quantitative, noninvasive methods for monitoring in vivo tumor progression in real time. Toward this goal, we performed whole-body fluorescence imaging and ultrasound imaging to evaluate and to compare these noninvasive imaging modalities for assessing tumor burden and tumor progression in an orthotopic mouse model of pancreatic cancer. The human pancreatic cancer cell line XPA-1, engineered for stable, high-level expression of red fluorescent protein (RFP), was implanted into the pancreas of nude mice using orthotopic implantation. The tumors were allowed to grow over a period of one to several weeks during which time the mice were imaged using both fluorescence imaging and ultrasound imaging to measure tumor burden and to monitor tumor growth. Whole-body fluorescence imaging and ultrasound imaging both allowed for the visualization and measurement of orthotopic pancreatic tumor implants in vivo. The imaging sessions were well-tolerated by the mice and yielded data which correlated well in the quantitative assessment of tumor burden. Whole-body fluorescence and two-dimensional ultrasound imaging showed a strong correlation for measurement of tumor size over a range of tumor sizes (R2 = 0.6627, P = 0.003 for an exposure time of 67 msec and R2 = 0.6553, P = 0.003 for an exposure time of 120 msec). Our findings suggest a complementary role for fluorescence imaging and ultrasound imaging in assessing tumor burden and tumor progression in orthotopic mouse models of human cancer

  14. Automated parasite faecal egg counting using fluorescence labelling, smartphone image capture and computational image analysis.

    Science.gov (United States)

    Slusarewicz, Paul; Pagano, Stefanie; Mills, Christopher; Popa, Gabriel; Chow, K Martin; Mendenhall, Michael; Rodgers, David W; Nielsen, Martin K

    2016-07-01

    Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay to promote good clinical practice. Using a fluorescent chitin-binding protein, we show that this structural carbohydrate is present and accessible in shells of ova of strongyle, ascarid, trichurid and coccidian parasites. Furthermore, we show that a cellular smartphone can be used as an inexpensive device to image fluorescent eggs and, by harnessing the computational power of the phone, to perform image analysis to count the eggs. Strongyle egg counts generated by the smartphone system had a significant linear correlation with manual McMaster counts (R(2)=0.98), but with a significantly lower coefficient of variation (P=0.0177). Furthermore, the system was capable of differentiating equine strongyle and ascarid eggs similar to the McMaster method, but with significantly lower coefficients of variation (Psmartphones as relatively sophisticated, inexpensive and portable medical diagnostic devices. PMID:27025771

  15. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    Science.gov (United States)

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

    2016-02-01

    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

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

    International Nuclear Information System (INIS)

    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. 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. 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. 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

  17. In vivo tomographic imaging with fluorescence and MRI using tumor-targeted dual-labeled nanoparticles

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2013-12-01

    Full Text Available Yue Zhang,1 Bin Zhang,1 Fei Liu,1,2 Jianwen Luo,1,3 Jing Bai1 1Department of Biomedical Engineering, School of Medicine, 2Tsinghua-Peking Center for Life Sciences, 3Center for Biomedical Imaging Research, Tsinghua University, Beijing, People's Republic of China Abstract: Dual-modality imaging combines the complementary advantages of different modalities, and offers the prospect of improved preclinical research. The combination of fluorescence imaging and magnetic resonance imaging (MRI provides cross-validated information and direct comparison between these modalities. Here, we report on the application of a novel tumor-targeted, dual-labeled nanoparticle (NP, utilizing iron oxide as the MRI contrast agent and near infrared (NIR dye Cy5.5 as the fluorescent agent. Results of in vitro experiments verified the specificity of the NP to tumor cells. In vivo tumor targeting and uptake of the NPs in a mouse model were visualized by fluorescence and MR imaging collected at different time points. Quantitative analysis was carried out to evaluate the efficacy of MRI contrast enhancement. Furthermore, tomographic images were also acquired using both imaging modalities and cross-validated information of tumor location and size between these two modalities was revealed. The results demonstrate that the use of dual-labeled NPs can facilitate the dual-modal detection of tumors, information cross-validation, and direct comparison by combing fluorescence molecular tomography (FMT and MRI. Keywords: dual-modality, fluorescence molecular tomography (FMT, magnetic resonance imaging (MRI, nanoparticle

  18. Random laser in biological tissues impregnated with a fluorescent anticancer drug

    Science.gov (United States)

    Lahoz, F.; Martín, I. R.; Urgellés, M.; Marrero-Alonso, J.; Marín, R.; Saavedra, C. J.; Boto, A.; Díaz, M.

    2015-04-01

    We have demonstrated that chemically modified anticancer drugs can provide random laser (RL) when infiltrated in a biological tissue. A fluorescent biomarker has been covalently bound to tamoxifen, which is one of the most frequently used drugs for breast cancer therapy. The light emitted by the drug-dye composite is scattered in tissue, which acts as a gain medium. Both non-coherent and coherent RL regimes have been observed. Moreover, the analysis of power Fourier transforms of coherent RL spectra indicates that the tissues show a dominant random laser cavity length of about 18 µm, similar to the average size of single cells. These results show that RL could be obtained from other drugs, if properly marked with a fluorescent tag, which could be appealing for new forms of combined opto-chemical therapies.

  19. Turn-off fluorescence sensor for the detection of ferric ion in water using green synthesized N-doped carbon dots and its bio-imaging.

    Science.gov (United States)

    Edison, Thomas Nesakumar Jebakumar Immanuel; Atchudan, Raji; Shim, Jae-Jin; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol; Lee, Yong Rok

    2016-05-01

    This paper reports turn-off fluorescence sensor for Fe(3+) ion in water using fluorescent N-doped carbon dots as a probe. A simple and efficient hydrothermal carbonization of Prunus avium fruit extract for the synthesis of fluorescent nitrogen-doped carbon dots (N-CDs) is described. This green approach proceeds quickly and provides good quality N-CDs. The mean size of synthesized N-CDs was approximately 7nm calculated from the high-resolution transmission electron microscopic images. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy revealed the presence of -OH, -NH2, -COOH, and -CO functional groups over the surface of CDs. The N-CDs showed excellent fluorescent properties, and emitted blue fluorescence at 411nm upon excitation at 310nm. The calculated quantum yield of the synthesized N-CDs is 13% against quinine sulfate as a reference fluorophore. The synthesized N-CDs were used as a fluorescent probe towards the selective and sensitive detection of biologically important Fe(3+) ions in water by fluorescence spectroscopy and for bio-imaging of MDA-MB-231 cells. The limit of detection (LOD) and the Stern-Volmer quenching constant for the synthesized N-CDs were 0.96μM and 2.0958×10(3)M of Fe(3+) ions. The green synthesized N-CDs are efficiently used as a promising candidate for the detection of Fe(3+) ions and bio-imaging. PMID:26994332

  20. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    DEFF Research Database (Denmark)

    Hojman, Pernille; Eriksen, Jens; Gehl, Julie

    2009-01-01

    DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly...... weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability....

  1. Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects

    OpenAIRE

    Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

    2010-01-01

    We demonstrate the use of a compressive sampling algorithm for on-chip fluorescent imaging of sparse objects over an ultra-large field-of-view (>8 cm2) without the need for any lenses or mechanical scanning. In this lensfree imaging technique, fluorescent samples placed on a chip are excited through a prism interface, where the pump light is filtered out by total internal reflection after exciting the entire sample volume. The emitted fluorescent light from the specimen is collected through a...

  2. Aminopeptidase N/CD13 targeting fluorescent probes: synthesis and application to tumor cell imaging.

    Science.gov (United States)

    Zhang, Zhouen; Harada, Hiroshi; Tanabe, Kazuhito; Hatta, Hiroshi; Hiraoka, Masahiro; Nishimoto, Sei-ichi

    2005-11-01

    A family of fluorescein-peptide conjugates (CNP1-3) for aminopeptidase N (APN/CD13) targeting fluorescent probes were designed and synthesized. Among the three conjugates, CNP1 bearing tumor-homing cyclic peptide CNGRC, could selectively label APN/CD13 over-expressing on the surface of tumor cells of HT-1080, as identified by means of fluorescent microscopic cell imaging. CNP1 was shown to be a promising fluorescent probe applicable to tumor-targeting molecular imaging. PMID:15885853

  3. Silica nanocapsules of fluorescent conjugated polymers and superparamagnetic nanocrystals for dual-mode cellular imaging.

    Science.gov (United States)

    Tan, Happy; Wang, Miao; Yang, Chang-Tong; Pant, Shilpa; Bhakoo, Kishore Kumar; Wong, Siew Yee; Chen, Zhi-Kuan; Li, Xu; Wang, John

    2011-06-01

    We describe here a facile and benign synthetic strategy to integrate the fluorescent behavior of conjugated polymers and superparamagnetic properties of iron oxide nanocrystals into silica nanocapsules, forming a new type of bifunctional magnetic fluorescent silica nanocapsule (BMFSN). The resultant BMFSNs are uniform, colloidally stable in aqueous medium, and exhibit the desired dual functionality of fluorescence and superparamagnetism in a single entity. Four conjugated polymers with different emissions were used to demonstrate the versatility of employing this class of fluorescent materials for the preparation of BMFSNs. The applicability of BMFSNs in cellular imaging was studied by incubating them with human liver cancer cells, the result of which demonstrated that the cells could be visualized by dual-mode fluorescence and magnetic resonance imaging. Furthermore, the superparamagnetic behavior of the BMFSNs was exploited for in vitro magnetic-guided delivery of the nanocapsules into the cancer cells, thereby highlighting their potential for targeting biomedical applications.

  4. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    Directory of Open Access Journals (Sweden)

    Hojman Pernille

    2009-04-01

    Full Text Available Abstract DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

  5. Direct analysis of biological samples by total reflection X-ray fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Lue M, Marco P. [Unidad de Analisis Instrumental, Departamento de Quimica y Suelos, Decanato de Agronomia, Universidad Centro-occidental Lisandro Alvarado, Apartado Postal 4076, Cabudare 3023 (Venezuela)]. E-mail: luemerumarco@yahoo.es; Hernandez-Caraballo, Edwin A. [Instituto Venezolano-Andino para la Investigacion Quimica (IVAIQUIM), Facultad de Ciencias, Universidad de los Andes, Merida 5101 (Venezuela)

    2004-08-31

    The technique of total reflection X-ray fluorescence (TXRF) is well suited for the direct analysis of biological samples due to the low matrix interferences and simultaneous multi-element nature. Nevertheless, biological organic samples are frequently analysed after digestion procedures. The direct determination of analytes requires shorter analysis time, low reactive consumption and simplifies the whole analysis process. On the other hand, the biological/clinical samples are often available in minimal amounts and routine studies require the analysis of large number of samples. To overcome the difficulties associated with the analysis of organic samples, particularly of solid ones, different procedures of sample preparation and calibration to approach the direct analysis have been evaluated: (1) slurry sampling, (2) Compton peak standardization, (3) in situ microwave digestion, (4) in situ chemical modification and (5) direct analysis with internal standardization. Examples of analytical methods developed by our research group are discussed. Some of them have not been previously published, illustrating alternative strategies for coping with various problems that may be encountered in the direct analysis by total reflection X-ray fluorescence spectrometry.

  6. Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

    Science.gov (United States)

    Lee, Sungmoo; Piao, Hong Hua; Sepheri-Rad, Masoud; Jung, Arong; Sung, Uhna; Song, Yoon-Kyu; Baker, Bradley J

    2016-02-04

    Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

  7. Fluorescence-enhanced optical tomography and nuclear imaging system for small animals

    Science.gov (United States)

    Tan, I.-Chih; Lu, Yujie; Darne, Chinmay; Rasmussen, John C.; Zhu, Banghe; Azhdarinia, Ali; Yan, Shikui; Smith, Anne M.; Sevick-Muraca, Eva M.

    2012-03-01

    Near-infrared (NIR) fluorescence is an alternative modality for molecular imaging that has been demonstrated in animals and recently in humans. Fluorescence-enhanced optical tomography (FEOT) using continuous wave or frequency domain photon migration techniques could be used to provide quantitative molecular imaging in vivo if it could be validated against "gold-standard," nuclear imaging modalities, using dual-labeled imaging agents. Unfortunately, developed FEOT systems are not suitable for incorporation with CT/PET/SPECT scanners because they utilize benchtop devices and require a large footprint. In this work, we developed a miniaturized fluorescence imaging system installed in the gantry of the Siemens Inveon PET/CT scanner to enable NIR transillumination measurements. The system consists of a CCD camera equipped with NIR sensitive intensifier, a diode laser controlled by a single board compact controller, a 2-axis galvanometer, and RF circuit modules for homodyne detection of the phase and amplitude of fluorescence signals. The performance of the FEOT system was tested and characterized. A mouse-shaped solid phantom of uniform optical properties with a fluorescent inclusion was scanned using CT, and NIR fluorescence images at several projections were collected. The method of high-order approximation to the radioactive transfer equation was then used to reconstruct the optical images. Dual-labeled agents were also used on a tumor bearing mouse to validate the results of the FEOT against PET/CT image. The results showed that the location of the fluorophore obtained from the FEOT matches the location of tumor obtained from the PET/CT images. Besides validation of FEOT, this hybrid system could allow multimodal molecular imaging (FEOT/PET/CT) for small animal imaging.

  8. Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

    Science.gov (United States)

    Kawanabe, Satoshi; Araki, Yoshie; Uchimura, Tomohiro; Imasaka, Totaro

    2015-06-01

    Fluorescence lifetime imaging microscopy was applied to evaluate the efficacy of anticancer drugs. A decrease in the fluorescence lifetime of the nucleus in apoptotic cancer cells stained by SYTO 13 dye was detected after treatment with antitumor antibiotics such as doxorubicin or epirubicin. It was confirmed that the change in fluorescence lifetime occurred earlier than morphological changes in the cells. We found that the fluorescence lifetime of the nucleus in the cells treated with epirubicin decreased more rapidly than that of the cells treated with doxorubicin. This implies that epirubicin was more efficacious than doxorubicin in the treatment of cancer cells. The change in fluorescence lifetime was, however, not indicated when the cells were treated with cyclophosphamide. The decrease in fluorescence lifetime was associated with the processes involving caspase activation and chromatin condensation. Therefore, this technique would provide useful information about apoptotic cells, particularly in the early stages.

  9. Janus particles for biological imaging and sensing.

    Science.gov (United States)

    Yi, Yi; Sanchez, Lucero; Gao, Yuan; Yu, Yan

    2016-06-21

    Janus particles, named after the two-faced Roman god Janus, have different surface makeups, structures or compartments on two sides. This review highlights recent advances in employing Janus particles as novel analytical tools for live cell imaging and biosensing. Unlike conventional particles used in analytical science, two-faced Janus particles provide asymmetry and directionality, and can combine different or even incompatible properties within a single particle. The broken symmetry enables imaging and quantification of rotational dynamics, revealing information beyond what traditional measurements offer. The spatial segregation of molecules on the surface of a single particle also allows analytical functions that would otherwise interfere with each other to be decoupled, opening up opportunities for novel multimodal analytical methods. We summarize here the development of Janus particles, a few general methods for their fabrication and, more importantly, the emerging and novel applications of Janus particles as multi-functional imaging probes and sensors. PMID:27052001

  10. Functional brain fluorescence plurimetry in rat by implantable concatenated CMOS imaging system.

    Science.gov (United States)

    Kobayashi, Takuma; Masuda, Hiroyuki; Kitsumoto, Chikara; Haruta, Makito; Motoyama, Mayumi; Ohta, Yasumi; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Shiosaka, Sadao; Ohta, Jun

    2014-03-15

    Measurement of brain activity in multiple areas simultaneously by minimally invasive methods contributes to the study of neuroscience and development of brain machine interfaces. However, this requires compact wearable instruments that do not inhibit natural movements. Application of optical potentiometry with voltage-sensitive fluorescent dye using an implantable image sensor is also useful. However, the increasing number of leads required for the multiple wired sensors to measure larger domains inhibits natural behavior. For imaging broad areas by numerous sensors without excessive wiring, a web-like sensor that can wrap the brain was developed. Kaleidoscopic potentiometry is possible using the imaging system with concatenated sensors by changing the alignment of the sensors. This paper describes organization of the system, evaluation of the system by a fluorescence imaging, and finally, functional brain fluorescence plurimetry by the sensor. The recorded data in rat somatosensory cortex using the developed multiple-area imaging system compared well with electrophysiology results.

  11. Fluorescence microscopy imaging of cells with a plasmonic dish integrally molded

    Science.gov (United States)

    Tawa, Keiko; Sasakawa, Chisato; Fujita, Tsuyoshi; Kiyosue, Kazuyuki; Hosokawa, Chie; Nishii, Junji; Oike, Makoto; Kakinuma, Norihiro

    2016-03-01

    A plastic dish with a wavelength-scale periodic structure at a bottom panel was integrally molded and coated with thin metal films. The integrally molded dish called plasmonic dish was applied to bioimaging under a fluorescence microscope. On the plasmonic substrate, the enhanced electric field based on a grating-coupled surface plasmon resonance (GC-SPR) can provide an enhanced fluorescence. In this study, two kinds of cells, human embryonic kidney (HEK) cells and neuronal cells, were observed in our plasmonic dish. Fluorescence images of HEK cells were above 10 times brighter than those obtained on a conventional glass-bottomed dish. Neuronal cells were successfully cultured for 10 d on the plasmonic dish integrally molded, and in fluorescence images with transmitted light, a higher contrast was obtained than in epifluorescence images. The plasmonic dish integrally molded, as well as that fabricated by the UV nanoimprint method, was also found to be useful for sensitive bioimaging.

  12. Fluorescence Modified Chitosan-Coated Magnetic Nanoparticles for High-Efficient Cellular Imaging

    Directory of Open Access Journals (Sweden)

    Nie Fang

    2009-01-01

    Full Text Available Abstract Labeling of cells with nanoparticles for living detection is of interest to various biomedical applications. In this study, novel fluorescent/magnetic nanoparticles were prepared and used in high-efficient cellular imaging. The nanoparticles coated with the modified chitosan possessed a magnetic oxide core and a covalently attached fluorescent dye. We evaluated the feasibility and efficiency in labeling cancer cells (SMMC-7721 with the nanoparticles. The nanoparticles exhibited a high affinity to cells, which was demonstrated by flow cytometry and magnetic resonance imaging. The results showed that cell-labeling efficiency of the nanoparticles was dependent on the incubation time and nanoparticles’ concentration. The minimum detected number of labeled cells was around 104by using a clinical 1.5-T MRI imager. Fluorescence and transmission electron microscopy instruments were used to monitor the localization patterns of the magnetic nanoparticles in cells. These new magneto-fluorescent nanoagents have demonstrated the potential for future medical use.

  13. CEA-targeted nanoparticles allow specific in vivo fluorescent imaging of colorectal cancer models.

    Science.gov (United States)

    Tiernan, James P; Ingram, Nicola; Marston, Gemma; Perry, Sarah L; Rushworth, Jo V; Coletta, P Louise; Millner, Paul A; Jayne, David G; Hughes, Thomas A

    2015-01-01

    Fluorescent imaging of colorectal tumor cells would improve tumor localization and allow intra-operative staging, facilitating stratification of surgical resections thereby improving patient outcomes. We aimed to develop and test fluorescent nanoparticles capable of allowing this in vivo. Dye-doped silica nanoparticles were synthesized. Anti-CEA (carcinoembryonic antigen) or control IgGs were conjugated to nanoparticles using various chemical strategies. Binding of CEA-targeted or control nanoparticles to colorectal cancer cells was quantified in vitro, and in vivo after systemic-delivery to murine xenografts. CEA-targeted, polyamidoamine dendrimer-conjugated, nanoparticles, but not control nanoparticles, allowed strong tumor-specific imaging. We are the first to demonstrate live, specific, in vivo imaging of colorectal cancer cells using antibody-targeted fluorescent nanoparticles. These nanoparticles have potential to allow intra-operative fluorescent visualization of tumor cells.

  14. Observations of fluorescent and biological aerosol at a high-altitude site in Central France

    Directory of Open Access Journals (Sweden)

    A. M. Gabey

    2013-01-01

    Full Text Available Total bacteria, fungal spore and yeast counts were compared with UV Light-Induced Fluorescence (UV-LIF measurements of ambient aerosol at the summit of the Puy de Dôme (pdD mountain in Central France (1465 m a.s.l, which represents a background elevated site. Bacteria, fungal spores and yeast were enumerated by epifluorescence microscopy (EFM and found to number 2.2 to 23 L−1 and 0.8 to 2 L−1, respectively. Bacteria counts on two successive nights were an order of magnitude larger than in the intervening day.

    A Wide Issue Bioaerosol Spectrometer, version 3 (WIBS-3 was used to perform UV-LIF measurements on ambient aerosol sized 0.8 to 20 μm. Mean total number concentration was 270 L−1 (σ = 66 L−1 found predominantly in a size mode at 2 μm for most of the campaign. Total concentration (fluorescent + non-fluorescent aerosol peaked at 500 L−1 with a size mode at 1 μm because of a change in air mass origin lasting around 48 h. The WIBS-3 features two excitation and fluorescence detection wavelengths corresponding to different biological molecules. The mean fluorescent particle concentration after short-wave (280 nm; Tryptophan excitation was 12 L−1 (σ = 6 L−1, and did not vary much through the campaign. In contrast the mean concentration of particles fluorescent after long-wave (370 nm; NADH excitation was 95 L−1 (σ = 25 L−1, and a nightly rise and subsequent fall of up to 100 L−1 formed a strong diurnal cycle in the latter. The fluorescent populations exhibited size modes at 3 μm and 2 to 3 μm, respectively. A hierarchical agglomerative cluster analysis algorithm was applied to the data and used to extract different particle factors. A cluster concentration time series representative of bacteria was identified. This was found to exhibit a diurnal cycle with a maximum peak appearing during the day.

  15. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Kyle C. [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Mito, Jeffrey K.; Javid, Melodi P. [Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States); Ferrer, Jorge M. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Kim, Yongbaek [Department of Clinical Pathology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Lee, W. David [The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Bawendi, Moungi G. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Brigman, Brian E. [Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina (United States); Kirsch, David G., E-mail: david.kirsch@duke.edu [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States)

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  16. Spectral and fluorescence imaging of immune system and tissue response to an immunogenic agent

    Science.gov (United States)

    Choe, Se-woon; Acharya, Abhinav; Keselowsky, Benjamin G.; Sorg, Brian S.

    2009-05-01

    Imaging of immune system and tissue response to immunogenic agents can be important to the development of new biomaterials. Additionally, quantitative functional imaging can be useful for testing and evaluation of methods to alter or control the immune system response to implanted materials. In this preliminary study, we employ spectral imaging and fluorescence imaging to measure immune system and tissue response to implanted immunogenic agents. Poly (D,L lactide-co-glycolide) (PLGA) with a 50:50 composition was used to create immunogenic microparticles (MPs). Lipopolysaccharide (LPS) encapsulated in the MPs was used to provoke a tissue immune response in mice and encapsulated fluorescein isothiocyanate (FITC) was used to fluorescently label the MPs for imaging. Control MPs did not contain LPS. The MPs were delivered at 50 particles/μL in a total volume of 20μL by subcutaneous injection in the skin of a nude mouse in a dorsal skin-fold window chamber preparation. Cultured immune cells from a mouse leukemic monocyte macrophage cell line were exogenously labeled with the fluorescent dye DiD in solution at a concentration of 8000cells/μL. Immediately after window chamber surgery and implantation of the MPs, 100μL of the fluorescent macrophage solution was administered via the tail vein. Fluorescence imaging was used to track MPs and macrophages while spectral imaging was used for imaging and measurement of hemoglobin saturation in the tissue microvasculature. Imaging was performed periodically over about three days. The spectral and fluorescence imaging combination enabled detailed observations of the macrophage response and functional effects on the tissue.

  17. Clinical multi-colour fluorescence imaging of malignant tumours - initial experience

    Energy Technology Data Exchange (ETDEWEB)

    Svanberg, K.; Wang, I. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Oncology; Colleen, S. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Urology; Idvall, I. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Pathology; Ingvar, C. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Surgery; Rydell, R. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Oto-Rhino-Laryngology; Jocham, D. [Luebeck Univ. (Germany). Abt. fuer Urologie; Diddens, H. [Luebeck Univ. (Germany). Medizinisches Laser Zentrum; Bown, S.; Gregory, G. [National Medical Laser Centre, Dept. of Surgery, Rayne Inst., London (United Kingdom); Montan, S. [Spectraphos AB, Ideon, Lund (Sweden); Andersson-Engels, S.; Svanberg, S. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Inst. of Technology (Sweden). Dept. of Physics

    1998-01-01

    The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation, ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s was mixed with a normal colour video image of the tissue. A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in vitro investigations of resected breast cancer. (orig./MG).

  18. Pattern recognition software and techniques for biological image analysis.

    Directory of Open Access Journals (Sweden)

    Lior Shamir

    Full Text Available The increasing prevalence of automated image acquisition systems is enabling new types of microscopy experiments that generate large image datasets. However, there is a perceived lack of robust image analysis systems required to process these diverse datasets. Most automated image analysis systems are tailored for specific types of microscopy, contrast methods, probes, and even cell types. This imposes significant constraints on experimental design, limiting their application to the narrow set of imaging methods for which they were designed. One of the approaches to address these limitations is pattern recognition, which was originally developed for remote sensing, and is increasingly being applied to the biology domain. This approach relies on training a computer to recognize patterns in images rather than developing algorithms or tuning parameters for specific image processing tasks. The generality of this approach promises to enable data mining in extensive image repositories, and provide objective and quantitative imaging assays for routine use. Here, we provide a brief overview of the technologies behind pattern recognition and its use in computer vision for biological and biomedical imaging. We list available software tools that can be used by biologists and suggest practical experimental considerations to make the best use of pattern recognition techniques for imaging assays.

  19. Recent trends in total reflection X-ray fluorescence spectrometry for biological applications

    International Nuclear Information System (INIS)

    This review is focused on the application of total reflection X-ray fluorescence (TXRF) spectrometry in the field of biological research. In the last decade, most papers were published by authors who applied laboratory-scale TXRF equipments. The application of synchrotron radiation as excitation source (SR-TXRF) shows a slowly increasing tendency. In the cited papers the micro-, trace and multielement capability of these TXRF techniques was demonstrated in the clinical and medical laboratory practice, as well as in various plant physiological studies. For speciation of elements in biological matrices, the TXRF was used as element specific detector following an off-line separation step (e.g., thin layer chromatography, high performance liquid chromatography), however, these off-line methods are not competitive with the on-line coupled HPLC-inductively coupled plasma mass spectrometry

  20. Fluorescence excitation and imaging of single molecules near dielectric-coated and bare surfaces: a theoretical study.

    Science.gov (United States)

    Axelrod, Daniel

    2012-08-01

    Microscopic fluorescent samples of interest to cell and molecular biology are commonly embedded in an aqueous medium near a solid surface that is coated with a thin film such as a lipid multilayer, collagen, acrylamide, or a cell wall. Both excitation and emission of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index and the fluorophore's orientation. For total internal reflection excitation, multiple reflections in the film can lead to resonance peaks in the evanescent intensity versus incidence angle curve. For emission, multiple reflections arising from the fluorophore's near field emission can create a distinct intensity pattern in both the back focal plane and the image plane of a high aperture objective. This theoretical analysis discusses how these features can be used to report film thickness and refractive index, and fluorophore axial position and orientation. PMID:22612666

  1. A compact gamma camera for biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, E L; Cella, J; Majewski, S; Popov, V; Qian, Jianguo; Saha, M S; Smith, M F; Weisenberger, A G; Welsh, R E

    2006-02-01

    A compact detector, sized particularly for imaging a mouse, is described. The active area of the detector is approximately 46 mm; spl times/ 96 mm. Two flat-panel Hamamatsu H8500 position-sensitive photomultiplier tubes (PSPMTs) are coupled to a pixellated NaI(Tl) scintillator which views the animal through a copper-beryllium (CuBe) parallel-hole collimator specially designed for {sup 125}I. Although the PSPMTs have insensitive areas at their edges and there is a physical gap, corrections for scintillation light collection at the junction between the two tubes results in a uniform response across the entire rectangular area of the detector. The system described has been developed to optimize both sensitivity and resolution for in-vivo imaging of small animals injected with iodinated compounds. We demonstrate an in-vivo application of this detector, particularly to SPECT, by imaging mice injected with approximately 10-15; spl mu/Ci of {sup 125}I.

  2. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    Directory of Open Access Journals (Sweden)

    Karolina Jahn

    Full Text Available For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR and Asante Calcium Green (ACG for two-photon (2P-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+-free and Ca(2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+-dependent way, unraveling in vitro dissociation constants K(D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns and long (2.44 ns decay time components attributable to the Ca(2+-free and Ca(2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D of 180 nM was determined. Thus, quantitative [Ca(2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  3. Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

    Science.gov (United States)

    Lai, Zhenhua; Lian, Christine; Ma, Jie; Yu, Jingyi; Gu, Zetong; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2014-02-01

    Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

  4. Fluorescence endoscopic imaging study of anastomotic recurrence of Crohn's disease after right ileocolonic resection

    Science.gov (United States)

    Mordon, Serge R.; Maunoury, Vincent; Klein, Olivier; Colombel, Jean-Frederic

    1995-12-01

    Crohn's disease is an inflammatory bowel disease of unknown etiology. Vasculitis is hypothesized but it was never demonstrated in vivo. This study aimed to evaluate the vascular mucosa perfusion using fluorescence imaging in 13 patients who had previously undergone eileocolonic resection and who agreed to participate in a prospective endoscopic study of anastomotic recurrence. This anastomotic recurrence rate is known to be high (73% after 1 year follow-up) and is characterized by ulcerations. The fluorescence study was started with an I.V. bolus injection of sodium fluorescein. The pre-anastomotic mucosa was endoscopically examined with blue light that stimulates fluorescein fluorescence. Fluorescence emission was recorded with an ultra-high-sensitivity camera connected to the endoscope via an interference filter (520 - 560 nm). A uniform fluorescence was observed a few seconds after the injection and lasted for 15 min in healthy subjects. In case of recurrence, the centers of the ulcerations displayed a very low fluorescence indicating localized ischemia. In contrast, the rims of the ulcers revealed brighter fluorescent images than those of normal mucosa. The anastomotic ulcerations of Crohn's disease recurrence exhibit a high fluorescence intensity at their margins indicating an increased mucosal blood flow and/or enhanced transcapillary diffusion. These findings support the hypothesis of a primary vasculitis in Crohn's disease.

  5. Nucleic Acid-Based Nanodevices in Biological Imaging.

    Science.gov (United States)

    Chakraborty, Kasturi; Veetil, Aneesh T; Jaffrey, Samie R; Krishnan, Yamuna

    2016-06-01

    The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand. PMID:27294440

  6. Improvement and biological applications of fluorescent probes for zinc, ZnAFs.

    Science.gov (United States)

    Hirano, Tomoya; Kikuchi, Kazuya; Urano, Yasuteru; Nagano, Tetsuo

    2002-06-12

    The development and cellular applications of novel fluorescent probes for Zn2+, ZnAF-1F, and ZnAF-2F are described. Fluorescein is used as a fluorophore of ZnAFs, because its excitation and emission wavelengths are in the visible range, which minimizes cell damage and autofluorescence by excitation light. N,N-Bis(2-pyridylmethyl)ethylenediamine, used as an acceptor for Zn2+, is attached directly to the benzoic acid moiety of fluorescein, resulting in very low quantum yields of 0.004 for ZnAF-1F and 0.006 for ZnAF-2F under physiological conditions (pH 7.4) due to the photoinduced electron-transfer mechanism. Upon the addition of Zn2+, the fluorescence intensity is quickly increased up to 69-fold for ZnAF-1F and 60-fold for ZnAF-2F. Apparent dissociation constants (K(d)) are in the nanomolar range, which affords sufficient sensitivity for biological applications. ZnAFs do not fluoresce in the presence of other biologically important cations such as Ca2+ and Mg2+, and are insensitive to change of pH. The complexes with Zn2+ of previously developed ZnAFs, ZnAF-1, and ZnAF-2 decrease in fluorescence intensity below pH 7.0 owing to protonation of the phenolic hydroxyl group of fluorescein, whose pKa value is 6.2. On the other hand, the Zn2+ complexes of ZnAF-1F and ZnAF-2F emit stable fluorescence around neutral and slightly acidic conditions because the pKa values are shifted to 4.9 by substitution of electron-withdrawing fluorine at the ortho position of the phenolic hydroxyl group. For application to living cells, the diacetyl derivative of ZnAF-2F, ZnAF-2F DA, was synthesized. ZnAF-2F DA can permeate through the cell membrane, and is hydrolyzed by esterase in the cytosol to yield ZnAF-2F, which is retained in the cells. Using ZnAF-2F DA, we could measure the changes of intracellular Zn2+ in cultured cells and hippocampal slices.

  7. Image overlay solution based on threshold detection for a compact near infrared fluorescence goggle system.

    Science.gov (United States)

    Gao, Shengkui; Mondal, Suman B; Zhu, Nan; Liang, RongGuang; Achilefu, Samuel; Gruev, Viktor

    2015-01-01

    Near infrared (NIR) fluorescence imaging has shown great potential for various clinical procedures, including intraoperative image guidance. However, existing NIR fluorescence imaging systems either have a large footprint or are handheld, which limits their usage in intraoperative applications. We present a compact NIR fluorescence imaging system (NFIS) with an image overlay solution based on threshold detection, which can be easily integrated with a goggle display system for intraoperative guidance. The proposed NFIS achieves compactness, light weight, hands-free operation, high-precision superimposition, and a real-time frame rate. In addition, the miniature and ultra-lightweight light-emitting diode tracking pod is easy to incorporate with NIR fluorescence imaging. Based on experimental evaluation, the proposed NFIS solution has a lower detection limit of 25 nM of indocyanine green at 27 fps and realizes a highly precise image overlay of NIR and visible images of mice in vivo. The overlay error is limited within a 2-mm scale at a 65-cm working distance, which is highly reliable for clinical study and surgical use.

  8. Red to far-red multispectral fluorescence image fusion for detection of fecal contamination on apples

    Science.gov (United States)

    This research developed a multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet/blue LED excitation for detection of fecal contamination on Golden Delicious apples. Using a hyperspectral line-scan imaging system consisting of an EMCCD camera, spectrograph, an...

  9. Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

    Directory of Open Access Journals (Sweden)

    Haibiao Gong

    Full Text Available Fluorescence in the near-infrared (NIR spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f. This property makes SNAP(f a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f-Beta-2 adrenergic receptor (SNAP(f-ADRβ2 fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

  10. A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis.

    Science.gov (United States)

    Yang, Tao; Wang, Sibo; Zhang, Xuyong; Xia, Jie; Guo, Jun; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-04-01

    Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes. PMID:27180584

  11. Formalin-induced fluorescence reveals cell shape and morphology in biological tissue samples.

    Directory of Open Access Journals (Sweden)

    Ulrich Leischner

    Full Text Available Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques.

  12. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    International Nuclear Information System (INIS)

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 106-3 x 108 labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 106 cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells. (orig.)

  13. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [Department of Radiology, UCSF Medical Center, University of California in San Francisco, 513 Parnassus Ave, CA 94143, San Francisco (United States); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Institute of Pathology, Technical University, Munich (Germany); Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Department of Radiology, Technical University, Munich (Germany); Pichler, Bernd [Department of Biomedical Engineering, University of California Davis, Davis (United States); Heinzmann, Ulrich [National Research Center for Environment and Health, Technical University, Munich (Germany); Oostendorp, Robert A.J. [3. Clinic of Internal Medicine, Laboratory of Stem Cell Physiology, Technical University, Munich (Germany)

    2004-09-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10{sup 6}-3 x 10{sup 8} labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10{sup 6} cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells

  14. Pinhole X-ray Fluorescence Imaging of Gadolinium Nanoparticles: A Preliminary Monte Carlo Study

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Seong Moon; Sung, Won Mo; Ye, Sung Joon [Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    X-ray fluorescence imaging is a modality for the element-specific imaging of a subject through analysis of characteristic x-rays produced by exploiting the interaction of high atomic number elements and incoming x-rays. Previous studies have utilized a polychromatic x-ray source to investigate the production of in vivo x-ray fluorescence images for the assessment of concentrations and locations of gold nanoparticles. However, previous efforts have so far been unable to detect low concentrations, such as 0.001% gold by weight, which is an expected concentration accumulated in tumors. We examined the feasibility of a monochromatic synchrotron x-rays implementation of pinhole x-ray fluorescence imaging by Monte Carlo simulations using MCNP5. In the current study, gadolinium (Gd) nanoparticles, which have been widely used as a contrast agent in magnetic resonance imaging and also as a dose enhancer in radiation therapy, were chosen for tumor targeting. Since a monochromatic x-ray source is used, the increased x-ray fluorescence signals allow the detection of low concentrations of Gd. Two different monochromatic x-ray beam energies, 50.5 keV near the Kedge energy (i.e., 50.207 keV) of Gd and 55 keV, were compared by their respective imaging results. Using Monte Carlo simulations the feasibility of imaging low concentrations of Gd nanoparticles (e.g., 0.001 wt%) with x-ray fluorescence using monochromatic synchrotron x-rays of two different energies was shown. In the case of imaging a single Gd column inserted in the center of a water phantom, the fluorescence signals from 0.05 wt% and 0.1 wt% Gd columns irradiated with a 50.5 keV photon beam were higher than those irradiated with 55 keV. Below 0.05 wt% region no significant differences were found.

  15. Pinhole X-ray Fluorescence Imaging of Gadolinium Nanoparticles: A Preliminary Monte Carlo Study

    International Nuclear Information System (INIS)

    X-ray fluorescence imaging is a modality for the element-specific imaging of a subject through analysis of characteristic x-rays produced by exploiting the interaction of high atomic number elements and incoming x-rays. Previous studies have utilized a polychromatic x-ray source to investigate the production of in vivo x-ray fluorescence images for the assessment of concentrations and locations of gold nanoparticles. However, previous efforts have so far been unable to detect low concentrations, such as 0.001% gold by weight, which is an expected concentration accumulated in tumors. We examined the feasibility of a monochromatic synchrotron x-rays implementation of pinhole x-ray fluorescence imaging by Monte Carlo simulations using MCNP5. In the current study, gadolinium (Gd) nanoparticles, which have been widely used as a contrast agent in magnetic resonance imaging and also as a dose enhancer in radiation therapy, were chosen for tumor targeting. Since a monochromatic x-ray source is used, the increased x-ray fluorescence signals allow the detection of low concentrations of Gd. Two different monochromatic x-ray beam energies, 50.5 keV near the Kedge energy (i.e., 50.207 keV) of Gd and 55 keV, were compared by their respective imaging results. Using Monte Carlo simulations the feasibility of imaging low concentrations of Gd nanoparticles (e.g., 0.001 wt%) with x-ray fluorescence using monochromatic synchrotron x-rays of two different energies was shown. In the case of imaging a single Gd column inserted in the center of a water phantom, the fluorescence signals from 0.05 wt% and 0.1 wt% Gd columns irradiated with a 50.5 keV photon beam were higher than those irradiated with 55 keV. Below 0.05 wt% region no significant differences were found

  16. The possibilities of improvement in the sensitivity of cancer fluorescence diagnostics by computer image processing

    Science.gov (United States)

    Ledwon, Aleksandra; Bieda, Robert; Kawczyk-Krupka, Aleksandra; Polanski, Andrzej; Wojciechowski, Konrad; Latos, Wojciech; Sieron-Stoltny, Karolina; Sieron, Aleksander

    2008-02-01

    Background: Fluorescence diagnostics uses the ability of tissues to fluoresce after exposition to a specific wavelength of light. The change in fluorescence between normal and progression to cancer allows to see early cancer and precancerous lesions often missed by white light. Aim: To improve by computer image processing the sensitivity of fluorescence images obtained during examination of skin, oral cavity, vulva and cervix lesions, during endoscopy, cystoscopy and bronchoscopy using Xillix ONCOLIFE. Methods: Function of image f(x,y):R2 --> R 3 was transformed from original color space RGB to space in which vector of 46 values refers to every point labeled by defined xy-coordinates- f(x,y):R2 --> R 46. By means of Fisher discriminator vector of attributes of concrete point analalyzed in the image was reduced according to two defined classes defined as pathologic areas (foreground) and healthy areas (background). As a result the highest four fisher's coefficients allowing the greatest separation between points of pathologic (foreground) and healthy (background) areas were chosen. In this way new function f(x,y):R2 --> R 4 was created in which point x,y corresponds with vector Y, H, a*, c II. In the second step using Gaussian Mixtures and Expectation-Maximisation appropriate classificator was constructed. This classificator enables determination of probability that the selected pixel of analyzed image is a pathologically changed point (foreground) or healthy one (background). Obtained map of probability distribution was presented by means of pseudocolors. Results: Image processing techniques improve the sensitivity, quality and sharpness of original fluorescence images. Conclusion: Computer image processing enables better visualization of suspected areas examined by means of fluorescence diagnostics.

  17. Synthesis and Application of an Aldazine-Based Fluorescence Chemosensor for the Sequential Detection of Cu2+ and Biological Thiols in Aqueous Solution and Living Cells

    Directory of Open Access Journals (Sweden)

    Hongmin Jia

    2016-01-01

    Full Text Available A fluorescence chemosensor, 2-hydroxy-1-naphthaldehyde azine (HNA was designed and synthesized for sequential detection of Cu2+ and biothiols. It was found that HNA can specifically bind to Cu2+ with 1:1 stoichiometry, accompanied with a dramatic fluorescence quenching and a remarkable bathochromic-shift of the absorbance peak in HEPES buffer. The generated HNA-Cu2+ ensemble displayed a “turn-on” fluorescent response specific for biothiols (Hcy, Cys and GSH based on the displacement approach, giving a remarkable recovery of fluorescence and UV-Vis spectra. The detection limits of HNA-Cu2+ to Hcy, Cys and GSH were estimated to be 1.5 μM, 1.0 μM and 0.8 μM, respectively, suggesting that HNA-Cu2+ is sensitive enough for the determination of thiols in biological systems. The biocompatibility of HNA towards A549 human lung carcinoma cell, was evaluated by an MTT assay. The capability of HNA-Cu2+ to detect biothiols in live A549 cells was then demonstrated by a microscopy fluorescence imaging assay.

  18. Fluorescence endoscopic imaging for evaluation of gastric mucosal blood flow: a preliminary study

    Science.gov (United States)

    Bocquillon, Nicolas; Mordon, Serge R.; Mathieu, D.; Maunoury, Vincent; Marechal, Xavier-Marie; Neviere, Remi; Wattel, Francis; Chopin, Claude

    1999-02-01

    Microcirculatory disorders of the gastrointestinal tract appear to be a major compound of the multiple organ dysfunction syndrome secondary to sepsis or septic shock. A better analysis of mucosal hypoperfusion in critically ill patients with sepsis may be helpful for the comprehension of this high mortality-associated syndrome. Fluorescence endoscopy has been recognized as a non-invasive method for both spatial and temporal evaluation of gastrointestinal mucosal perfusion. We performed this imaging technique during routine gastric endoscopy in patients with sepsis criteria. The study included gastric observation and appearance time of gastric fluorescence after an intravenous 10% sodium - fluorescein bolus. Qualitative analysis of high fluorescence areas was compared with mucosal blood flow measurements by laser - Doppler flowmetry. We concluded that the fluorescence endoscopic imaging in critically ill patients with sepsis may reveal spacial and temporal differences in the mucosal microcirculation distribution.

  19. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    International Nuclear Information System (INIS)

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases

  20. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  1. Development of fluorescence based handheld imaging devices for food safety inspection

    Science.gov (United States)

    Lee, Hoyoung; Kim, Moon S.; Chao, Kuanglin; Lefcourt, Alan M.; Chan, Diane E.

    2013-05-01

    For sanitation inspection in food processing environment, fluorescence imaging can be a very useful method because many organic materials reveal unique fluorescence emissions when excited by UV or violet radiation. Although some fluorescence-based automated inspection instrumentation has been developed for food products, there remains a need for devices that can assist on-site inspectors performing visual sanitation inspection of the surfaces of food processing/handling equipment. This paper reports the development of an inexpensive handheld imaging device designed to visualize fluorescence emissions and intended to help detect the presence of fecal contaminants, organic residues, and bacterial biofilms at multispectral fluorescence emission bands. The device consists of a miniature camera, multispectral (interference) filters, and high power LED illumination. With WiFi communication, live inspection images from the device can be displayed on smartphone or tablet devices. This imaging device could be a useful tool for assessing the effectiveness of sanitation procedures and for helping processors to minimize food safety risks or determine potential problem areas. This paper presents the design and development including evaluation and optimization of the hardware components of the imaging devices.

  2. Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

    Science.gov (United States)

    Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

    2014-01-01

    Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

  3. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

    Science.gov (United States)

    Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

    2013-01-01

    Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

  4. Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue

    Science.gov (United States)

    Davis, Scott C.; Pogue, Brian W.; Springett, Roger; Leussler, Christoph; Mazurkewitz, Peter; Tuttle, Stephen B.; Gibbs-Strauss, Summer L.; Jiang, Shudong S.; Dehghani, Hamid; Paulsen, Keith D.

    2008-06-01

    A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1nM in a 70mm diameter homogeneous phantom, and detection is feasible to near 10pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

  5. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    International Nuclear Information System (INIS)

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing

  6. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, B-M.; Stoller, P.; Reiser, K.; Eichler, J.; Yan, M.; Rubenchik, A.; Da Silva, L.

    2000-03-06

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.

  7. Ratiometric fluorescence imaging of free Zn2+ in brain

    Science.gov (United States)

    Thompson, Richard B.; Suh, Sang W.; Frederickson, Christopher J.

    2001-05-01

    Recently, the function of zinc in the axonal boutons of hippocampal neurons has come under increased scrutiny as evidence has emerged of a putative role for this metal ion in neural damage following insults such as ischemia, blunt force trauma, and seizure. Indeed, the nonpathological role of free zinc in the brain remains cryptic after more than 40 years. We have used a biosensing approach to determine free zinc ion concentrations by fluorescence lifetime, intensity, intensity ratio, or anisotropy changes caused by binding of zinc to variants of a protein, apocarbonic anhydrase II (apo-CA). This approach permits real time measurement of zinc down to picomolar levels, with no perceptible interference from other divalent metal ions abundant in serum and tissue, such as calcium and magnesium. Recently, we used apo-CA together with a fluorescent ligand whose binding is metal-dependent to obtain the first fluorescence micrographs of zinc release from a rat hippocampus model in response to electrical stimulus. In our view, elucidation of the zinc fluxes in neural tissue ultimately requires quantitation, as in the case of calcium. Recent results will be shown.

  8. Fluorescence Imaging of Interscapular Brown Adipose Tissue in Living Mice†

    OpenAIRE

    Rice, Douglas R.; White, Alexander G.; Leevy, W. Matthew; Smith, Bradley D.

    2015-01-01

    Brown adipose tissue (BAT) plays a key role in energy expenditure and heat generation and is a promising target for diagnosing and treating obesity, diabetes and related metabolism disorders. While several nuclear and magnetic resonance imaging methods are established for detecting human BAT, there are no convenient protocols for high throughput imaging of BAT in small animal models. Here we disclose a simple but effective method for non-invasive optical imaging of interscapular BAT in mice u...

  9. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging1

    OpenAIRE

    Li, Cong; Greenwood, Tiffany R; Glunde, Kristine

    2008-01-01

    Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of thes...

  10. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging

    OpenAIRE

    Cong Li; Greenwood, Tiffany R; Kristine Glunde

    2008-01-01

    Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of thes...

  11. New image colocalization coefficient for fluorescence microscopy to quantify (bio-)molecular interactions

    OpenAIRE

    Herce, HD; Casas-Delucchi, CS; Cardoso, MC

    2013-01-01

    The spatial relationship, or degree of colocalization, between two or more types of molecules in live cells is commonly detected using fluorescence microscopy. This spatial distribution can be used to estimate the interaction between fluorescently labelled molecules. These interactions are usually quantified by analysing the correlation and/or the overlap between images, using the Pearson's and Manders’ coefficients, respectively. However, the correlation and overlap coefficients are paramete...

  12. X-ray fluorescence imaging of the hippocampal formation after manganese exposure.

    Science.gov (United States)

    Robison, Gregory; Zakharova, Taisiya; Fu, Sherleen; Jiang, Wendy; Fulper, Rachael; Barrea, Raul; Zheng, Wei; Pushkar, Yulia

    2013-11-01

    Manganese (Mn) intoxication results in neurological conditions similar, but not identical, to idiopathic Parkinson's disease. While the mechanism(s) by which Mn exposure leads to neurotoxic effects remains unclear, studies by magnetic resonance imaging demonstrate a high Mn accumulation in the hippocampal formation (HPCf) of the brain. Metal quantification using this method is not possible. Using X-ray fluorescence imaging, we measured the distribution of Mn in the HPCf for a rodent model of chronic Mn exposure and quantitatively compared it with distributions of other biologically relevant metals. We found considerable increases in average Mn concentrations in all analyzed areas and we identified the dentate gyrus (DG) and the cornus ammonis 3 (CA3) layer as areas accumulating the highest Mn content (∼1.2 μg Mn per g tissue). The DG is significantly enriched with iron (Fe), while the CA3 layer has high zinc (Zn) content. Additionally, significant spatial correlations were found for Mn-Zn concentrations across the HPCf substructures and for Mn-Fe concentrations in the DG. Combined results support that at least two mechanisms may be responsible for Mn transport and/or storage in the brain, associated with either Fe or Zn. Subcellular resolution images of metal distribution in cells of the CA3 show diffuse Mn distributions consistent with Mn localization in both the cytoplasm and nucleus. Mn was not increased in localized intracellular Fe or copper accumulations. A consistent Mn-Zn correlation both at the tissue (40 μm × 40 μm) and cellular (0.3 μm × 0.3 μm) levels suggests that a Zn transport/storage mechanism in the HPCf is likely associated with Mn accumulation. PMID:23999853

  13. 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

  14. Simulation of air shower image in fluorescence light based on energy deposits derived from CORSIKA

    OpenAIRE

    Gora, D.; Heck, D.; Homola, P.; Klages, H.; Pekala, J.; Risse, M.; Wilczynska, B.; Wilczynski, H.

    2004-01-01

    Spatial distributions of energy deposited by an extensive air shower in the atmosphere through ionization, as obtained from the CORSIKA simulation program, are used to find the fluorescence light distribution in the optical image of the shower. The shower image derived in this way is somewhat smaller than that obtained from the NKG lateral distribution of particles in the shower. The size of the image shows a small dependence on the primary particle type.

  15. Rapid Cancer Fluorescence Imaging Using A γ-Glutamyltranspeptidase-Specific Probe For Primary Lung Cancer

    OpenAIRE

    Hino, Haruaki; Kamiya, Mako; Kitano, Kentaro; Mizuno, Kazue; Tanaka, Sayaka; Nishiyama, Nobuhiro; Kataoka, Kazunori; Urano, Yasuteru; Nakajima, Jun

    2016-01-01

    BACKGROUND: We set out to examine the activity of γ-glutamyltranspeptidase (GGT) in lung cancer and the validity of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) for intraoperative imaging of primary lung cancer. METHODS: GGT activities and mRNA expression levels of GGT1 (one of the GGT subtypes) in five human lung cancer cell lines were examined by fluorescence imaging and quantitative reverse transcription polymerase chain reaction. In vivo imaging of an orthotopic A549 xenograft mod...

  16. Hyperspectral fluorescence lidar imaging at the Colosseum, Rome: elucidating past conservation interventions.

    Science.gov (United States)

    Palombi, L; Lognoli, D; Raimondi, V; Cecchi, G; Hällström, J; Barup, K; Conti, C; Grönlund, R; Johansson, A; Svanberg, S

    2008-05-12

    Fluorescence lidar techniques offer considerable potential for remote, non-invasive diagnostics of stone cultural heritage in the outdoor environment. Here we present the results of a joint Italian-Swedish experiment, deploying two hyperspectral fluorescence lidar imaging systems, for the documentation of past conservation interventions on the Colosseum, Rome. Several portions of the monument were scanned and we show that it was possible to discriminate among masonry materials, reinforcement structures and protective coatings inserted during past conservation interventions, on the basis of their fluorescence signatures, providing useful information for a first quick, large-scale in situ screening of the monument. PMID:18545382

  17. Preliminary experiment of fluorescent X-ray computed tomography to detect dual agents for biological study.

    Science.gov (United States)

    Yu, Q; Takeda, T; Yuasa, T; Hasegawa, Y; Wu, J; Thet-Thet-Lwin; Hyodo, K; Dilmanian, F A; Itai, Y; Akatsuka, T

    2001-05-01

    The simultaneous observation of various information, such as blood flow, tissue metabolism and distribution of receptors, is quite important in order to understand the functional state of biomedical objects. The simultaneous detectability of contrast agents by fluorescent X-ray computed tomography (FXCT) with synchrotron radiation is examined in this study. The system consisted of a silicon (111) double-crystal monochromator, an X-ray slit system, a scanning table, a PIN diode, a highly purified germanium detector and an X-ray charge-coupled device (CCD) camera. The monochromatic X-ray beam energy was adjusted to 37.0 keV and collimated into a pencil beam of 1 x 1 mm. The fluorescent spectra of the K alpha lines for iodine and xenon were detected simultaneously. FXCT could image the distribution of both iodine and xenon agents in a phantom clearly and the contrast ratio was significantly better than that of transmission X-ray computed tomography images. PMID:11486409

  18. Improved tumor identification using dual tracer molecular imaging in fluorescence guided brain surgery

    Science.gov (United States)

    Xu, Xiaochun; Torres, Veronica; Straus, David; Brey, Eric M.; Byrne, Richard W.; Tichauer, Kenneth M.

    2015-03-01

    Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional "single-tracer" molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.

  19. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

    Science.gov (United States)

    Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

    2013-02-01

    There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

  20. Cisplatin Prodrug-Conjugated Gold Nanocluster for Fluorescence Imaging and Targeted Therapy of the Breast Cancer

    Science.gov (United States)

    Zhou, Fangyuan; Feng, Bing; Yu, Haijun; Wang, Dangge; Wang, Tingting; Liu, Jianping; Meng, Qingshuo; Wang, Siling; Zhang, Pengcheng; Zhang, Zhiwen; Li, Yaping

    2016-01-01

    Theranostic nanomedicine has emerged as a promising modality for cancer diagnosis and treatment. In this study, we report the fabrication of fluorescence gold nanoclusters (GNC) conjugated with a cisplatin prodrug and folic acid (FA) (FA-GNC-Pt) for fluorescence imaging and targeted chemotherapy of breast cancer. The physio-chemical properties of FA-GNC-Pt nanoparticles are thoroughly characterized by fluorescence/UV-Vis spectroscopic measurement, particle size and zeta-potential examination. We find that FA-modification significantly accelerated the cellular uptake and increased the cytotoxicity of GNC-Pt nanoparticles in murine 4T1 breast cancer cells. Fluorescence imaging in vivo using 4T1 tumor bearing nude mouse model shows that FA-GNC-Pt nanoparticles selectively accumulate in the orthotopic 4T1 tumor and generate strong fluorescence signal due to the tumor targeting effect of FA. Moreover, we demonstrate that FA-GNC-Pt nanoparticles significantly inhibit the growth and lung metastasis of the orthotopically implanted 4T1 breast tumors. All these data imply a good potential of the GNC-based theranostic nanoplatform for fluorescence tumor imaging and cancer therapy. PMID:27022415

  1. Improving Automated Annotation of Benthic Survey Images Using Wide-band Fluorescence

    Science.gov (United States)

    Beijbom, Oscar; Treibitz, Tali; Kline, David I.; Eyal, Gal; Khen, Adi; Neal, Benjamin; Loya, Yossi; Mitchell, B. Greg; Kriegman, David

    2016-03-01

    Large-scale imaging techniques are used increasingly for ecological surveys. However, manual analysis can be prohibitively expensive, creating a bottleneck between collected images and desired data-products. This bottleneck is particularly severe for benthic surveys, where millions of images are obtained each year. Recent automated annotation methods may provide a solution, but reflectance images do not always contain sufficient information for adequate classification accuracy. In this work, the FluorIS, a low-cost modified consumer camera, was used to capture wide-band wide-field-of-view fluorescence images during a field deployment in Eilat, Israel. The fluorescence images were registered with standard reflectance images, and an automated annotation method based on convolutional neural networks was developed. Our results demonstrate a 22% reduction of classification error-rate when using both images types compared to only using reflectance images. The improvements were large, in particular, for coral reef genera Platygyra, Acropora and Millepora, where classification recall improved by 38%, 33%, and 41%, respectively. We conclude that convolutional neural networks can be used to combine reflectance and fluorescence imagery in order to significantly improve automated annotation accuracy and reduce the manual annotation bottleneck.

  2. In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

    Science.gov (United States)

    Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

    2014-03-01

    The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

  3. Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

    Science.gov (United States)

    Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

    2015-10-01

    Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

  4. Full-angle optical imaging of near-infrared fluorescent probes implanted in small animals

    Institute of Scientific and Technical Information of China (English)

    Gang Hu; Junjie Yao; Jing Bai

    2008-01-01

    To provide a valuable experimental platform for in vivo biomedical research of small animal model with fluorescence mediated approach, we developed a whole-body near-infrared fluorescence molecular imaging system as described in this paper. This system is based on a sensitive CCD camera and has the ability to achieve 360° full-angle source illuminations and projections capture of the targets to obtain the dense sampling by performing rotational scan. The measurement accuracy is validated from cylinder phantom experiments by the comparison between the experimental data and theoretical predictions. Finally, we also present typical in vivo images of fluorescent tube implanted into the mouse body. The results are promising and have proved the system imaging performance for macroscopic optical biomedical research.

  5. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques.

    Science.gov (United States)

    Wagner, D; Börgardts, M; Grünzweig, C; Lehmann, E; Müller, T J J; Egelhaaf, S U; Hermes, H E

    2015-09-01

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied. PMID:26429447

  6. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

    Science.gov (United States)

    Wagner, D.; Börgardts, M.; Grünzweig, C.; Lehmann, E.; Müller, T. J. J.; Egelhaaf, S. U.; Hermes, H. E.

    2015-09-01

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied.

  7. Assessment of the quality of durum wheat products by spectrofluorometry and fluorescence video image analysis

    Science.gov (United States)

    Novales, Bruno; Abecassis, Joel; Bertrand, Dominique; Devaux, Marie-Francoise; Robert, Paul

    1995-01-01

    Because assessment of Durum wheat semolina purity by standard ash-test has been widely criticized, we attempted to characterize products of a semolina mill by spectrofluorometry and fluorescence imaging. A collection of milled wheat products ranging from very pure semolina to brans were chosen for this study. Multidimensional statistical analyses (Principal component analyses) were applied to the spectral and image data. Maps showing a classification of the products according to purity were obtained without biochemical calibration. Principal component regression was applied to the data in order to test the relationship of aleurone fluorescence to ash content. Both spectrofluorometry and fluorescence imaging gave similar results with good determination coefficients (r2 equals 0.97 and 0.92) for the study of a single wheat variety. Products obtained from different wheat varieties were more difficult to compare.

  8. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, D.; Egelhaaf, S. U.; Hermes, H. E. [Condensed Matter Physics Laboratory, Heinrich Heine University, 40225 Düsseldorf (Germany); Börgardts, M.; Müller, T. J. J. [Institute for Organic and Macromolecular Chemistry, Heinrich Heine University, 40225 Düsseldorf (Germany); Grünzweig, C.; Lehmann, E. [Neutron Imaging and Activation Group, Paul Scherrer Institute, 5232 Villigen (Switzerland)

    2015-09-15

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied.

  9. A two-photon fluorescent turn-on probe for imaging of SO2 derivatives in living cells and tissues.

    Science.gov (United States)

    Zhu, Xiaoyan; Zhu, Longming; Liu, Hong-Wen; Hu, Xiaoxiao; Peng, Rui-Zi; Zhang, Jing; Zhang, Xiao-Bing; Tan, Weihong

    2016-09-21

    SO2 and its derivatives (bisulfite/sulfite) play crucial roles in several physiological processes. Therefore, development of reliable analytical methods for monitoring SO2 and its derivatives in biological systems is very significant. In this paper, a FRET-based two-photon fluorescent turn-on probe, A-HCy, was proposed for specific detection of SO2 derivatives through the bisulfite/sulfite-promoted Michael addition reaction. In this FRET system, an acedan (2-acetyl-6-dialkylaminonaphthalene) moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite. A-HCy exhibited excellent selectivity and rapid response to HSO3(-) with a detection limit of 0.24 μM. More importantly, probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO2 derivatives in living cells and tissues, suggesting its great potential for practical application in biological systems. PMID:27590555

  10. Chromosome translocations as biological dosimeter identified by in situ fluorescent hybridization (FISH)

    International Nuclear Information System (INIS)

    Chromosomal aberration analysis in lymphocytes is used for dose assessment by workers overexposed to ionizing radiation. The conventional method is based on the dicentric metaphase chromosomes and/or micronucleus scoring, which gives thoroughly satisfactory results for accidental and recent exposures. Nevertheless, when evaluating acute doses occurring in the past or accidents where the time elapsed between exposure and analysis is unknown, the translocations scoring could be the method to use. This is because translocations retain the cell's reproductive potential so that they persist over time. For a long time the detection of translocations for dosimetric purposes was impractical as a routine method until situ fluorescent hybridization (FISH) techniques appeared. This method arising from molecular biology and applied to biological dosimetry has greatly simplified detection and although it is expensive, requiring special infrastructure, its availability is a sign of the state of development for these techniques in the region. The use of translocations scoring for biological dosimetry, as for the dicentric scoring, requires calibration curves for the different types of ionizing radiation. Therefore, the aim of this work was to prepare a dose-effect curve for Co-60 γ rays by chromosome translocations analysis. The study material was peripheral lymphocytes from a clinically healthy donor, irradiated and cultured in vitro and the detection of translocations in chromosomes 1, 2 and 4. The results showed a dose effect curve linear quadratic, accordingly with other authors

  11. Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence

    NARCIS (Netherlands)

    Laptenok, S.; Borst, J.W.; Mullen, K.M.; Stokkum, van I.H.M.; Visser, A.J.W.G.; Amerongen, van H.

    2010-01-01

    A methodology is described for the quantitative determination of Förster resonance energy transfer (FRET) in live cells using the rise time of acceptor fluorescence as determined with fluorescence lifetime imaging microscopy (FLIM). An advantage of this method is that only those molecules that are i

  12. Evaluating the use of fluorescent imaging for the quantification of dental fluorosis

    Directory of Open Access Journals (Sweden)

    McGrady Michael G

    2012-11-01

    Full Text Available Abstract Background The quantification of fluorosis using fluorescence imaging (QLF hardware and stain analysis software has been demonstrated in selected populations with good correlation between fluorescent image metrics and TF Index scores from photographs. The aim of this study was to evaluate the ability of QLF to quantify fluorosis in a population of subjects (aged 11–13 participating in an epidemiological caries and fluorosis survey in fluoridated and non-fluoridated communities in Northern England. Methods Fluorescent images of the maxillary incisors were captured together with standardized photographs were scored blind for fluorosis using the TF Index. Subjects were excluded from the analysis if there were restorations or caries on the maxillary central incisors. Results Data were available for 1774 subjects (n=905 Newcastle, n=869 Manchester. The data from the fluorescence method demonstrated a significant correlation with TF Index scores from photographs (Kendall’s tau = 0.332 p Conclusions Despite confounding factors the fluorescence imaging system may provide a useful objective, blinded system for the assessment of enamel fluorosis when used adjunctively with photographic scoring.

  13. Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

    Science.gov (United States)

    Inagaki, Yoshinori; Matsumoto, Yasuhiko; Ishii, Masaki; Uchino, Keiro; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2015-06-10

    In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

  14. In vivo macroscopic HPD fluorescence reflectance imaging on small animals bearing surface ARO/NPA tumor

    Science.gov (United States)

    Autiero, Maddalena; Celentano, Luigi; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Montesi, Maria C.; Riccio, Patrizia; Russo, Paolo; Roberti, Giuseppe

    2005-08-01

    Recently multimodal imaging systems have been devised because the combination of different imaging modalities results in the complementarity and integration of the techniques and in a consequent improvement of the diagnostic capabilities of the multimodal system with respect to each separate imaging modality. We developed a simple and reliable HematoPorphyrin (HP) mediated Fluorescence Reflectance Imaging (FRI) system that allows for in vivo real time imaging of surface tumors with a large field of view. The tumor cells are anaplastic human thyroid carcinoma-derived ARO cells, or human papillary thyroid carcinoma-derived NPA cells. Our measurements show that the optical contrast of the tumor region image is increased by a simple digital subtraction of the background fluorescence and that HP fluorescence emissivity of ARO tumors is about 2 times greater than that of NPA tumors, and about 4 times greater than that of healthy tissues. This is also confirmed by spectroscopic measurements on histological sections of tumor and healthy tissues. It was shown also the capability of this system to distinguish the tumor type on the basis of the different intensity of the fluorescence emission, probably related to the malignancy degree. The features of this system are complementary with those ones of a pixel radionuclide detection system, which allows for relatively time expensive, narrow field of view measurements, and applicability to tumors also deeply imbedded in tissues. The fluorescence detection could be used as a large scale and quick analysis tool and could be followed by narrow field, higher resolution radionuclide measurements on previously determined highly fluorescent regions.

  15. Iodinated oil-loaded, fluorescent mesoporous silica-coated iron oxide nanoparticles for magnetic resonance imaging/computed tomography/fluorescence trimodal imaging

    Directory of Open Access Journals (Sweden)

    Xue S

    2014-05-01

    Full Text Available Sihan Xue,1 Yao Wang,1 Mengxing Wang,2 Lu Zhang,1 Xiaoxia Du,2 Hongchen Gu,1 Chunfu Zhang1,31School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, 2Shanghai Key Laboratory of Magnetic Resonance, Department of Physics, East China Normal University, 3State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: In this study, a novel magnetic resonance imaging (MRI/computed tomography (CT/fluorescence trifunctional probe was prepared by loading iodinated oil into fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (i-fmSiO4@SPIONs. Fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs were prepared by growing fluorescent dye-doped silica onto superparamagnetic iron oxide nanoparticles (SPIONs directed by a cetyltrimethylammonium bromide template. As prepared, fmSiO4@SPIONs had a uniform size, a large surface area, and a large pore volume, which demonstrated high efficiency for iodinated oil loading. Iodinated oil loading did not change the sizes of fmSiO4@SPIONs, but they reduced the MRI T2 relaxivity (r2 markedly. I-fmSiO4@SPIONs were stable in their physical condition and did not demonstrate cytotoxic effects under the conditions investigated. In vitro studies indicated that the contrast enhancement of MRI and CT, and the fluorescence signal intensity of i-fmSiO4@SPION aqueous suspensions and macrophages, were intensified with increased i-fmSiO4@SPION concentrations in suspension and cell culture media. Moreover, for the in vivo study, the accumulation of i-fmSiO4@SPIONs in the liver could also be detected by MRI, CT, and fluorescence imaging. Our study demonstrated that i-fmSiO4@SPIONs had great potential for MRI/C/fluorescence trimodal imaging.Keywords: multifunctional probe, SPIONs, mesoporous silica

  16. Silver Nanoclusters with Specific Ion Recognition Modulated by Ligand Passivation toward Fluorimetric and Colorimetric Copper Analysis and Biological Imaging

    Science.gov (United States)

    Sun, Zongzhao; Li, Shuying; Jiang, Yao; Qiao, Yuchun; Zhang, Liyan; Xu, Lulu; Liu, Jinghui; Qi, Wei; Wang, Hua

    2016-02-01

    Silver nanoclusters were synthesized and passivated by glutathione (GSH) ligand, with high aqueous stability and powerful red fluorescence and UV-vis yellow colour. Importantly, the specific recognition of the AgNCs was modulated from Hg2+ ions to Cu2+ ions upon the GSH passivation, of which the unique GSH-Cu2+ chelating reaction could conduct the fluorescence quenching of AgNCs. Strong UV-vis absorbance of GSH-passivated AgNCs could also be realized depending on the Cu2+ levels. Moreover, the Cu2+-induced loss of fluorescence and UV-vis absorbance of GSH-passivated AgNCs could be well restored by using stronger Cu2+ chelating agent. A simultaneous and reversible fluorimetric and colorimetric sensing method was thereby developed for probing Cu2+ ions in blood with high sensitivity and selectivity. Subsequently, the fluorescence-trackable imaging for live tissues and cells was demonstrated towards the analysis Cu2+ ions using GSH-passivated AgNCs as the fluorescent probes. This study indicates that the use of functional ligands like GSH could not only modulate the specific ion recognition of AgNCs, but also endow them the high aqueous stability and powerful red fluorescence towards the wide applications for ion sensing and biological imaging in the complicated media like blood.

  17. Compact fluorescence and white-light imaging system for intraoperative visualization of nerves

    Science.gov (United States)

    Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; tan Hehir, Cristina

    2012-02-01

    Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

  18. A compact fluorescence and white light imaging system for intraoperative visualization of nerves

    Science.gov (United States)

    Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; Tan Hehir, Cristina

    2012-03-01

    Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

  19. 3D Imaging of Transition Metals in the Zebrafish Embryo by X-ray Fluorescence Microtomography

    Science.gov (United States)

    Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan; Yi, Hong; Will, Fabian; Richter, Heiko; Shin, Chong Hyun; Fahrni, Christoph J.

    2014-01-01

    Synchrotron X-ray fluorescence (SXRF) microtomography has emerged as a powerful technique for the 3D visualization of the elemental distribution in biological samples. The mechanical stability, both of the instrument and the specimen, is paramount when acquiring tomographic projection series. By combining the progressive lowering of temperature method (PLT) with femtosecond laser sectioning, we were able to embed, excise, and preserve a zebrafish embryo at 24 hours post fertilization in an X-ray compatible, transparent resin for tomographic elemental imaging. Based on a data set comprised of 60 projections, acquired with a step size of 2 μm during 100 hours of beam time, we reconstructed the 3D distribution of zinc, iron, and copper using the iterative maximum likelihood expectation maximization (MLEM) reconstruction algorithm. The volumetric elemental maps, which entail over 124 million individual voxels for each transition metal, revealed distinct elemental distributions that could be correlated with characteristic anatomical features at this stage of embryonic development. PMID:24992831

  20. Temperature-modulated fluorescence tomography in a turbid media

    OpenAIRE

    Lin, Yuting; Bolisay, Linden; Ghijsen, Michael; Kwong, Tiffany C.; Gulsen, Gultekin

    2012-01-01

    High scattering in biological tissues makes fluorescence tomography inverse problem very challenging in thick medium. We describe an approach termed “temperature-modulated fluorescence tomography” that can acquire fluorescence images at focused ultrasound resolution. By utilizing recently emerged temperature sensitive fluorescence contrast agents, this technique provides fluorescence images with high resolution prior to any reconstruction process. We demonstrate that this technique is well su...

  1. Method and apparatus to image biological interactions in plants

    Science.gov (United States)

    Weisenberger, Andrew; Bonito, Gregory M.; Reid, Chantal D.; Smith, Mark Frederick

    2015-12-22

    A method to dynamically image the actual translocation of molecular compounds of interest in a plant root, root system, and rhizosphere without disturbing the root or the soil. The technique makes use of radioactive isotopes as tracers to label molecules of interest and to image their distribution in the plant and/or soil. The method allows for the study and imaging of various biological and biochemical interactions in the rhizosphere of a plant, including, but not limited to, mycorrhizal associations in such regions.

  2. Development of a noncontact 3-D fluorescence tomography system for small animal in vivo imaging

    Science.gov (United States)

    Zhang, Xiaofeng; Badea, Cristian; Jacob, Mathews; Johnson, G. Allan

    2009-02-01

    Fluorescence imaging is an important tool for tracking molecular-targeting probes in preclinical studies. It offers high sensitivity, but nonetheless low spatial resolution compared to other leading imaging methods such CT and MRI. We demonstrate our methodological development in small animal in vivo whole-body imaging using fluorescence tomography. We have implemented a noncontact fluid-free fluorescence diffuse optical tomography system that uses a raster-scanned continuous-wave diode laser as the light source and an intensified CCD camera as the photodetector. The specimen is positioned on a motorized rotation stage. Laser scanning, data acquisition, and stage rotation are controlled via LabVIEW applications. The forward problem in the heterogeneous medium is based on a normalized Born method, and the sensitivity function is determined using a Monte Carlo method. The inverse problem (image reconstruction) is performed using a regularized iterative algorithm, in which the cost function is defined as a weighted sum of the L-2 norms of the solution image, the residual error, and the image gradient. The relative weights are adjusted by two independent regularization parameters. Our initial tests of this imaging system were performed with an imaging phantom that consists of a translucent plastic cylinder filled with tissue-simulating liquid and two thin-wall glass tubes containing indocyanine green. The reconstruction is compared to the output of a finite element method-based software package NIRFAST and has produced promising results.

  3. An exciton-polariton laser based on biologically produced fluorescent protein

    Science.gov (United States)

    Dietrich, Christof P.; Steude, Anja; Tropf, Laura; Schubert, Marcel; Kronenberg, Nils M.; Ostermann, Kai; Höfling, Sven; Gather, Malte C.

    2016-01-01

    Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. PMID:27551686

  4. An exciton-polariton laser based on biologically produced fluorescent protein.

    Science.gov (United States)

    Dietrich, Christof P; Steude, Anja; Tropf, Laura; Schubert, Marcel; Kronenberg, Nils M; Ostermann, Kai; Höfling, Sven; Gather, Malte C

    2016-08-01

    Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. PMID:27551686

  5. CMOS image sensor with lateral electric field modulation pixels for fluorescence lifetime imaging with sub-nanosecond time response

    Science.gov (United States)

    Li, Zhuo; Seo, Min-Woong; Kagawa, Keiichiro; Yasutomi, Keita; Kawahito, Shoji

    2016-04-01

    This paper presents the design and implementation of a time-resolved CMOS image sensor with a high-speed lateral electric field modulation (LEFM) gating structure for time domain fluorescence lifetime measurement. Time-windowed signal charge can be transferred from a pinned photodiode (PPD) to a pinned storage diode (PSD) by turning on a pair of transfer gates, which are situated beside the channel. Unwanted signal charge can be drained from the PPD to the drain by turning on another pair of gates. The pixel array contains 512 (V) × 310 (H) pixels with 5.6 × 5.6 µm2 pixel size. The imager chip was fabricated using 0.11 µm CMOS image sensor process technology. The prototype sensor has a time response of 150 ps at 374 nm. The fill factor of the pixels is 5.6%. The usefulness of the prototype sensor is demonstrated for fluorescence lifetime imaging through simulation and measurement results.

  6. Potential of biological images for radiation therapy of cancer

    International Nuclear Information System (INIS)

    Full text: Recent technical advances in 3D conformal and intensity modulated radiotherapy (3DCRT and IMRT) based, on patient-specific CT and MRI images, have the potential of delivering exquisitely conformal dose distributions to the target volume while avoiding critical structures. Emerging clinical results in terms of reducing treatment-related morbidity and increasing local control appear promising. Recent developments in imaging have suggested that biological images may further positively impact cancer diagnosis, characterization and therapy. While in the past radiological images are largely anatomical, the new types of images can provide metabolic, biochemical, physiological, functional and molecular (genotypic and phenotypic) information. For radiation therapy, images that give information about factors (e.g. tumor hypoxia, Tpot) that influence radiosensitivity and treatment outcome can be regarded as radiobiological images. The ability of IMRT to 'paint' (in 2D) or 'sculpt' (in 3D) the dose, and produce exquisitely conformal dose distributions begs the '64 million dollar question' as to how to paint or sculpt, and whether biological imaging may provide the pertinent information. Can this new approach provide 'radiobiological phenotypes' non-invasively, and incrementally improve upon the predictive assays of radiobiological characteristics such as proliferative activity (Tpot - the potential doubling time), radiosensitivity (SF2 - the surviving fraction at a dose of 2 Gy), energy status (relative to sublethal damage repair), pH (a possible surrogate of hypoxia), tumor hypoxia, etc. as prognosticator(s) of radiation treatment outcome. Important for IMRT, the spatial (geometrical) distribution of the radiobiological phenotypes provide the basis for dose distribution design to conform to both the physical (geometrical) and the biological attributes. Copyright (2001) Australasian College of Physical Scientists and Engineers in Medicine

  7. Laser induced fluorescence as a diagnostic tool integrated into a scanning fiber endoscope for mouse imaging

    Science.gov (United States)

    Brown, Christopher M.; Maggio-Price, Lillian; Seibel, Eric J.

    2007-02-01

    Scanning fiber endoscope (SFE) technology has shown promise as a minimally invasive optical imaging tool. To date, it is capable of capturing full-color 500-line images, at 15 Hz frame rate in vivo, as a 1.6 mm diameter endoscope. The SFE uses a singlemode optical fiber actuated at mechanical resonance to scan a light spot over tissue while backscattered or fluorescent light at each pixel is detected in time series using several multimode optical fibers. We are extending the capability of the SFE from a RGB reflectance imaging device to a diagnostic tool by imaging laser induced fluorescence (LIF) in tissue, allowing for correlation of endogenous fluorescence to tissue state. Design of the SFE for diagnostic imaging is guided by a comparison of single point spectra acquired from an inflammatory bowel disease (IBD) model to tissue histology evaluated by a pathologist. LIF spectra were acquired by illuminating tissue with a 405 nm light source and detecting intrinsic fluorescence with a multimode optical fiber. The IBD model used in this study was mdr1a-/- mice, where IBD was modulated by infection with Helicobacter bilis. IBD lesions in the mouse model ranged from mild to marked hyperplasia and dysplasia, from the distal colon to the cecum. A principle components analysis (PCA) was conducted on single point spectra of control and IBD tissue. PCA allowed for differentiation between healthy and dysplastic tissue, indicating that emission wavelengths from 620 - 650 nm were best able to differentiate diseased tissue and inflammation from normal healthy tissue.

  8. Colocalization of fluorescence and Raman microscopic images for the identification of subcellular compartments: a validation study.

    Science.gov (United States)

    Krauß, Sascha D; Petersen, Dennis; Niedieker, Daniel; Fricke, Inka; Freier, Erik; El-Mashtoly, Samir F; Gerwert, Klaus; Mosig, Axel

    2015-04-01

    A major promise of Raman microscopy is the label-free detailed recognition of cellular and subcellular structures. To this end, identifying colocalization patterns between Raman spectral images and fluorescence microscopic images is a key step to annotate subcellular components in Raman spectroscopic images. While existing approaches to resolve subcellular structures are based on fluorescence labeling, we propose a combination of a colocalization scheme with subsequent training of a supervised classifier that allows label-free resolution of cellular compartments. Our colocalization scheme unveils statistically significant overlapping regions by identifying correlation between the fluorescence color channels and clusters from unsupervised machine learning methods like hierarchical cluster analysis. The colocalization scheme is used as a pre-selection to gather appropriate spectra as training data. These spectra are used in the second part as training data to establish a supervised random forest classifier to automatically identify lipid droplets and nucleus. We validate our approach by examining Raman spectral images overlaid with fluorescence labelings of different cellular compartments, indicating that specific components may indeed be identified label-free in the spectral image. A Matlab implementation of our colocalization software is available at . PMID:25679809

  9. Near-field measurements of vegetation by laser-induced fluorescence imaging

    Science.gov (United States)

    Sowinska, Malgorzata; Cunin, Bernard; Deruyver, Aline; Heisel, Francine; Miehe, Joseph-Albert; Langsdorf, Gabriele; Lichtenthaler, Hartmut K.

    1999-12-01

    In this paper, a validation of a new UV-A laser-induced fluorescence imaging system implemented in an all-road car for near-field remote sensing of vegetation will be presented. It has been developed as a part of a European Community Program INTERREG II and is consisting of three main parts: excitation, detection and control units. The excitation source is a frequency tripled Nd:YAG laser and the laser spot size is adjusted via a variable beam expander. Fluorescence images are recorded at four characteristic fluorescence bands: 440, 520, 690 and 740 nm with a gated intensified digital CCD camera. The laser head and camera are situated on a directed in site and azimuth platform which can be high up to 6 meters. The platform positioning, localization and distance detection, spot size determination and adjustment, focus, sharpness, selection of the filter, laser and camera synchronization, gain of the intensifier, real time visualization of images, acquisition time are controlled by a newly developed software which allows also image storage, analysis and treatment. Examples of remote sensing fluorescence images from several plant species recorded at a distance of 10 - 30 m will be given and discussed further in this paper.

  10. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    Science.gov (United States)

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  11. Endoscopic fluorescence imaging for early assessment of anastomotic recurrence of Crohn's disease

    Science.gov (United States)

    Mordon, Serge R.; Maunoury, Vincent; Geboes, K.; Klein, Olivier; Desreumaux, P.; Debaert, A.; Colombel, Jean-Frederic

    1999-02-01

    Crohn's disease is an inflammatory bowel disease of unknown etiology. The mechanism of the initial mucosal alterations is still unclear: ulcerations overlying lymphoid follicles and/or vasculitis have been proposed as the early lesions. We have developed a new and original method combining endoscopy of fluorescence angiography for identifying the early pathological lesions, occurring in the neo-terminal ileum after right ileocolonic resection. The patient population consisted of 10 subjects enrolled in a prospective protocol of endoscopic follow-up at 3 and 12 months after surgery. Fluorescence imaging showed small spots giving a bright fluorescence distributed singly in mucosa which appeared normal in routine endoscopy. Histopathological examination demonstrated that the fluorescence of small spots originated from small, usually superficial, erosive lesions. In several cases, these erosive lesions occurred over lymphoid follicles. Endoscopic fluorescence imaging provides a suitable means of investigating the initial aspect of the Crohn's disease process in displaying some correlative findings between fluorescent aspects and early pathological mucosal alterations.

  12. 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.

  13. Fluorescent non-conjugated polymer dots for targeted cell imaging

    Science.gov (United States)

    Sun, Bin; Zhao, Bin; Wang, Dandan; Wang, Yibo; Tang, Qi; Zhu, Shoujun; Yang, Bai; Sun, Hongchen

    2016-05-01

    Through the chemical crosslinking of the sub-fluorophore, linear non-conjugated polymers can possess strong photoluminescence (PL), which is a very important fluorescence behavior and the non-conjugated polymer dots (PDs) are efficient bio-fluorophores for bio-based applications. Herein, the new type of non-conjugated polyethyleneimine (PEI) PDs was further modified by targeting molecules (folic acid) for a new generation of bio-fluorophores. The free folic acid can quench the PL of PDs by energy transfer, while the conjugated folic acid@PDs (FA@PDs) can still maintain their PL properties to a certain degree. The FA@PDs also possess lower toxicity compared with free PDs, which is possibly due to blocking of the amino groups. Moreover, we investigated the targeted bioimaging applications of the FA@PDs, which gave a very important direction for application of these types of materials.Through the chemical crosslinking of the sub-fluorophore, linear non-conjugated polymers can possess strong photoluminescence (PL), which is a very important fluorescence behavior and the non-conjugated polymer dots (PDs) are efficient bio-fluorophores for bio-based applications. Herein, the new type of non-conjugated polyethyleneimine (PEI) PDs was further modified by targeting molecules (folic acid) for a new generation of bio-fluorophores. The free folic acid can quench the PL of PDs by energy transfer, while the conjugated folic acid@PDs (FA@PDs) can still maintain their PL properties to a certain degree. The FA@PDs also possess lower toxicity compared with free PDs, which is possibly due to blocking of the amino groups. Moreover, we investigated the targeted bioimaging applications of the FA@PDs, which gave a very important direction for application of these types of materials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01909a

  14. A biological phantom for evaluation of CT image reconstruction algorithms

    Science.gov (United States)

    Cammin, J.; Fung, G. S. K.; Fishman, E. K.; Siewerdsen, J. H.; Stayman, J. W.; Taguchi, K.

    2014-03-01

    In recent years, iterative algorithms have become popular in diagnostic CT imaging to reduce noise or radiation dose to the patient. The non-linear nature of these algorithms leads to non-linearities in the imaging chain. However, the methods to assess the performance of CT imaging systems were developed assuming the linear process of filtered backprojection (FBP). Those methods may not be suitable any longer when applied to non-linear systems. In order to evaluate the imaging performance, a phantom is typically scanned and the image quality is measured using various indices. For reasons of practicality, cost, and durability, those phantoms often consist of simple water containers with uniform cylinder inserts. However, these phantoms do not represent the rich structure and patterns of real tissue accurately. As a result, the measured image quality or detectability performance for lesions may not reflect the performance on clinical images. The discrepancy between estimated and real performance may be even larger for iterative methods which sometimes produce "plastic-like", patchy images with homogeneous patterns. Consequently, more realistic phantoms should be used to assess the performance of iterative algorithms. We designed and constructed a biological phantom consisting of porcine organs and tissue that models a human abdomen, including liver lesions. We scanned the phantom on a clinical CT scanner and compared basic image quality indices between filtered backprojection and an iterative reconstruction algorithm.

  15. Infrared spectroscopic imaging microscopy: Applications to biological systems

    International Nuclear Information System (INIS)

    The coupling of imaging modalities with spectroscopic techniques adds additional dimensions to sample analysis in both the spectroscopic and spatial domains. The particular ability of infrared (IR) imaging to explore the spatial distribution of chemically distinct species on length scales ranging from microns to kilometers demonstrates the versatility and diversity of spectroscopic imaging. In this paper, we focus on the further development of our Fourier-transform (FT) based mid-IR spectroscopic imaging technique which combines the analytical capabilities of mid-IR spectroscopy with the morphological information obtained from optical imaging. The seamless combination of spectroscopy for molecular analysis with the power of visualization represents the future of infrared microscopy. Our spectroscopic imaging instrument integrates several infrared focal-plane arrays with a Michelson step-scan interferometer, generating high-fidelity and high spectral resolution mid-infrared spectroscopic images. The instrumentation produces multidimensional, chemically specific images, while simultaneously obtaining high resolution spectra for each detector pixel. The spatial resolution of the images approaches the diffraction limit for mid-infrared wavelengths, while the spectral resolution is determined by the interferometer, and can be 4 cm-1 or higher. Data derived from a variety of materials, particularly biological samples, illustrate the capabilities of the technique for readily visualizing chemical complexity and for providing statistical data on sample heterogeneity

  16. Photoacoustic tomography of human hepatic malignancies using intraoperative indocyanine green fluorescence imaging.

    Science.gov (United States)

    Miyata, Akinori; Ishizawa, Takeaki; Kamiya, Mako; Shimizu, Atsushi; Kaneko, Junichi; Ijichi, Hideaki; Shibahara, Junji; Fukayama, Masashi; Midorikawa, Yutaka; Urano, Yasuteru; Kokudo, Norihiro

    2014-01-01

    Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous) was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10) under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases), photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical advances are needed

  17. Photoacoustic tomography of human hepatic malignancies using intraoperative indocyanine green fluorescence imaging.

    Directory of Open Access Journals (Sweden)

    Akinori Miyata

    Full Text Available Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10 under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases, photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical

  18. 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.

  19. Evaluation of Copper Oxide Nanoparticles Toxicity Using Chlorophyll a Fluorescence Imaging in Lemna gibba

    Directory of Open Access Journals (Sweden)

    François Perreault

    2010-01-01

    Full Text Available Copper oxide nanoparticles (CuO NPs, used in antifouling paints of boats, are released in the environment and can induce toxicity to aquatic organisms. In this report, we used chlorophyll a fluorescence imaging to evaluate CuO NPs toxicity in Lemna gibba. This approach allowed to evaluate the differential effect of CuO NPs on photosynthesis of whole L. gibba plants. Exposure to 0.1 to 0.4 g/L CuO NPs during 48h induced strong inhibition of photosynthetic processes resulting in a decrease of plant growth. By using fluorescence imaging, different photosynthetic parameters were evaluated simultaneously in microplate conditions. Imaging of FO fluorescence yield showed the decrease of leaf photosynthetic active surface for whole plants exposed to CuO NPs. This method showed that CuO NPs inhibited photosystem II maximal, photosystem II operational quantum yields, and photochemical quenching of fluorescence associated with electron transport. Nonphotochemical fluorescence quenching as an indicator of energy dissipation not used in photosynthesis was shown to be increased by the effect of CuO NPs. Such approach in microplate conditions provides synchronous high repetition measurements for numerous plants. This study may give a reliable methodological approach to evaluate toxicity risk of NPs in aquatic ecosystems.

  20. Biodistribution and imaging of fluorescently-tagged iron oxide nanoparticles in a breast cancer mouse model

    Science.gov (United States)

    Tate, Jennifer A.; Savellano, Mark D.; Hoopes, P. Jack

    2013-02-01

    Iron oxide nanoparticle (IONP) hyperthermia is an emerging treatment that shows great potential as a cancer therapy both alone and in synergy with conventional modalities. Pre-clinical studies are attempting to elucidate the mechanisms of action and distributions of IONP in various in vitro and in vivo models, however these studies would greatly benefit from real-time imaging of IONP locations both in cellular and in mammalian systems. To this end, fluorescently-tagged IONP (fIONP) have been employed for real time tracking and co-registration of IONP with iron content. Starch-coated IONP were fluorescently-tagged, purified and analyzed for fluorescent signal at various concentrations. fIONP were incubated with MTGB cells for varying times and cellular uptake analyzed using confocal microscopy, flow cytometry and inductively-coupled plasma mass spectrometry (ICP-MS). fIONP were also injected into a bilateral mouse tumor model for radiation modification of tumor tissue and enhanced fIONP deposition assessed using a Xenogen IVIS fluorescent imager. Results demonstrated that fIONP concentrations in vitro correlated with ICPMS iron readings. fIONP could be tracked in vitro as well as in tissue samples from an in vivo model. Future work will employ whole animal fluorescent imaging to track the biodistribution of fIONP over time.

  1. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

    Science.gov (United States)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

    2016-03-01

    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  2. 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.

  3. Intracellular Dual Fluorescent Lightup Bioprobes for Image-Guided Photodynamic Cancer Therapy.

    Science.gov (United States)

    Han, Haijie; Jin, Qiao; Wang, Haibo; Teng, Wenzhuo; Wu, Jina; Tong, Hongxin; Chen, Tingting; Ji, Jian

    2016-07-01

    An intracellular dual fluorescent light-up bioprobe with aggregation-induced emission features and endogenously producing photosensitizer protoporphyrin IX (PpIX) abilities is designed and synthesized. The bioprobe is nonemissive in physiological environment. However, the bioprobe can selectively light up cancer cells with blue fluorescence of tetraphenylene (TPE) and red fluorescence of PpIX, owing to the release of TPE and methyl aminolevulinate after targeted internalization by cancer cells. Moreover, upon endogenous generation and accumulation of PpIX in cancer cells, efficient photodynamic ablation of cancer cells after light irradiation is demonstrated with easy regulation for optimal therapeutic efficacy. The design of such dual fluorescent light-up bioprobes might provide a new opportunity for targeted and image-guided photodynamic cancer therapy. PMID:27322139

  4. Tracking graphene by fluorescence imaging: a tool for detecting multiple populations of graphene in solution

    Science.gov (United States)

    Guidetti, G.; Cantelli, A.; Mazzaro, R.; Ortolani, L.; Morandi, V.; Montalti, M.

    2016-04-01

    Most methods used for the characterization of graphene produced by liquid phase exfoliation require the deposition of the liquid sample on a substrate and subsequent drying. Because of this or other post-synthetic treatments, the reliability of the data in describing the actual features of the graphene particles in the pristine solution becomes questionable. Hence there is a need for new methods that permit the study of graphene directly in solution. Fluorescence imaging is at present the most convenient and sensitive method to visualize nanosized objects in solution. Here we report the development of a new method for visualizing and tracking exfoliated graphene directly in solution using a conventional set-up for fluorescence microscopy. We functionalized a fluorescent surfactant typically used for exfoliating graphite in aqueous phase (Pluronic P123) with two different fluorophores, in order to make graphene detectable by fluorescence microscopy. The photophysical interactions between the fluorescent surfactant and graphene were investigated at the bulk level. Finally, fluorescence microscopy allowed us to track the carbon particles produced and to identify two different populations of particles with sizes of 265 +/- 25 and 1100 +/- 200 nm respectively. The correlation of these results with TEM and DLS data is discussed.Most methods used for the characterization of graphene produced by liquid phase exfoliation require the deposition of the liquid sample on a substrate and subsequent drying. Because of this or other post-synthetic treatments, the reliability of the data in describing the actual features of the graphene particles in the pristine solution becomes questionable. Hence there is a need for new methods that permit the study of graphene directly in solution. Fluorescence imaging is at present the most convenient and sensitive method to visualize nanosized objects in solution. Here we report the development of a new method for visualizing and tracking

  5. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil

    OpenAIRE

    Boschi, F.; Fontanella, M.; Calderan, L; Sbarbati, A

    2011-01-01

    Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical propert...

  6. Concentrations, size distributions and temporal variations of fluorescent biological aerosol particles in southern tropical India

    Science.gov (United States)

    Valsan, Aswathy; Krishna R, Ravi; CV, Biju; Huffman, Alex; Poschl, Ulrich; Gunthe, Sachin

    2015-04-01

    Biological aerosols constitute a wide range of dead and alive biological materials and structures that are suspended in the atmosphere. They play an important role in the atmospheric physical, chemical and biological processes and health of living being by spread of diseases among humans, plants, and, animals. The atmospheric abundance, sources, physical properties of PBAPs as compared to non-biological aerosols, however, is poorly characterized. The Indian tropical region, where large fraction of the world's total population is residing, experiences a distinctive meteorological phenomenon by means of Indian Summer Monsoon (IMS). Thus, the properties and characteristics of biological aerosols are also expected to be very diverse over the Indian subcontinent depending upon the seasons. Here we characterize the number concentration and size distribution of Fluorescent Biological Aerosol Particles (FBAP) at a high altitude continental site, Munnar (10.09 N, 77.06 E; 1605 m asl) in South India during the South-West monsoon, which constitute around 80 percent of the annual rainfall in Munnar. Continuous three months measurements (from 01 June 2014 to 21 Aug 2104) FBAPs were carried out at Munnar using Ultra Violet Aerodynamic Particle Sizer (UVAPS) during IMS. The mean number and mass concentration of coarse FBAP averaged over the entire campaign was 1.7 x 10-2 cm-3 and 0.24 µg m-3 respectively, which corresponds to 2 percent and 6 percent of total aerosol particle number and mass concentration. In agreement to other previous measurements the number size distribution of FBAP also peaks at 3.2 micron indicating the strong presence of fungal spores. This was also supported by the Scanning Electron Microscopic analysis of bioaerosols on filter paper. They also displayed a strong diurnal cycle with maximum concentration occurring at early morning hours. During periods of heavy and continuous rain where the wind is consistently blowing from South-West direction it was

  7. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    KechengLi; DouglasW.Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. With the thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  8. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    Kecheng Li; Douglas W. Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. Withthe thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration, and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  9. Rapid assessment of different oxygenic phototrophs and single-cell photosynthesis with multicolour variable chlorophyll fluorescence imaging

    DEFF Research Database (Denmark)

    Trampe, Erik Christian Løvbjerg; Kolbowski, J.; Schreiber, U.;

    2011-01-01

    We present a new system for microscopic multicolour variable chlorophyll fluorescence imaging of aquatic phototrophs. The system is compact and portable and enables microscopic imaging of photosynthetic performance of individual cells and chloroplasts using different combinations of blue, green...

  10. Multi-Dimensional Time-Correlated Single Photon Counting (TCSPC) Fluorescence Lifetime Imaging Microscopy (FLIM) to Detect FRET in Cells

    OpenAIRE

    Duncan, R. R.; Bergmann, A; Cousin, M. A.; Apps, D. K.; Shipston, M. J.

    2004-01-01

    We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This tec...

  11. Development of a Regional Coral Observation Method by a Fluorescence Imaging LIDAR Installed in a Towable Buoy

    OpenAIRE

    Masahiko Sasano; Motonobu Imasato; Hiroya Yamano; Hiroyuki Oguma

    2016-01-01

    Coral bleaching and mortality is predicted to increase under global climate change. A new observation technique is required to monitor regional coral conditions. To this end, we developed a light detection and ranging (LIDAR) system installed in a towable buoy for boat observations, which acquires continuous fluorescent images of the seabed during day-time. Most corals have innate fluorescent proteins in their tissue, and they emit fluorescence by ultraviolet excitation. This fluorescence dis...

  12. Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice.

    Science.gov (United States)

    Wahl, Daniel J; Jian, Yifan; Bonora, Stefano; Zawadzki, Robert J; Sarunic, Marinko V

    2016-01-01

    Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina.

  13. Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice.

    Science.gov (United States)

    Wahl, Daniel J; Jian, Yifan; Bonora, Stefano; Zawadzki, Robert J; Sarunic, Marinko V

    2016-01-01

    Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina. PMID:26819812

  14. Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime.

    Science.gov (United States)

    Battisti, Antonella; Panettieri, Silvio; Abbandonato, Gerardo; Jacchetti, Emanuela; Cardarelli, Francesco; Signore, Giovanni; Beltram, Fabio; Bizzarri, Ranieri

    2013-07-01

    The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress. PMID:23780224

  15. Highly photostable zinc selective molecular marker bearing flexible pivotal unit: opto-fluorescence enhancement effect and imaging applications in living systems.

    Science.gov (United States)

    Sinha, Sougata; Gaur, Pankaj; Dev, Sagarika; Mukherjee, Trinetra; Mathew, Jomon; Mukhopadhyay, Subhrakanti; Ghosh, Subrata

    2015-05-28

    Novel molecular probes for imaging zinc in biological systems are gaining interest as they help in understanding the role of zinc in regulating various bio-events. In this regard, a new C2-symmetric molecular system has been developed and successfully applied as light-up material for signaling divalent zinc with green emission. The fluorescence enhancement was highly zinc specific and this newly developed probe bears a submicromolar detection capability. While probe and the ensemble -Zn(2+) exhibited remarkably high photostability, light-triggered fluorescence enhancement was observed in the case of -Zn(2+). The nature of the -Zn(2+) complex and the associated spectral shift are further supported by theoretical calculations. As the present probe absorbs in the visible region and emits in the green, it was preferred as a potential material for imaging zinc in biological systems including animal and plant cells such as pollen grains and fish egg cells. Such fluorescence imaging of zinc revealed the efficacy of the probe in detection and localization of zinc in various biological systems.

  16. Detecting thermal phase transitions in corneal stroma by fluorescence micro-imaging analysis

    Science.gov (United States)

    Matteini, P.; Rossi, F.; Ratto, F.; Bruno, I.; Nesi, P.; Pini, R.

    2008-02-01

    Thermal modifications induced in corneal stroma were investigated by the use of fluorescence microscopy. Freshly extracted porcine corneas were immersed for 5 minutes in a water bath at temperatures in the 35-90°C range and stored in formalin. The samples were then sliced in 200-μm-thick transversal sections and analyzed under a stereomicroscope to assess corneal shrinkage. Fluorescence images of the thermally treated corneal samples were acquired using a slow-scan cooled CCD camera, after staining the slices with Indocyanine Green (ICG) fluorescent dye which allowed to detect fluorescence signal from the whole tissue. All measurements were performed using an inverted epifluorescence microscope equipped with a mercury lamp. The thermally-induced modifications to the corneal specimens were evaluated by studying the grey level distribution in the fluorescence images. For each acquired image, Discrete Fourier Transform (DFT) and entropy analyses were performed. The spatial distribution of DFT absolute value indicated the spatial orientation of the lamellar planes, while entropy was used to study the image texture, correlated to the stromal structural transitions. As a result, it was possible to indicate a temperature threshold value (62°C) for high thermal damage, resulting in a disorganization of the lamellar planes and in full agreement with the measured temperature for corneal shrinkage onset. Analysis of the image entropy evidenced five strong modifications in stromal architecture at temperatures of ~45°C, 53°C, 57°C, 66°C, 75°C. The proposed procedure proved to be an effective micro-imaging method capable of detecting subtle changes in corneal tissue subjected to thermal treatment.

  17. 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.

  18. Fluorescent Properties of Au Nanoclusters and Their Biological Applications%金团簇的荧光性质及其生物应用

    Institute of Scientific and Technical Information of China (English)

    任娟; 代雯乐; 付欣; 苏梦; 蔡晶晶; 马占芳

    2011-01-01

    金纳米团簇作为一类新型纳米材料具有独特的光学特性。当金纳米团簇颗粒的尺寸小到与电子的费米波长(〈1nm)相当时,由于量子尺寸效应,金颗粒会受激发射出荧光。作为一种新型荧光材料,金纳米团簇具有发光颜色随团簇尺寸可调、荧光不易猝灭等许多优势。本文主要综述了金纳米团簇的荧光性质及其在生物标记、生物成像以及生物检测等方面的应用。%Au nanoclusters have emerged as a class of materials with unique optical properties.When the size of Au nanocluster decrease to comparable to the Fermi wavelength(1nm) of an electron,it exhibits fluorescent characteristics while they are excited,which resulted from its quantum effect.As a novel fluorescent material,Au nanoclusters show size-dependent emission,a slower photobleaching rate and very good stability.The fluorescent properties of Au nanoclusters and their applications in the fields of biological labeling,biological imaging,and biodetection were mainly reviewed.

  19. Projection imaging of photon beams using Čerenkov-excited fluorescence

    OpenAIRE

    Glaser, Adam K.; Davis, Scott C.; Voigt, William H.A.; Zhang, Rongxiao; Pogue, Brian W.; Gladstone, David J.

    2013-01-01

    Full 3D beam profiling and quality assurance (QA) of therapeutic megavoltage linear accelerator (LINAC) x-ray photon beams is not routinely performed due to the slow point-by-point measurement nature of conventional scanning ionization chamber systems. In this study we explore a novel optical-based dose imaging approach using a standard commercial camera, water tank, and fluorescent dye, which when excited by the Čerenkov emission induced by the radiation beam, allows 2D projection imaging in...

  20. Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging

    OpenAIRE

    Xi, Jiefeng; Chen, Yongping; Zhang, Yuying; Murari, Kartikeya; Li, Ming-Jun; Li, Xingde

    2012-01-01

    We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence to-mography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound...

  1. 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.

  2. 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.

  3. pH within pores in plant fiber cell walls assessed by Fluorescence Ratio Imaging

    DEFF Research Database (Denmark)

    Hidayat, Budi Juliman; Thygesen, Lisbeth Garbrecht; Johansen, Katja S.

    2013-01-01

    The pH within cell wall pores of filter paper fibers and hemp fibers was assessed by Fluorescence Ratio Imaging (FRIM). It was found that the Donnan effect affected the pH measured within the fibers. When the conductivity of the added liquid was low (0. 7 mS), pH values were lower within the cell...

  4. Fixed fluorescent images of an 80 MeV proton pencil beam

    NARCIS (Netherlands)

    Warman, J. M.; de Haas, M. P.; Luthjens, L. H.; Denkova, A. G.; Kavatsyuk, O.; van Goethem, M. -J.; Kiewiet, H. H.; Brandenburg, S.

    2013-01-01

    We have used an organic radio-fluorogenic gel to make fixed fluorescent images of the track of an 80 MeV proton pencil beam NB this is not a scintillation effect; rather a small fraction of the molecules of the medium are converted permanently from a non-emissive to an emissive form. The spatial res

  5. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N.; Wientjes, Emilie; Amerongen, van Herbert

    2016-01-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was ac

  6. Portable hyperspectral fluorescence imaging system for detection of biofilms on stainless steel surfaces

    Science.gov (United States)

    A rapid nondestructive technology is needed to detect bacterial contamination on the surfaces of food processing equipment to reduce public health risks. A portable hyperspectral fluorescence imaging system was used to evaluate potential detection of microbial biofilm on stainless steel typically u...

  7. Detection of fecal contamination on beef meat surfaces using handheld fluorescence imaging device (HFID)

    Science.gov (United States)

    Current meat inspection in slaughter plants, for food safety and quality attributes including potential fecal contamination, is conducted through by visual examination human inspectors. A handheld fluorescence-based imaging device (HFID) was developed to be an assistive tool for human inspectors by ...

  8. Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

    NARCIS (Netherlands)

    Putman, Constant A.J.; Hansma, Helen G.; Gaub, Hermann E.; Hansma, Paul K.

    1992-01-01

    The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microsc

  9. Morphological segmentation of multiprobe fluorescence images for immunophenotyping in melanoma tissue sections

    Science.gov (United States)

    Dow, Alasdair I.; Shafer, Steven A.; Waggoner, Alan S.

    1993-08-01

    A fundamental task in studying the action of cancer chemotherapy is to determine the quantity and spatial relationship of tumor-infiltrating lymphocyte populations. Classically this is performed by staining thin tissue sections with antibodies by immunoperoxidase amplification. The staining technique is practically limited to locating a single cell type per tissue section. Full immunophenotyping requires successive staining of serial sections, using statistical analysis to correlate the results. This paper describes a system that brings together multi- parameter fluorescence imaging and morphological segmentation techniques to provide a fast, accurate, and automatic analysis of the lymphocyte infiltrate in tissue sections. With fluorescence techniques a single section can be stained with up to four distinct fluorescently labelled antibodies to determine cell phenotypes. To harness this potential computer vision techniques are required to analyze the images. A routine based on the water shed algorithm has been developed that segments the nuclei image with an accuracy of greater than 90%. By matching the nuclei boundaries to the local peak fluorescence, cell boundary estimates are obtained in the antigen images. By then extracting two measurements from the boundary signal the cells can be classified according to their antigen expression. Determining cell expression of multiple antigens simultaneously provides a more detailed and accurate picture of the tumor infiltrate than single parameter analysis, and increases understanding of the immune response associated with the chemotherapy.

  10. Detection of organic residues on poultry processing equipment surfaces by LED-induced fluorescence imaging

    Science.gov (United States)

    Organic residues on equipment surfaces in poultry processing plants can generate cross- contamination and increase the risk of unsafe food for consumers. This research was aimed to investigate the potential of LED-induced fluorescence imaging technique for rapid inspection of stainless steel proces...

  11. Planar laser-induced fluorescence fuel imaging during gas-turbine relight

    DEFF Research Database (Denmark)

    Read, Robert; Rogerson, J.W.; Hochgreb, S.

    2013-01-01

    This experimental study investigates the influence of fuel distribution on ignition outcome during high-altitude relight of a gas turbine. Planar laser-induced fluorescence is used to image fuel inside a lean direct-injection combustor under realistic conditions. A novel apparatus is developed...

  12. Monitoring cashew seedlings during interactions with the fungus Lasiodiplodia theobromae using chlorophyll fluorescence imaging

    NARCIS (Netherlands)

    Muniz, C.R.; Freire, F.C.O.; Viana, F.M.P.; Cardoso, J.E.; Sousa, C.A.F.; Guedes, M.I.F.; Schoor, van der R.; Jalink, H.

    2014-01-01

    The chlorophyll (Chl) fluorescence imaging technique was applied to cashew seedlings inoculated with the fungus Lasiodiplodia theobromae to assess any disturbances in the photosynthetic apparatus of the plants before the onset of visual symptoms. Two-month-old cashew plants were inoculated with myce

  13. Innovations of wide-field optical-sectioning fluorescence microscopy: toward high-speed volumetric bio-imaging with simplicity

    Science.gov (United States)

    Yu, Jiun-Yann

    Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere. The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths. Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often

  14. A new self-made digital slide scanner and microscope for im