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Sample records for coincidence fluorescence imaging

  1. Entangled-photon coincidence fluorescence imaging.

    Science.gov (United States)

    Scarcelli, Giuliano; Yun, Seok H

    2008-09-29

    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.

  2. Coincidence velocity map imaging using a single detector

    Science.gov (United States)

    Zhao, Arthur; Sándor, Péter; Weinacht, Thomas

    2017-07-01

    We demonstrate a single-detector velocity map imaging setup which is capable of rapidly switching between coincidence and non-coincidence measurements. By rapidly switching the extraction voltages on the electrostatic lenses, both electrons and ions can be collected in coincidence with a single detector. Using a fast camera as the 2D detector avoids the saturation problem associated with traditional delay line detectors and allows for easy transitions between coincidence and non-coincidence data collection modes. This is a major advantage in setting up a low-cost and versatile coincidence apparatus. We present both coincidence and non-coincidence measurements of strong field atomic and molecular ionization.

  3. 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.

  4. Fluorescent atom coincidence spectroscopy of extremely neutron-deficient barium isotopes

    Science.gov (United States)

    Wells, S. A.; Evans, D. E.; Griffith, J. A. R.; Eastham, D. A.; Groves, J.; Smith, J. R. H.; Tolfree, D. W. L.; Warner, D. D.; Billowes, J.; Grant, I. S.; Walker, P. M.

    1988-09-01

    Fluorescent atom coincidence spectroscopy (FACS) has been used to measure the nuclear mean square radii and moments of the extremely neutron-deficient isotopes 120-124Ba. At N=65 an abrupt change in nuclear mean square charge radii is observed which can be understood in terms of the occupation of the spin-orbit partner g7/25/2[413] neutron and g9/29/2[404] proton orbitals and the consequent enhancement of the n-p interaction.

  5. Fluorescent atom coincidence spectroscopy of extremely neutron-deficient barium isotopes

    Energy Technology Data Exchange (ETDEWEB)

    Wells, S.A.; Evans, D.E.; Griffith, J.A.R.; Eastham, D.A.; Groves, J.; Smith, J.R.H.; Tolfree, D.W.L.; Warner, D.D.; Billowes, J.; Grant, I.S.

    1988-09-01

    Fluorescent atom coincidence spectroscopy (FACS) has been used to measure the nuclear mean square radii and moments of the extremely neutron-deficient isotopes /sup 120-124/Ba. At N=65 an abrupt change in nuclear mean square charge radii is observed which can be understood in terms of the occupation of the spin-orbit partner g/sub 7/2/ 5/2(413) neutron and g/sub 9/2/ 9/2(404) proton orbitals and the consequent enhancement of the n-p interaction.

  6. Coincidence ion imaging with a fast frame camera.

    Science.gov (United States)

    Lee, Suk Kyoung; Cudry, Fadia; Lin, Yun Fei; Lingenfelter, Steven; Winney, Alexander H; Fan, Lin; Li, Wen

    2014-12-01

    A new time- and position-sensitive particle detection system based on a fast frame CMOS (complementary metal-oxide semiconductors) camera is developed for coincidence ion imaging. The system is composed of four major components: a conventional microchannel plate/phosphor screen ion imager, a fast frame CMOS camera, a single anode photomultiplier tube (PMT), and a high-speed digitizer. The system collects the positional information of ions from a fast frame camera through real-time centroiding while the arrival times are obtained from the timing signal of a PMT processed by a high-speed digitizer. Multi-hit capability is achieved by correlating the intensity of ion spots on each camera frame with the peak heights on the corresponding time-of-flight spectrum of a PMT. Efficient computer algorithms are developed to process camera frames and digitizer traces in real-time at 1 kHz laser repetition rate. We demonstrate the capability of this system by detecting a momentum-matched co-fragments pair (methyl and iodine cations) produced from strong field dissociative double ionization of methyl iodide.

  7. Fluorescence lifetime imaging microscopy (FLIM).

    NARCIS (Netherlands)

    van Munster, E.B.; Gadella, Th.W.J.; Rietdorf, J.

    2005-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM systems have been implemented both in the frequency domain, using sinusoidally intensity-modulated excitation light and modulated

  8. Fluorescence Live Cell Imaging

    OpenAIRE

    Ettinger, Andreas; Wittmann, Torsten

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein (FP) tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio and to provide a suitable environment for ...

  9. 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…

  10. Development of Simultaneous Beta-and-Coincidence-Gamma Imager for Plant Imaging Research

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    Tai, Yuan-Chuan [Washington Univ., St. Louis, MO (United States). School of Medicine

    2016-09-30

    The goal of this project is to develop a novel imaging system that can simultaneously acquire beta and coincidence gamma images of positron sources in thin objects such as leaves of plants. This hybrid imager can be used to measure carbon assimilation in plants quantitatively and in real-time after C-11 labeled carbon-dioxide is administered. A better understanding of carbon assimilation, particularly under the increasingly elevated atmospheric CO2 level, is extremely critical for plant scientists who study food crop and biofuel production. Phase 1 of this project is focused on the technology development with 3 specific aims: (1) develop a hybrid detector that can detect beta and gamma rays simultaneously; (2) develop an imaging system that can differentiate these two types of radiation and acquire beta and coincidence gamma images in real-time; (3) develop techniques to quantify radiotracer distribution using beta and gamma images. Phase 2 of this project is to apply technologies developed in phase 1 to study plants using positron-emitting radionuclide such as 11C to study carbon assimilation in biofuel plants.

  11. Fluorescence imaging agents in cancerology.

    Science.gov (United States)

    Paganin-Gioanni, Aurélie; Bellard, Elisabeth; Paquereau, Laurent; Ecochard, Vincent; Golzio, Muriel; Teissié, Justin

    2010-09-01

    One of the major challenges in cancer therapy is to improve early detection and prevention using novel targeted cancer diagnostics. Detection requests specific recognition. Tumor markers have to be ideally present on the surface of cancer cells. Their targeting with ligands coupled to imaging agents make them visible/detectable. Fluorescence imaging is a newly emerging technology which is becoming a complementary medical method for cancer diagnosis. It allows detection with a high spatio-temporal resolution of tumor markers in small animals and in clinical studies. In this review, we focus on the recent outcome of basic studies in the design of new approaches (probes and devices) used to detect tumor cells by fluorescence imaging.

  12. A simultaneous beta and coincidence-gamma imaging system for plant leaves.

    Science.gov (United States)

    Ranjbar, Homayoon; Wen, Jie; Mathews, Aswin J; Komarov, Sergey; Wang, Qiang; Li, Ke; O'Sullivan, Joseph A; Tai, Yuan-Chuan

    2016-05-07

    Positron emitting isotopes, such as (11)C, (13)N, and (18)F, can be used to label molecules. The tracers, such as (11)CO2, are delivered to plants to study their biological processes, particularly metabolism and photosynthesis, which may contribute to the development of plants that have a higher yield of crops and biomass. Measurements and resulting images from PET scanners are not quantitative in young plant structures or in plant leaves due to poor positron annihilation in thin objects. To address this problem we have designed, assembled, modeled, and tested a nuclear imaging system (simultaneous beta-gamma imager). The imager can simultaneously detect positrons ([Formula: see text]) and coincidence-gamma rays (γ). The imaging system employs two planar detectors; one is a regular gamma detector which has a LYSO crystal array, and the other is a phoswich detector which has an additional BC-404 plastic scintillator for beta detection. A forward model for positrons is proposed along with a joint image reconstruction formulation to utilize the beta and coincidence-gamma measurements for estimating radioactivity distribution in plant leaves. The joint reconstruction algorithm first reconstructs beta and gamma images independently to estimate the thickness component of the beta forward model and afterward jointly estimates the radioactivity distribution in the object. We have validated the physics model and reconstruction framework through a phantom imaging study and imaging a tomato leaf that has absorbed (11)CO2. The results demonstrate that the simultaneously acquired beta and coincidence-gamma data, combined with our proposed joint reconstruction algorithm, improved the quantitative accuracy of estimating radioactivity distribution in thin objects such as leaves. We used the structural similarity (SSIM) index for comparing the leaf images from the simultaneous beta-gamma imager with the ground truth image. The jointly reconstructed images yield SSIM indices of 0

  13. 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.

  14. Blue fluorescent cGMP sensor for multiparameter fluorescence imaging.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Cyclic GMP (cGMP regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP and Ca(2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP. We cotransfected them and loaded a red fluorescent probe for Ca(2+ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca(2+, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.

  15. Combined fluorescence and phosphorescence lifetime imaging

    Energy Technology Data Exchange (ETDEWEB)

    Shcheslavskiy, V. I. [Becker & Hickl GmbH, Nahmitzer Damm 30, Berlin 12277 (Germany); Institute of Biomedical Technologies, Nizhny Novgorod State Medical Academy, Minin and Pozharsky Square, 10/1, Nizhny Novgorod 603005 (Russian Federation); Neubauer, A.; Becker, W. [Becker & Hickl GmbH, Nahmitzer Damm 30, Berlin 12277 (Germany); Bukowiecki, R.; Dinter, F. [Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Str. 10, Berlin 13092 (Germany)

    2016-02-29

    We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

  16. Fluorescence lifetime imaging using light emitting diodes

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Gordon T; Munro, Ian; Poher, Vincent; French, Paul M W; Neil, Mark A A [Blackett Laboratory, Imperial College London, South Kensington Campus, London SW7 2AZ (United Kingdom); Elson, Daniel S [Institute of Biomedical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ (United Kingdom); Hares, Jonathan D [Kentech Instruments Ltd, Unit 9, Hall Farm Workshops, South Moreton, Didcot, Oxfordshire, OX11 9AG (United Kingdom)], E-mail: gordon.kennedy@imperial.ac.uk

    2008-05-07

    We demonstrate flexible use of low cost, high-power light emitting diodes as illumination sources for fluorescence lifetime imaging (FLIM). Both time-domain and frequency-domain techniques have been implemented at wavelengths spanning the range 450-640 nm. Additionally, we demonstrate optically sectioned fluorescence lifetime imaging by combining structured illumination with frequency-domain FLIM.

  17. Ion-ion coincidence imaging at high event rate using an in-vacuum pixel detector

    Science.gov (United States)

    Long, Jingming; Furch, Federico J.; Durá, Judith; Tremsin, Anton S.; Vallerga, John; Schulz, Claus Peter; Rouzée, Arnaud; Vrakking, Marc J. J.

    2017-07-01

    A new ion-ion coincidence imaging spectrometer based on a pixelated complementary metal-oxide-semiconductor detector has been developed for the investigation of molecular ionization and fragmentation processes in strong laser fields. Used as a part of a velocity map imaging spectrometer, the detection system is comprised of a set of microchannel plates and a Timepix detector. A fast time-to-digital converter (TDC) is used to enhance the ion time-of-flight resolution by correlating timestamps registered separately by the Timepix detector and the TDC. In addition, sub-pixel spatial resolution (principle experiment on strong field dissociative double ionization of carbon dioxide molecules (CO2), using a 400 kHz repetition rate laser system. The experimental results demonstrate that the spectrometer can detect multiple ions in coincidence, making it a valuable tool for studying the fragmentation dynamics of molecules in strong laser fields.

  18. Bringing aerospace images into coincidence with subpixel accuracy by the local-correlation method

    Science.gov (United States)

    Potapov, A. S.; Malyshev, I. A.; Lutsiv, V. R.

    2004-05-01

    This paper proposes a local-correlation method that makes it possible to bring aerospace images into coincidence with subpixel accuracy after preliminary rough juxtaposition by transforming them relative to each other by uniform projective transformation and by additional mutual local displacements. The basis of the method is to establish a correspondence between the points of a pair of images by means of a phase correlation of individual segments of the images. The dissemination of information concerning measured shifts from reference points for which the correspondence has been found on a pair of images, as well as the use of analysis with variable spatial resolution, makes the method workable when the errors of the preliminary superposition are greater than the size of the correlation window. This paper presents the results of an experimental verification of the approach, using actual pairs of aerospace images as an example.© 2004

  19. Double Photon Emission Coincidence Imaging using GAGG-SiPM pixel detectors

    Science.gov (United States)

    Shimazoe, K.; Uenomachi, M.; Mizumachi, Y.; Takahashi, H.; Masao, Y.; Shoji, Y.; Kamada, K.; Yoshikawa, A.

    2017-12-01

    Single photon emission computed tomography(SPECT) is a useful medical imaging modality using single photon detection from radioactive tracers, such as 99Tc and 111In, however further development of increasing the contrast in the image is still under investigation. A novel method (Double Photon Emission CT / DPECT) using a coincidence detection of two cascade gamma-rays from 111In is proposed and characterized in this study. 111In, which is well-known and commonly used as a SPECT tracer, emits two cascade photons of 171 keV and 245 keV with a short delay of approximately 85 ns. The coincidence detection of two gamma-rays theoretically determines the position in a single point compared with a line in single photon detection and increases the signal to noise ratio drastically. A fabricated pixel detector for this purpose consists of 8 × 8 array of high-resolution type 1.5 mm thickness Ce:GAGG (3.9% @ 662 keV, 6.63g/cm3, C&A Co. Ce:Gd3Ga2.7Al2.3O12 2.5 × 2.5 × 1.5 mm3) crystals coupled a 3 mm pixel SiPM array (Hamamatsu MPPC S13361-2050NS-08). The signal from each pixel is processed and readout using time over threshold (TOT) based parallel processing circuit to extract the energy and timing information. The coincidence was detected by FPGA with the frequency of 400 MHz. Two pixel detectors coupled to parallel-hole collimators are located at the degree of 90 to determine the position and coincidence events (time window =1 μs) are detected and used for making back-projection image. The basic principle of DPECT is characterized including the detection efficiency and timing resolution.

  20. Recent Progress in Fluorescent Imaging Probes

    Science.gov (United States)

    Pak, Yen Leng; Swamy, K. M. K.; Yoon, Juyoung

    2015-01-01

    Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn2+, Hg2+, Cu2+ and Au3+, and anions including cyanide and adenosine triphosphate (ATP). PMID:26402684

  1. Boronic acids for fluorescence imaging of carbohydrates.

    Science.gov (United States)

    Sun, Xiaolong; Zhai, Wenlei; Fossey, John S; James, Tony D

    2016-02-28

    "Fluorescence imaging" is a particularly exciting and rapidly developing area of research; the annual number of publications in the area has increased ten-fold over the last decade. The rapid increase of interest in fluorescence imaging will necessitate the development of an increasing number of molecular receptors and binding agents in order to meet the demand in this rapidly expanding area. Carbohydrate biomarkers are particularly important targets for fluorescence imaging given their pivotal role in numerous important biological events, including the development and progression of many diseases. Therefore, the development of new fluorescent receptors and binding agents for carbohydrates is and will be increasing in demand. This review highlights the development of fluorescence imaging agents based on boronic acids a particularly promising class of receptors given their strong and selective binding with carbohydrates in aqueous media.

  2. Imaging photoelectron circular dichroism of chiral molecules by femtosecond multiphoton coincidence detection.

    Science.gov (United States)

    Lehmann, C Stefan; Ram, N Bhargava; Powis, Ivan; Janssen, Maurice H M

    2013-12-21

    Here, we provide a detailed account of novel experiments employing electron-ion coincidence imaging to discriminate chiral molecules. The full three-dimensional angular scattering distribution of electrons is measured after photoexcitation with either left or right circular polarized light. The experiment is performed using a simplified photoelectron-photoion coincidence imaging setup employing only a single particle imaging detector. Results are reported applying this technique to enantiomers of the chiral molecule camphor after three-photon ionization by circularly polarized femtosecond laser pulses at 400 nm and 380 nm. The electron-ion coincidence imaging provides the photoelectron spectrum of mass-selected ions that are observed in the time-of-flight mass spectra. The coincident photoelectron spectra of the parent camphor ion and the various fragment ions are the same, so it can be concluded that fragmentation of camphor happens after ionization. We discuss the forward-backward asymmetry in the photoelectron angular distribution which is expressed in Legendre polynomials with moments up to order six. Furthermore, we present a method, similar to one-photon electron circular dichroism, to quantify the strength of the chiral electron asymmetry in a single parameter. The circular dichroism in the photoelectron angular distribution of camphor is measured to be 8% at 400 nm. The electron circular dichroism using femtosecond multiphoton excitation is of opposite sign and about 60% larger than the electron dichroism observed before in near-threshold one-photon ionization with synchrotron excitation. We interpret our multiphoton ionization as being resonant at the two-photon level with the 3s and 3p Rydberg states of camphor. Theoretical calculations are presented that model the photoelectron angular distribution from a prealigned camphor molecule using density functional theory and continuum multiple scattering X alpha photoelectron scattering calculations

  3. Multicolor in vivo targeted imaging to guide real-time surgery of HER2-positive micrometastases in a two-tumor coincident model of ovarian cancer.

    Science.gov (United States)

    Longmire, Michelle; Kosaka, Nobuyuki; Ogawa, Mikako; Choyke, Peter L; Kobayashi, Hisataka

    2009-06-01

    One of the primary goals of oncological molecular imaging is to accurately identify and characterize malignant tissues in vivo. Currently, molecular imaging relies on targeting a single molecule that while overexpressed in malignancy, is often also expressed at lower levels in normal tissue, resulting in reduced tumor to background ratios. One approach to increasing the specificity of molecular imaging in cancer is to use multiple probes each with distinct fluorescence to target several surface antigens simultaneously, in order to identify tissue expression profiles, rather than relying on the expression of a single target. This next step forward in molecular imaging will rely on characterization of tissue based on fluorescence and therefore will require the ability to simultaneously identify several optical probes each attached to different targeting ligands. We created a novel 'coincident' ovarian cancer mouse model by coinjecting each animal with two distinct cell lines, HER2+/red fluorescent protein (RFP)- SKOV3 and HER2-/RFP+ SHIN3-RFP, in order to establish a model of disease in which animals simultaneously bore tumors with two distinct phenotypes (HER2+/RFP-, HER2-/RFP+), which could be utilized for multicolor imaging. The HER2 receptor of the SKOV3 cell line was targeted with a trastuzumab-rhodamine green conjugate to create green tumor implants, whereas the RFP plasmid of the SHIN3 cells created red tumor implants. We demonstrate that real-time in vivo multicolor imaging is feasible and that fluorescence characteristics can then serve to guide the surgical removal of disease.

  4. Hyperspectral small animal fluorescence imaging: spectral selection imaging

    Science.gov (United States)

    Leavesley, Silas; Jiang, Yanan; Patsekin, Valery; Hall, Heidi; Vizard, Douglas; Robinson, J. Paul

    2008-02-01

    Molecular imaging is a rapidly growing area of research, fueled by needs in pharmaceutical drug-development for methods for high-throughput screening, pre-clinical and clinical screening for visualizing tumor growth and drug targeting, and a growing number of applications in the molecular biology fields. Small animal fluorescence imaging employs fluorescent probes to target molecular events in vivo, with a large number of molecular targeting probes readily available. The ease at which new targeting compounds can be developed, the short acquisition times, and the low cost (compared to microCT, MRI, or PET) makes fluorescence imaging attractive. However, small animal fluorescence imaging suffers from high optical scattering, absorption, and autofluorescence. Much of these problems can be overcome through multispectral imaging techniques, which collect images at different fluorescence emission wavelengths, followed by analysis, classification, and spectral deconvolution methods to isolate signals from fluorescence emission. We present an alternative to the current method, using hyperspectral excitation scanning (spectral selection imaging), a technique that allows excitation at any wavelength in the visible and near-infrared wavelength range. In many cases, excitation imaging may be more effective at identifying specific fluorescence signals because of the higher complexity of the fluorophore excitation spectrum. Because the excitation is filtered and not the emission, the resolution limit and image shift imposed by acousto-optic tunable filters have no effect on imager performance. We will discuss design of the imager, optimizing the imager for use in small animal fluorescence imaging, and application of spectral analysis and classification methods for identifying specific fluorescence signals.

  5. 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

  6. Analysis of Coincident HICO and Airborne Hyperspectral Images Over Lake Erie Western Basin HABs

    Science.gov (United States)

    Cline, Michael T., Jr.

    Harmful algal blooms (HABs) produce waterborne toxins that pose a significant threat to people, livestock, and wildlife. Nearly 40 million people in both Canada and the U.S. depend on Great Lakes water. In the summer of 2014, in the Lake Erie Western Basin, an HAB of the cyanobacteria Microcystis was so severe that a do-not-drink advisory was in effect for the greater Toledo area, Ohio. This advisory applied to the water supply to over 400,000 people from a single water intake. Bloom intensity, composition, and spatial variability were investigated by comparing coincidental hyperspectral data from NASA's HICO, and NASA GRC's HSI airborne sensor, with on-lake ASD radiometer measurements and in situ water quality testing as ground reference data. Coincident data sets were obtained with HICO only on one day in 2014, however all other datasets coincide four times in 2015. Remote sensing data were atmospherically corrected using the empirical line method, utilizing dark reference spectra from a nearby asphalt parking lot measured from ASD and HSI radiometers. Cyanobacteria Index (CI) images were created from processed images using the Wynne (2010) algorithm, previously used for MODIS and MERIS imagery. This algorithm-generated CI images provide reliable results for both ground level (R2=0.921), airborne (R2=0.7981), and satellite imagery (R2=0.7794) for seven sampling points. The ability to robustly atmospherically correct and generate useful CI maps from airborne and satellite sensors can provide a time- and cost-effective method for HABs analysis. Timely processing of these high spatial and spectral resolution remote sensing data can aid in management of water intake resources. These results will help to improve methods leading to HABs mapping by testing different algal retrieval algorithms and atmospheric correction techniques using a three tiered hyperspectral sensor approach utilizing satellite, airborne, and ground level sensors, coupled with water quality

  7. Design of a coincidence processing board for a dual-head PET scanner for breast imaging

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, J.D. [Departamento de Ingenieria Electronica, University Politecnica de Valencia, Camino de Vera s/n 46022, Valencia (Spain)]. E-mail: jormarp1@doctor.upv.es; Toledo, J. [Departamento de Ingenieria Electronica, University Politecnica de Valencia, Camino de Vera s/n 46022, Valencia (Spain); Esteve, R. [Departamento de Ingenieria Electronica, University Politecnica de Valencia, Camino de Vera s/n 46022, Valencia (Spain); Sebastia, A. [Departamento de Ingenieria Electronica, University Politecnica de Valencia, Camino de Vera s/n 46022, Valencia (Spain); Mora, F.J. [Departamento de Ingenieria Electronica, University Politecnica de Valencia, Camino de Vera s/n 46022, Valencia (Spain); Benlloch, J.M. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain); Fernandez, M.M. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain); Gimenez, M. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain); Gimenez, E.N. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain); Lerche, Ch.W. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain); Pavon, N. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain); Sanchez, F. [Instituto de Fisica Corpuscular, CSIC-UV, Valencia (Spain)

    2005-07-01

    This paper describes the design of a coincidence processing board for a dual-head Positron Emission Tomography (PET) scanner for breast imaging. The proposed block-oriented data acquisition system relies on a high-speed DSP processor for fully digital trigger and on-line event processing that surpasses the performance of traditional analog coincidence detection systems. A mixed-signal board has been designed and manufactured. The analog section comprises 12 coaxial inputs (six per head) which are digitized by means of two 8-channel 12-bit 40-MHz ADCs in order to acquire the scintillation pulse, the charge division signals and the depth of interaction within the scintillator. At the digital section, a state-of-the-art FPGA is used as deserializer and also implements the DMA interface to the DSP processor by storing each digitized channel into a fast embedded FIFO memory. The system incorporates a high-speed USB 2.0 interface to the host computer.

  8. Fluorescence imaging of early lung cancer

    Science.gov (United States)

    Lam, Stephen; MacAulay, Calum E.; Le Riche, Jean C.; Ikeda, Norihiko; Palcic, Branko

    1995-01-01

    The performance of a fluorescence imaging device was compared with conventional white-light bronchoscopy in 100 patients with lung cancer, 46 patients with resected State I nonsmall cell lung cancer, 10 patients with head and neck cancer, and 67 volunteers who had smoked at least one pack of cigarettes per day for twenty-five years or more. Using differences in tissue autofluorescence between premalignant, malignant and normal tissues, fluorescence bronchoscopy was found to detect more than twice as many moderate-severe dysplasia and carcinoma in situ sites than conventional white-light bronchoscopy. The use of fluorescence imaging to detect small peripheral lung nodules was investigated in a micro metastatic lung model of mice implanted with Lewis lung tumor cells. Fluorescence imaging was found to be able to detect small malignant lung lesions. The use of (delta) -aminolevulinic acid (ALA) to enhance fluorescence detection of CIS was investigated in a patient after oral administration of 60 mg/kg of ALA four hours prior to bronchoscopy, although ALA enhanced the tumor's visibility, multiple sites of false positive fluorescence were observed in areas of inflammation or metaplasia.

  9. Radar Coincidence Imaging for Off-Grid Target Using Frequency-Hopping Waveforms

    Directory of Open Access Journals (Sweden)

    Xiaoli Zhou

    2016-01-01

    Full Text Available Radar coincidence imaging (RCI is a high-resolution staring imaging technique without the limitation of the target relative motion. To achieve better imaging performance, sparse reconstruction is commonly used. While its performance is based on the assumption that the scatterers are located at the prediscretized grid-cell centers, otherwise, off-grid emerges and the performance of RCI degrades significantly. In this paper, RCI using frequency-hopping (FH waveforms is considered. The off-grid effects are analyzed, and the corresponding constrained Cramér-Rao bound (CCRB is derived based on the mean square error (MSE of the “oracle” estimator. For off-grid RCI, the process is composed of two stages: grid matching and off-grid error (OGE calibration, where two-dimension (2D band-excluded locally optimized orthogonal matching pursuit (BLOOMP and alternating iteration minimization (AIM algorithms are proposed, respectively. Unlike traditional sparse recovery methods, BLOOMP realizes the recovery in the refinement grids by overwhelming the shortages of coherent dictionary and is robust to noise and OGE. AIM calibration algorithm adaptively adjusts the OGE and, meanwhile, seeks the optimal target reconstruction result.

  10. System for fluorescence lifetime imaging microscopy

    Science.gov (United States)

    van Geest, Lambertus K.; Boddeke, Frank R.; van Dijk, Pieter W.; Kamp, Arjen F.; van der Oord, Cornelius J. R.; Stoop, Karel W. J.

    1999-05-01

    One of the promising recent developments in fluorescence microscopy is fluorescence lifetime imaging microscopy (FLIM). In this technique the fluorescence lifetime (ns range) of molecules is expressed in the image rather than the intensity of the light emitted by these molecules. This physical property is of interest as it gives information about the local environment of the molecule, such as molecular concentration of O2, Ca2+, pH, and conjugation. We develop an affordable, robust and easy-to-use FLIM workstation which is completely automated and does not need any difficult calibration procedure. The system consists of a standard fluorescence microscope, a modulated excitation light source, a camera, a modulation signal generator and acquisition/processing software. The camera contains an Intensified CCD of which the image intensifier gain is modulated. Depending on the application different light sources can be selected. The current light source contains a 12 mW modulated laser-diode emitting at 635 nm. A homodyne detection scheme with modulation frequencies of 1 to 100 MHz is applied, aiming at a resolution of 0.1 ns or better. High level image acquisition strategies are implemented in software, along with the low level image processing routines for lifetime estimation, calibration and correction. An evaluation of the system and its critical components will be presented in this paper.

  11. CRF-PEPICO: Double velocity map imaging photoelectron photoion coincidence spectroscopy for reaction kinetics studies

    Science.gov (United States)

    Sztáray, Bálint; Voronova, Krisztina; Torma, Krisztián G.; Covert, Kyle J.; Bodi, Andras; Hemberger, Patrick; Gerber, Thomas; Osborn, David L.

    2017-07-01

    Photoelectron photoion coincidence (PEPICO) spectroscopy could become a powerful tool for the time-resolved study of multi-channel gas phase chemical reactions. Toward this goal, we have designed and tested electron and ion optics that form the core of a new PEPICO spectrometer, utilizing simultaneous velocity map imaging for both cations and electrons, while also achieving good cation mass resolution through space focusing. These optics are combined with a side-sampled, slow-flow chemical reactor for photolytic initiation of gas-phase chemical reactions. Together with a recent advance that dramatically increases the dynamic range in PEPICO spectroscopy [D. L. Osborn et al., J. Chem. Phys. 145, 164202 (2016)], the design described here demonstrates a complete prototype spectrometer and reactor interface to carry out time-resolved experiments. Combining dual velocity map imaging with cation space focusing yields tightly focused photoion images for translationally cold neutrals, while offering good mass resolution for thermal samples as well. The flexible optics design incorporates linear electric fields in the ionization region, surrounded by dual curved electric fields for velocity map imaging of ions and electrons. Furthermore, the design allows for a long extraction stage, which makes this the first PEPICO experiment to combine ion imaging with the unimolecular dissociation rate constant measurements of cations to detect and account for kinetic shifts. Four examples are shown to illustrate some capabilities of this new design. We recorded the threshold photoelectron spectrum of the propargyl and the iodomethyl radicals. While the former agrees well with a literature threshold photoelectron spectrum, we have succeeded in resolving the previously unobserved vibrational structure in the latter. We have also measured the bimolecular rate constant of the CH2I + O2 reaction and observed its product, the smallest Criegee intermediate, CH2OO. Finally, the second

  12. 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.

  13. Molecular Probes for Fluorescence Lifetime Imaging

    Science.gov (United States)

    Sarder, Pinaki; Maji, Dolonchampa; Achilefu, Samuel

    2015-01-01

    Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely rely on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems, and outlines areas of potential high impact in the future. PMID:25961514

  14. An apertureless near-field microscope for fluorescence imaging

    OpenAIRE

    Yang, T. J.; Lessard, Guillaume A.; Quake, Stephen R.

    2000-01-01

    We describe an apertureless near field microscope for imaging fluorescent samples. Optical contrast is generated by exploiting fluorescent quenching near a metallized atomic force microscope tip. This microscope has been used to image fluorescent latex beads with subdiffraction limit resolution. The use of fluorescence allows us to prove that the contrast mechanism is indeed spectroscopic in origin.

  15. Imaging efficacy of a targeted imaging agent for fluorescence endoscopy

    Science.gov (United States)

    Healey, A. J.; Bendiksen, R.; Attramadal, T.; Bjerke, R.; Waagene, S.; Hvoslef, A. M.; Johannesen, E.

    2008-02-01

    Colorectal cancer is a major cause of cancer death. A significant unmet clinical need exists in the area of screening for earlier and more accurate diagnosis and treatment. We have identified a fluorescence imaging agent targeted to an early stage molecular marker for colorectal cancer. The agent is administered intravenously and imaged in a far red imaging channel as an adjunct to white light endoscopy. There is experimental evidence of preclinical proof of mechanism for the agent. In order to assess potential clinical efficacy, imaging was performed with a prototype fluorescence endoscope system designed to produce clinically relevant images. A clinical laparoscope system was modified for fluorescence imaging. The system was optimised for sensitivity. Images were recorded at settings matching those expected with a clinical endoscope implementation (at video frame rate operation). The animal model was comprised of a HCT-15 xenograft tumour expressing the target at concentration levels expected in early stage colorectal cancer. Tumours were grown subcutaneously. The imaging agent was administered intravenously at a dose of 50nmol/kg body weight. The animals were killed 2 hours post administration and prepared for imaging. A 3-4mm diameter, 1.6mm thick slice of viable tumour was placed over the opened colon and imaged with the laparoscope system. A receiver operator characteristic analysis was applied to imaging results. An area under the curve of 0.98 and a sensitivity of 87% [73, 96] and specificity of 100% [93, 100] were obtained.

  16. Developing an imaging bi-spectrometer for fluorescent materials

    Science.gov (United States)

    Mohammadi, Mahnaz

    Fluorescent effects have been observed for thousands of years. Stokes, in 1852, began the science of fluorescence culminating in his law of fluorescence, which explained that fluorescence emission occurs at longer wavelengths than the excitation wavelength. This phenomenon is observed extensively in the art world. Daylight fluorescent colors known as Day-GloRTM have become an artistic medium since the 1960s. Modern artists exploit these saturated and brilliant colors to glitter their painting. Multipsectral imaging as a noninvasive technique has been used for archiving by museums and cultural-heritage institutions for about a decade. The complex fluorescence phenomenon has been often ignored in the multispectral projects. The ignored fluorescence results in errors in digital imaging of artwork containing fluorescent colors. The illuminant-dependency of the fluorescence radiance makes the fluorescence colorimetry and consequently spectral imaging more complex. In this dissertation an abridged imaging bi-spectrometer for artwork containing both fluorescent and non-fluorescent colors was developed. The method developed included two stages of reconstruction of the spectral reflected radiance factor and prediction of the fluorescent radiance factor. The estimation of the reflected radiance factor as a light source independent component was achieved by imaging with a series of short-wavelength cutoff filters placed in the illumination path. The fluorescent radiance factor, a light source dependent component, was estimated based on a proposed model, the abridged two-monochromator method. The abridged two-monochromator method was developed for reconstructing the bi-spectral matrix of a fluorescent color based on a calibrated UV-fluorescence imaging. In this way, one could predict the fluorescence radiance factor under any desired illuminant and consequently a better color evaluation and rendering could be obtained. Furthermore, this method easily fitted in a general system

  17. Active mask segmentation of fluorescence microscope images.

    Science.gov (United States)

    Srinivasa, Gowri; Fickus, Matthew C; Guo, Yusong; Linstedt, Adam D; Kovacević, Jelena

    2009-08-01

    We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the "contour" to that of "inside and outside," or masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively, as well as quantitatively.

  18. Total Internal Reflection Fluorescence Microscopy Imaging-Guided Confocal Single-Molecule Fluorescence Spectroscopy

    OpenAIRE

    Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

    2013-01-01

    We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscop...

  19. From iron pentacarbonyl to the iron ion by imaging photoelectron photoion coincidence.

    Science.gov (United States)

    Russell, Eileen M; Cudjoe, Elvis; Mastromatteo, Michael E; Kercher, James P; Sztáray, Bálint; Bodi, Andras

    2013-06-06

    The dissociation dynamics of internal energy selected iron pentacarbonyl cations, Fe(CO)5(+), have been investigated using the imaging photoelectron photoion coincidence (iPEPICO) spectrometer at the Swiss Light Source. The molecular ion loses all five carbonyl ligands in sequential dissociations in the 8.5-20 eV photon energy range. The Fe(CO)5(+) parent ion is metastable at the onset of the first dissociation reaction on the time scale of the experiment. The slightly asymmetric and broad daughter ion time-of-flight distributions indicate parent ion lifetimes in the microsecond range, and are used to obtain an experimental dissociation rate curve. Further carbonyl losses were found to be fast at threshold. The fractional parent and daughter ion abundances as a function of the photon energy, that is, breakdown diagram, as well as the dissociation rates for the first CO loss were modeled using the statistical Rice-Ramsperger-Kassel-Marcus (RRKM) and statistical adiabatic channel model (SSACM) theories. The excess energy redistribution in the products was also taken into account in a statistical framework. The 0 K dissociative photoionization thresholds for the five carbonyl-loss channels were found to be 9.015 ± 0.024 eV, 10.199 ± 0.027 eV, 10.949 ± 0.033 eV, 12.282 ± 0.39 eV, and 13.821 ± 0.045 eV for the processes leading to Fe(CO)4(+), Fe(CO)3(+), Fe(CO)2(+), Fe(CO)(+), and Fe(+), respectively. The iron cation thermochemistry is well-known, and these onsets connect the bare metal ion to the other fragment ions as well as to the gas phase neutral Fe(CO)5.

  20. Fluorescence imaging to quantify crop residue cover

    Science.gov (United States)

    Daughtry, C. S. T.; Mcmurtrey, J. E., III; Chappelle, E. W.

    1994-01-01

    Crop residues, the portion of the crop left in the field after harvest, can be an important management factor in controlling soil erosion. Methods to quantify residue cover are needed that are rapid, accurate, and objective. Scenes with known amounts of crop residue were illuminated with long wave ultraviolet (UV) radiation and fluorescence images were recorded with an intensified video camera fitted with a 453 to 488 nm band pass filter. A light colored soil and a dark colored soil were used as background for the weathered soybean stems. Residue cover was determined by counting the proportion of the pixels in the image with fluorescence values greater than a threshold. Soil pixels had the lowest gray levels in the images. The values of the soybean residue pixels spanned nearly the full range of the 8-bit video data. Classification accuracies typically were within 3(absolute units) of measured cover values. Video imaging can provide an intuitive understanding of the fraction of the soil covered by residue.

  1. Fluorescence lifetime imaging microscopy (FLIM) data analysis with TIMP

    NARCIS (Netherlands)

    Laptenok, S.; Mullen, K.M.; Borst, J.W.; Stokkum, van I.H.M.; Apanasovich, V.V.; Visser, A.J.W.G.

    2007-01-01

    Fluorescence Lifetime Imaging Microscopy (FLIM) allows fluorescence lifetime images of biological objects to be collected at 250 nm spatial resolution and at (sub-)nanosecond temporal resolution. Often ncomp kinetic processes underlie the observed fluorescence at all locations, but the intensity of

  2. Fluorescent multiple chemical sensing using time-domain fluorescence lifetime imaging

    OpenAIRE

    Nagl, Stefan

    2008-01-01

    This thesis describes applications of fluorescence lifetime imaging in multiple chemical sensing approaches. Using fluorescence lifetime as an analytical parameter allows extracting more information out of probes than fluorescence intensity measurements and it is therefore attractive in order to quantitate multiple species. It leads to better data quality as fluorescence lifetime measurements are not or less affected by many sources of noise in fluorescence signals such as straylight and othe...

  3. Modelling of microcracks image treated with fluorescent dye

    Science.gov (United States)

    Glebov, Victor; Lashmanov, Oleg U.

    2015-06-01

    The main reasons of catastrophes and accidents are high level of wear of equipment and violation of the production technology. The methods of nondestructive testing are designed to find out defects timely and to prevent break down of aggregates. These methods allow determining compliance of object parameters with technical requirements without destroying it. This work will discuss dye penetrant inspection or liquid penetrant inspection (DPI or LPI) methods and computer model of microcracks image treated with fluorescent dye. Usually cracks on image look like broken extended lines with small width (about 1 to 10 pixels) and ragged edges. The used method of inspection allows to detect microcracks with depth about 10 or more micrometers. During the work the mathematical model of image of randomly located microcracks treated with fluorescent dye was created in MATLAB environment. Background noises and distortions introduced by the optical systems are considered in the model. The factors that have influence on the image are listed below: 1. Background noise. Background noise is caused by the bright light from external sources and it reduces contrast on the objects edges. 2. Noises on the image sensor. Digital noise manifests itself in the form of randomly located points that are differing in their brightness and color. 3. Distortions caused by aberrations of optical system. After passing through the real optical system the homocentricity of the bundle of rays is violated or homocentricity remains but rays intersect at the point that doesn't coincide with the point of the ideal image. The stronger the influence of the above-listed factors, the worse the image quality and therefore the analysis of the image for control of the item finds difficulty. The mathematical model is created using the following algorithm: at the beginning the number of cracks that will be modeled is entered from keyboard. Then the point with random position is choosing on the matrix whose size is

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

    OpenAIRE

    Patrick eKaifosh; Jeffrey eZaremba; Nathan eDanielson; Attila eLosonczy

    2014-01-01

    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-scannin...

  5. Laser induced fluorescence imaging system for localization of nasopharyngeal carcinoma

    Science.gov (United States)

    Liu, Lina; Xie, Shusen

    2007-11-01

    A laser induced fluorescence imaging system for localization of Nasopharyngeal Carcinoma is developed. In this fluorescence imaging system, the fluorescence intensity with information of detected objection is gained by an image intensifier, which makes color information of the fluorescence image eliminated and the result is a monochrome image of the fluorescence with thermally induced noise. The monochrome fluorescence image is sent to a CCD and captured by an image board, which is controlled by a computer. Image processing is carried out to improve the image quality and therefore improve the system's ability to differentiate carcinomas from normal tissue. Gaussian smoothing is implemented in order to reduce the noise. Image binarizing process is realized to obtain an optimal threshold of the image. Image pixels with grey value below this threshold are assigned as diseased and those above are normal. A pseudo color processing is then accomplished to get better visual perception and understanding of the image, greatly increasing the detail resolution of the grey image. The processed image is then displayed on the screen of the computer in real time. The real time laser induced fluorescence imaging system with the image processing methods developed is efficient for localization of the nasopharyngeal carcinoma.

  6. Synchrotron-based double imaging photoelectron/photoion coincidence spectroscopy of radicals produced in a flow tube: OH and OD

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, Gustavo A.; Tang, Xiaofeng; Gil, Jean-François; Nahon, Laurent [Synchrotron SOLEIL, L’Orme des Merisiers, St. Aubin, BP 48, 91192 Gif sur Yvette (France); Ward, Michael; Batut, Sebastien; Fittschen, Christa [PC2A, Université de Lille 1, UMR CNRS-USTL 8522, Cité Scientifique Bât. C11, F-59655 Villeneuve d’Ascq (France); Taatjes, Craig A.; Osborn, David L. [Combustion Research Facility, Mail Stop 9055, Sandia National Laboratories, Livermore, California 94551-0969 (United States); Loison, Jean-Christophe [ISM, Université Bordeaux 1, CNRS, 351 cours de la Libération, 33405 Talence Cedex (France)

    2015-04-28

    We present a microwave discharge flow tube coupled with a double imaging electron/ion coincidence device and vacuum ultraviolet (VUV) synchrotron radiation. The system has been applied to the study of the photoelectron spectroscopy of the well-known radicals OH and OD. The coincidence imaging scheme provides a high selectivity and yields the spectra of the pure radicals, removing the ever-present contributions from excess reactants, background, or secondary products, and therefore obviating the need for a prior knowledge of all possible byproducts. The photoelectron spectra encompassing the X{sup 3}Σ{sup −} ground state of the OH{sup +} and OD{sup +} cations have been extracted and the vibrational constants compared satisfactorily to existing literature values. Future advantages of this approach include measurement of high resolution VUV spectroscopy of radicals, their absolute photoionization cross section, and species/isomer identification in chemical reactions as a function of time.

  7. A framework for creating realistic synthetic fluorescence microscopy image sequences

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

    Full Text Available Fluorescence microscopy imaging is an important tool in modern biological research, allowing insights into the processes of biological systems. Automated image analysis algorithms help in extracting information from these images. Validation...

  8. Coincidence velocity map imaging using Tpx3Cam, a time stamping optical camera with 1.5 ns timing resolution

    Science.gov (United States)

    Zhao, Arthur; van Beuzekom, Martin; Bouwens, Bram; Byelov, Dmitry; Chakaberia, Irakli; Cheng, Chuan; Maddox, Erik; Nomerotski, Andrei; Svihra, Peter; Visser, Jan; Vrba, Vaclav; Weinacht, Thomas

    2017-11-01

    We demonstrate a coincidence velocity map imaging apparatus equipped with a novel time-stamping fast optical camera, Tpx3Cam, whose high sensitivity and nanosecond timing resolution allow for simultaneous position and time-of-flight detection. This single detector design is simple, flexible, and capable of highly differential measurements. We show detailed characterization of the camera and its application in strong field ionization experiments.

  9. Carbon Quantum Dots for Zebrafish Fluorescence Imaging.

    Science.gov (United States)

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-07-02

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model.

  10. Ion beam induced fluorescence imaging in biological systems

    Science.gov (United States)

    Bettiol, Andrew A.; Mi, Zhaohong; Vanga, Sudheer Kumar; Chen, Ce-belle; Tao, Ye; Watt, Frank

    2015-04-01

    Imaging fluorescence generated by MeV ions in biological systems such as cells and tissue sections requires a high resolution beam (fluorescent probe that has a high quantum efficiency and low bleaching rate. For cutting edge applications in bioimaging, the fluorescence imaging technique needs to break the optical diffraction limit allowing for sub-cellular structure to be visualized, leading to a better understanding of cellular function. In a nuclear microprobe this resolution requirement can be readily achieved utilizing low beam current techniques such as Scanning Transmission Ion Microscopy (STIM). In recent times, we have been able to extend this capability to fluorescence imaging through the development of a new high efficiency fluorescence detection system, and through the use of new novel fluorescent probes that are resistant to ion beam damage (bleaching). In this paper we demonstrate ion beam induced fluorescence imaging in several biological samples, highlighting the advantages and challenges associated with using this technique.

  11. Bacterial imaging with photostable upconversion fluorescent nanoparticles.

    Science.gov (United States)

    Ong, Li Ching; Ang, Lei Yin; Alonso, Sylvie; Zhang, Yong

    2014-03-01

    Autofluorescence, photodamage and photobleaching are often encountered when using downconverting fluorophores and fluorescent proteins for bacteria labeling. These caveats represent a serious limitation when trying to map bacteria dissemination for prolonged periods. Upconversion nanoparticles (UCNs), which are able to convert low energy near-infrared (NIR) excitation light into higher energy visible or NIR light, can address these limitations. These particles' unique optical properties translate into attractive advantages of minimal autofluorescence, reduced photodamage, deeper tissue penetration and prolonged photostability. Here, we report a UCN-based bacteria labeling strategy using Escherichia coli as prototypic bacteria. A comparative analysis highlighted the superior photostability of UCN-labeled bacteria over green fluorescent protein-expressing bacteria. Infection study of UCN-labeled bacteria in dendritic cells indicated co-localization of the UCN signal with bacterial position for up to 6 h post-infection. Furthermore, long-term monitoring of the same infected cells demonstrated the potential to utilize photostable UCN-based imaging for bacterial trafficking purposes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Dynamic fluorescence imaging with molecular agents for cancer detection

    Science.gov (United States)

    Kwon, Sun Kuk

    Non-invasive dynamic optical imaging of small animals requires the development of a novel fluorescence imaging modality. Herein, fluorescence imaging is demonstrated with sub-second camera integration times using agents specifically targeted to disease markers, enabling rapid detection of cancerous regions. The continuous-wave fluorescence imaging acquires data with an intensified or an electron-multiplying charge-coupled device. The work presented in this dissertation (i) assessed dose-dependent uptake using dynamic fluorescence imaging and pharmacokinetic (PK) models, (ii) evaluated disease marker availability in two different xenograft tumors, (iii) compared the impact of autofluorescence in fluorescence imaging of near-infrared (NIR) vs. red light excitable fluorescent contrast agents, (iv) demonstrated dual-wavelength fluorescence imaging of angiogenic vessels and lymphatics associated with a xenograft tumor model, and (v) examined dynamic multi-wavelength, whole-body fluorescence imaging with two different fluorescent contrast agents. PK analysis showed that the uptake of Cy5.5-c(KRGDf) in xenograft tumor regions linearly increased with doses of Cy5.5-c(KRGDf) up to 1.5 nmol/mouse. Above 1.5 nmol/mouse, the uptake did not increase with doses, suggesting receptor saturation. Target to background ratio (TBR) and PK analysis for two different tumor cell lines showed that while Kaposi's sarcoma (KS1767) exhibited early and rapid uptake of Cy5.5-c(KRGDf), human melanoma tumors (M21) had non-significant TBR differences and early uptake rates similar to the contralateral normal tissue regions. The differences may be due to different compartment location of the target. A comparison of fluorescence imaging with NIR vs. red light excitable fluorescent dyes demonstrates that NIR dyes are associated with less background signal, enabling rapid tumor detection. In contrast, animals injected with red light excitable fluorescent dyes showed high autofluorescence. Dual

  13. Patterned Fluorescence Images with Indigo Precursors in Polymer Film

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Bora; Oh, Eun Hae; Lee, Chan Woo; Kim, Jongman [Hanyang Univ., Seoul (Korea, Republic of)

    2013-04-15

    We have developed a new strategy for the generation of patterned fluorescence images in polymer film. A fluorescent acetyl protected indole 6 was transformed to a nonfluorescent indigo dye 7 by UV irradiation. In addition, a t-Boc protected fluorescent indigo molecule 8 was also converted to a nonfluorescent indigo derivative 7 under a chemical amplification condition. Photomasked UV irradiation of the precursor molecules allowed efficient generation of patterned fluorescence images in polymer film. The strategy described in current investigation is believed to be an important addition to the fluorescent patterning technology.

  14. Multimodal quantitative phase and fluorescence imaging of cell apoptosis

    Science.gov (United States)

    Fu, Xinye; Zuo, Chao; Yan, Hao

    2017-06-01

    Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

  15. Optical mammography combined with fluorescence imaging: lesion detection using scatterplots

    NARCIS (Netherlands)

    Leproux, Anaïs; van der Voort, Marjolein; van der Mark, Martin B.; Harbers, Rik; van de Ven, Stephanie M. W. Y.; van Leeuwen, Ton G.

    2011-01-01

    Using scatterplots of 2 or 3 parameters, diffuse optical tomography and fluorescence imaging are combined to improve detectability of breast lesions. Small or low contrast phantom-lesions that were missed in the optical and fluorescence images were detected in the scatterplots. In patient

  16. 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/r...

  17. Advances in fluorescence labeling strategies for dynamic cellular imaging.

    Science.gov (United States)

    Dean, Kevin M; Palmer, Amy E

    2014-07-01

    Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

  18. 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.

  19. Development and characterization of a multiple-coincidence ion-momentum imaging spectrometer

    Energy Technology Data Exchange (ETDEWEB)

    Laksman, J.; Céolin, D.; Månsson, E. P.; Sorensen, S. L.; Gisselbrecht, M. [Department of Synchrotron Radiation Research, Lund University, Box 118, S-221 00 Lund (Sweden)

    2013-12-15

    The design and performance of a high-resolution momentum-imaging spectrometer for ions which is optimized for experiments using synchrotron radiation is presented. High collection efficiency is achieved by a focusing electrostatic lens; a long drift tube improves mass resolution and a position-sensitive detector enables measurement of the transverse momentum of ions. The optimisation of the lens for particle momentum measurement at the highest resolution is described. We discuss the overall performance of the spectrometer and present examples demonstrating the momentum resolution for both kinetics and for angular measurements in molecular fragmentation for carbon monoxide and fullerenes. Examples are presented that confirm that complete space-time focussing is possible for a two-field three-dimensional imaging spectrometer.

  20. Intraoperative pulmonary neoplasm identification using near-infrared fluorescence imaging.

    Science.gov (United States)

    Kim, Hyun Koo; Quan, Yu Hua; Choi, Byeong Hyeon; Park, Ji-Ho; Han, Kook Nam; Choi, Yeonho; Kim, Beop-Min; Choi, Young Ho

    2016-05-01

    Near-infrared (NIR) fluorescence imaging provides surgeons with real-time visual information during surgery. The purpose of this pilot trial was to evaluate the safety and feasibility of the intraoperative detection of pulmonary neoplasms with NIR fluorescence imaging after low-dose indocyanine green (ICG) injection. Eleven consecutive patients who were scheduled to undergo resection of pulmonary neoplasms were enrolled in this study. ICG (1 mg/kg) was administered intravenously 1 day before surgery, and the retrieved surgical specimens were examined for fluorescence signalling by using NIR fluorescence imaging system on a back table in the operating room. We analysed the fluorescence intensity, pathology, size, depth from the pleural surface and metabolic activity of the pulmonary neoplasms. Fluorescence signalling was detected in all specimens except in one from a patient with primary lung cancer. Two false-positive cases that presented no residual tumour with obstructive pneumonitis, after concurrent chemoradiation therapy for primary lung cancer before the operation, were identified, and their fluorescence intensity was 8.6 ± 0.4. The mean fluorescence intensity of the eight pulmonary tumours was 3.4 ± 1.9, and these tumours did not differ in pathology, size, depth from the pleural surface or metabolic activity. NIR fluorescence imaging could safely identify pulmonary neoplasms after the systemic injection of ICG. In addition, low-dose ICG is sufficient for NIR fluorescence imaging of pulmonary neoplasms. However, because the passive accumulation of ICG could not be used to discriminate tumours with inflammation, tumour-targeted fluorescence should be developed to solve this problem in the future. © The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

  1. Fluorescent imaging of cancerous tissues for targeted surgery

    Science.gov (United States)

    Bu, Lihong; Shen, Baozhong; Cheng, Zhen

    2014-01-01

    To maximize tumor excision and minimize collateral damage is the primary goal of cancer surgery. Emerging molecular imaging techniques have to “image-guided surgery” developing into “molecular imaging-guided surgery”, which is termed “targeted surgery” in this review. Consequently, the precision of surgery can be advanced from tissue-scale to molecule-scale, enabling “targeted surgery” to be a component of “targeted therapy”. Evidence from numerous experimental and clinical studies has demonstrated significant benefits of fluorescent imaging in targeted surgery with preoperative molecular diagnostic screening. Fluorescent imaging can help to improve intraoperative staging and enable more radical cytoreduction, detect obscure tumor lesions in special organs, highlight tumor margins, better map lymph node metastases, and identify important normal structures intraoperatively. Though limited tissue penetration of fluorescent imaging and tumor heterogeneity are two major hurdles for current targeted surgery, multimodality imaging and multiplex imaging may provide potential solutions to overcome these issues, respectively. Moreover, though many fluorescent imaging techniques and probes have been investigated, targeted surgery remains at a proof-of-principle stage. The impact of fluorescent imaging on cancer surgery will likely be realized through persistent interdisciplinary amalgamation of research in diverse fields. PMID:25064553

  2. 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...

  3. Microglia specific fluorescent probes for live cell imaging.

    Science.gov (United States)

    Leong, Cheryl; Lee, Sung Chan; Ock, Jiyeon; Li, Xin; See, Peter; Park, Sung Jin; Ginhoux, Florent; Yun, Seong-Wook; Chang, Young-Tae

    2014-02-04

    Small molecule fluorescent probes offer significant advantages over conventional antibody and fluorescent protein labeling techniques. Here we present and , dyes that label live microglia specifically. They may be applied to the isolation and imaging of live microglia when investigating their role in neuroinflammatory diseases.

  4. Studying membrane properties using Fluorescence Lifetime Imaging Microscopy (FLIM)

    NARCIS (Netherlands)

    Stöckl, M.T.; Bizzarri, R.; Subramaniam, Vinod; Mely, Y.; Duportail, G.

    2012-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to investigate the structure and composition of biological membranes. A wide variety of fluorescent probes suitable for FLIM experiments have been described. These compounds differ strongly in the details of their incorporation into

  5. 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.

  6. Multi-spectral fluorescence imaging for cultural heritage

    Science.gov (United States)

    Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo; Toniolo, Lucia

    2007-07-01

    A portable fluorescence multi-spectral imaging system was developed and applied to the analysis of artistic surfaces. The imaging apparatus exploits UV lamps for excitation and a liquid crystal tunable filter coupled to a low-noise CCD as the image detector. A sequence of images is acquired by sweeping the transmission band of the filter in order to slice the fluorescence emission in many narrow bands within the visible region of the e.m. spectrum. In this way, the fluorescence spectrum is recorded in each point of the analyzed area. The main features of the system will be discussed, together with its application to the analysis of the fluorescence properties of binders and pigment typically used in mural paintings. Finally, the study of the conservation of Renaissance frescoes recently restored will be presented.

  7. 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.

  8. An overview of the web-based Google Earth coincident imaging tool

    Science.gov (United States)

    Chander, Gyanesh; Kilough, B.; Gowda, S.

    2010-01-01

    The Committee on Earth Observing Satellites (CEOS) Visualization Environment (COVE) tool is a browser-based application that leverages Google Earth web to display satellite sensor coverage areas. The analysis tool can also be used to identify near simultaneous surface observation locations for two or more satellites. The National Aeronautics and Space Administration (NASA) CEOS System Engineering Office (SEO) worked with the CEOS Working Group on Calibration and Validation (WGCV) to develop the COVE tool. The CEOS member organizations are currently operating and planning hundreds of Earth Observation (EO) satellites. Standard cross-comparison exercises between multiple sensors to compare near-simultaneous surface observations and to identify corresponding image pairs are time-consuming and labor-intensive. COVE is a suite of tools that have been developed to make such tasks easier.

  9. Soft fluorescent nanomaterials for biological and biomedical imaging.

    Science.gov (United States)

    Peng, Hong-Shang; Chiu, Daniel T

    2015-07-21

    Soft fluorescent nanomaterials have attracted recent attention as imaging agents for biological applications, because they provide the advantages of good biocompatibility, high brightness, and easy biofunctionalization. Here, we provide a survey of recent developments in fluorescent soft nano-sized biological imaging agents. Various soft fluorescent nanoparticles (NPs) (including dye-doped polymer NPs, semiconducting polymer NPs, small-molecule organic NPs, nanogels, micelles, vesicles, and biomaterial-based NPs) are summarized from the perspectives of preparation methods, structure, optical properties, and surface functionalization. Based on both optical and functional properties of the nano-sized imaging agents, their applications are then reviewed in terms of in vitro imaging, in vivo imaging, and cellular-process imaging, by means of specific or nonspecific targeting.

  10. Soft fluorescent nanomaterials for biological and biomedical imaging

    Science.gov (United States)

    Peng, Hong-Shang; Chiu, Daniel T.

    2015-01-01

    Soft fluorescent nanomaterials have attracted recent attention as imaging agents for biological applications, because they provide the advantages of good biocompatibility, high brightness, and easy biofunctionalization. Here, we provide a survey of recent developments in fluorescent soft nano-sized biological imaging agents. Various soft fluorescent nanoparticles (NPs) (including dye-doped polymer NPs, semiconducting polymer NPs, small-molecule organic NPs, nanogels, micelles, vesicles, and biomaterial-based NPs) are summarized from the perspectives of preparation method, structure, optical property, and surface functionalization. Based on both optical and functional properties of the nano-sized imaging agents, their applications are then reviewed in terms of in vitro imaging, in vivo imaging, and cellular-process imaging, by means of specific or nonspecific targeting. PMID:25531691

  11. 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.

  12. Fluorescence Image Analyzer - FLIMA: software for quantitative analysis of fluorescence in situ hybridization.

    Science.gov (United States)

    Silva, H C M; Martins-Júnior, M M C; Ribeiro, L B; Matoso, D A

    2017-03-30

    The Fluorescence Image Analyzer (FLIMA) software was developed for the quantitative analysis of images generated by fluorescence in situ hybridization (FISH). Currently, the images of FISH are examined without a coefficient that enables a comparison between them. Through GD Graphics Library, the FLIMA software calculates the amount of pixels on image and recognizes each present color. The coefficient generated by the algorithm shows the percentage of marks (probes) hybridized on the chromosomes. This software can be used for any type of image generated by a fluorescence microscope and is able to quantify digoxigenin probes exhibiting a red color, biotin probes exhibiting a green color, and double-FISH probes (digoxigenin and biotin used together), where the white color is displayed.

  13. Near-infrared fluorescence imaging for colonic cancer detection

    Science.gov (United States)

    Shao, Xiaozhuo; Mo, Jianhua; Zheng, Wei; Huang, Zhiwei

    2008-02-01

    Near-infrared (NIR) fluorescence imaging is a novel optical technique with an ability of probing larger volume of tissues or lesions located in deep tissue areas. An integrated fluorescence and reflectance imaging system was developed to evaluate its potential for cancer diagnosis. The results show that the NIR autofluorescence intensity of normal colon tissues is significantly higher than that of cancer, and the diagnostic accuracy of 92.8% can be achieved using NIR autofluorescence/reflectance imaging. This work demonstrates that NIR autofluorescence/NIR reflectance imaging technique has potential for colonic cancer diagnosis and detection.

  14. Ion beam induced fluorescence imaging in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Bettiol, Andrew A.; Mi, Zhaohong; Vanga, Sudheer Kumar; Chen, Ce-belle; Tao, Ye; Watt, Frank

    2015-04-01

    Imaging fluorescence generated by MeV ions in biological systems such as cells and tissue sections requires a high resolution beam (<100 nm), a sensitive detection system and a fluorescent probe that has a high quantum efficiency and low bleaching rate. For cutting edge applications in bioimaging, the fluorescence imaging technique needs to break the optical diffraction limit allowing for sub-cellular structure to be visualized, leading to a better understanding of cellular function. In a nuclear microprobe this resolution requirement can be readily achieved utilizing low beam current techniques such as Scanning Transmission Ion Microscopy (STIM). In recent times, we have been able to extend this capability to fluorescence imaging through the development of a new high efficiency fluorescence detection system, and through the use of new novel fluorescent probes that are resistant to ion beam damage (bleaching). In this paper we demonstrate ion beam induced fluorescence imaging in several biological samples, highlighting the advantages and challenges associated with using this technique.

  15. A study on a portable fluorescence imaging system

    Science.gov (United States)

    Chang, Han-Chao; Wu, Wen-Hong; Chang, Chun-Li; Huang, Kuo-Cheng; Chang, Chung-Hsing; Chiu, Shang-Chen

    2011-09-01

    The fluorescent reaction is that an organism or dye, excited by UV light (200-405 nm), emits a specific frequency of light; the light is usually a visible or near infrared light (405-900 nm). During the UV light irradiation, the photosensitive agent will be induced to start the photochemical reaction. In addition, the fluorescence image can be used for fluorescence diagnosis and then photodynamic therapy can be given to dental diseases and skin cancer, which has become a useful tool to provide scientific evidence in many biomedical researches. However, most of the methods on acquiring fluorescence biology traces are still stay in primitive stage, catching by naked eyes and researcher's subjective judgment. This article presents a portable camera to obtain the fluorescence image and to make up a deficit from observer competence and subjective judgment. Furthermore, the portable camera offers the 375nm UV-LED exciting light source for user to record fluorescence image and makes the recorded image become persuasive scientific evidence. In addition, when the raising the rate between signal and noise, the signal processing module will not only amplify the fluorescence signal up to 70 %, but also decrease the noise significantly from environmental light on bill and nude mouse testing.

  16. Chlorophyll Fluorescence Imaging Uncovers Photosynthetic Fingerprint of Citrus Huanglongbing

    Directory of Open Access Journals (Sweden)

    Haiyan Cen

    2017-08-01

    Full Text Available Huanglongbing (HLB is one of the most destructive diseases of citrus, which has posed a serious threat to the global citrus production. This research was aimed to explore the use of chlorophyll fluorescence imaging combined with feature selection to characterize and detect the HLB disease. Chlorophyll fluorescence images of citrus leaf samples were measured by an in-house chlorophyll fluorescence imaging system. The commonly used chlorophyll fluorescence parameters provided the first screening of HLB disease. To further explore the photosynthetic fingerprint of HLB infected leaves, three feature selection methods combined with the supervised classifiers were employed to identify the unique fluorescence signature of HLB and perform the three-class classification (i.e., healthy, HLB infected, and nutrient deficient leaves. Unlike the commonly used fluorescence parameters, this novel data-driven approach by using the combination of the mean fluorescence parameters and image features gave the best classification performance with the accuracy of 97%, and presented a better interpretation for the spatial heterogeneity of photochemical and non-photochemical components in HLB infected citrus leaves. These results imply the potential of the proposed approach for the citrus HLB disease diagnosis, and also provide a valuable insight for the photosynthetic response to the HLB disease.

  17. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    Science.gov (United States)

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

  18. Computational framework for simulating fluorescence microscope images with cell populations.

    Science.gov (United States)

    Lehmussola, Antti; Ruusuvuori, Pekka; Selinummi, Jyrki; Huttunen, Heikki; Yli-Harja, Olli

    2007-07-01

    Fluorescence microscopy combined with digital imaging constructs a basic platform for numerous biomedical studies in the field of cellular imaging. As the studies relying on analysis of digital images have become popular, the validation of image processing methods used in automated image cytometry has become an important topic. Especially, the need for efficient validation has arisen from emerging high-throughput microscopy systems where manual validation is impractical. We present a simulation platform for generating synthetic images of fluorescence-stained cell populations with realistic properties. Moreover, we show that the synthetic images enable the validation of analysis methods for automated image cytometry and comparison of their performance. Finally, we suggest additional usage scenarios for the simulator. The presented simulation framework, with several user-controllable parameters, forms a versatile tool for many kinds of validation tasks, and is freely available at http://www.cs.tut.fi/sgn/csb/simcep.

  19. [Study of applying fluorescence spectrum imaging to quantitative assay of proteins in bio-chip].

    Science.gov (United States)

    Chao, Ke-Fu; Zhang, You-Lin; Kong, Xiang-Gui; Li, Bing; Zeng, Qing-Hui; Song, Kai; Sun, Ya-Juan

    2008-07-01

    In the present work, the amount and the activity of the goat anti-human IgG, to bind the human IgG labelled with fluorescein isothiocyanate (FITC), immobilized on silicon surfaces modified with APTES and APTES-Glu, respectively, were studied using the fluorescence spectrum imaging (FSI), the results of which were compared with that of ellipsometry. It is shown that the amount of the human IgG labeled with FITC on APTES-Glu measured using FSI is 2.8 times higher than that on APTES, which is nearly coincident with the 2.2 times obtained using ellipsometry, showing that the activity of the goat anti-human IgG on APTES-Glu is higher than that on APTES. It is reasoned that the FSI is used in the fluorescence immunoassay the for measurement of quasi-quantification or quantification.

  20. 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.

  1. 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.

  2. Design of a fluorescence-lifetime imaging microscope workstation

    Science.gov (United States)

    Boddeke, Frank R.; van Geest, Lambertus K.; Young, Ian T.

    1997-05-01

    One of the promising recent developments in fluorescence microscopy is fluorescence lifetime imaging microscopy. This type of microscopy images the lifetime of fluorescence molecules (in the nano second range) rather than the amount of light emitted by these molecules. This physical property is of interest while it gives information about the local environment of the molecule, such as molecular concentration of O2, Ca2+, pH, and conjugation. Our goal is to design a affordable, robust and easy-to-use FLIM workstation which is completely automated and does not need any difficult calibration. Therefore we are developing a workstation which applies a homodyne detection scheme (frequency range: 1 - 100 MHz) with use of an intensity modulated laser-diode (635 nm) and a gain modulated intensified CCD camera to image fluorescence lifetimes in the range of 1 - 100 ns. Using these components it is possible to make a FLIM workstation based on a normal fluorescence microscope by just replacing the light source and image detector. The FLIM image acquisition procedure in software allows automatic optical measurements of fluorescence lifetimes in different ranges and mixtures of lifetimes by adjusting the modulation frequency.

  3. Wide Field-of-View Fluorescence Imaging of Coral Reefs

    Science.gov (United States)

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

    2015-01-01

    Coral reefs globally are declining rapidly because of both local and global stressors. Improved monitoring tools are urgently needed to understand the changes that are occurring at appropriate temporal and spatial scales. Coral fluorescence imaging tools have the potential to improve both ecological and physiological assessments. Although fluorescence imaging is regularly used for laboratory studies of corals, it has not yet been used for large-scale in situ assessments. Current obstacles to effective underwater fluorescence surveying include limited field-of-view due to low camera sensitivity, the need for nighttime deployment because of ambient light contamination, and the need for custom multispectral narrow band imaging systems to separate the signal into meaningful fluorescence bands. Here we describe the Fluorescence Imaging System (FluorIS), based on a consumer camera modified for greatly increased sensitivity to chlorophyll-a fluorescence, and we show high spectral correlation between acquired images and in situ spectrometer measurements. This system greatly facilitates underwater wide field-of-view fluorophore surveying during both night and day, and potentially enables improvements in semi-automated segmentation of live corals in coral reef photographs and juvenile coral surveys. PMID:25582836

  4. Imaging tumor hypoxia by near-infrared fluorescence tomography.

    Science.gov (United States)

    Biswal, Nrusingh C; Pavlik, Christopher; Smith, Michael B; Aguirre, Andres; Xu, Yan; Zanganeh, Saeid; Kuhn, Liisa T; Claffey, Kevin P; Zhu, Quing

    2011-06-01

    We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.

  5. 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.

  6. Fluorescence Lifetime Imaging Microscopy (FLIM Data Analysis with TIMP

    Directory of Open Access Journals (Sweden)

    Sergey Laptenok

    2007-01-01

    Full Text Available Fluorescence Lifetime Imaging Microscopy (FLIM allows fluorescence lifetime images of biological objects to be collected at 250 nm spatial resolution and at (sub-nanosecond temporal resolution. Often ncomp kinetic processes underlie the observed fluorescence at all locations, but the intensity of the fluorescence associated with each process varies per-location, i.e., per-pixel imaged. Then the statistical challenge is global analysis of the image: use of the fluorescence decay in time at all locations to estimate the ncomp lifetimes associated with the kinetic processes, as well as the amplitude of each kinetic process at each location. Given that typical FLIM images represent on the order of 102 timepoints and 103 locations, meeting this challenge is computationally intensive. Here the utility of the TIMP package for R to solve parameter estimation problems arising in FLIM image analysis is demonstrated. Case studies on simulated and real data evidence the applicability of the partitioned variable projection algorithm implemented in TIMP to the problem domain, and showcase options included in the package for the visual validation of models for FLIM data.

  7. High-Resolution Fluorescence Microscope Imaging of Erythroblast Structure.

    Science.gov (United States)

    Smith, Alyson S; Nowak, Roberta B; Fowler, Velia M

    2018-01-01

    During erythropoiesis, erythroblasts undergo dramatic morphological changes to produce mature erythrocytes. Many unanswered questions regarding the molecular mechanisms behind these changes can be addressed with high-resolution fluorescence imaging. Immunofluoresence staining enables localization of specific molecules, organelles, and membrane components in intact cells at different phases of erythropoiesis. Confocal laser scanning microscopy can provide high-resolution, three-dimensional images of stained structures, which can be used to dissect the molecular mechanisms driving erythropoiesis. The sample preparation, staining procedure, imaging parameters, and image analysis methods used directly affect the quality of the confocal images and the amount and accuracy of information that they can provide. Here, we describe methods to dissect erythropoietic tissues from mice, to perform immunofluorescence staining and confocal imaging of various molecules, organelles and structures of interest in erythroblasts, and to present and quantitatively analyze the data obtained in these fluorescence images.

  8. 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.

  9. Dissociation of internal energy-selected methyl bromide ion revealed from threshold photoelectron-photoion coincidence velocity imaging

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Xiaofeng [Hefei National Laboratory for Physical Sciences at the Microscale and Department of Chemical Physics, University of Science and Technology of China, Hefei 230026 (China); National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029 (China); Zhou, Xiaoguo, E-mail: xzhou@ustc.edu.cn, E-mail: yanbing@jlu.edu.cn; Liu, Shilin [Hefei National Laboratory for Physical Sciences at the Microscale and Department of Chemical Physics, University of Science and Technology of China, Hefei 230026 (China); Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China); Sun, Zhongfa [Hefei National Laboratory for Physical Sciences at the Microscale and Department of Chemical Physics, University of Science and Technology of China, Hefei 230026 (China); Liu, Fuyi; Sheng, Liusi [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029 (China); Yan, Bing, E-mail: xzhou@ustc.edu.cn, E-mail: yanbing@jlu.edu.cn [Institute of Atomic and Molecular Physics, Jilin University, Changchun 130012 (China)

    2014-01-28

    Dissociative photoionization of methyl bromide (CH{sub 3}Br) in an excitation energy range of 10.45–16.90 eV has been investigated by using threshold photoelectron-photoion coincidence (TPEPICO) velocity imaging. The coincident time-of-flight mass spectra indicate that the ground state X{sup 2}E of CH{sub 3}Br{sup +} is stable, and both A{sup 2}A{sub 1} and B{sup 2}E ionic excited states are fully dissociative to produce the unique fragment ion of CH{sub 3}{sup +}. From TPEPICO 3D time-sliced velocity images of CH{sub 3}{sup +} dissociated from specific state-selected CH{sub 3}Br{sup +} ion, kinetic energy release distribution (KERD) and angular distribution of CH{sub 3}{sup +} fragment ion are directly obtained. Both spin-orbit states of Br({sup 2}P) atom can be clearly observed in fast dissociation of CH{sub 3}Br{sup +}(A{sup 2}A{sub 1}) ion along C–Br rupture, while a KERD of Maxwell-Boltzmann profile is obtained in dissociation of CH{sub 3}Br{sup +}(B{sup 2}E) ion. With the aid of the re-calculated potential energy curves of CH{sub 3}Br{sup +} including spin-orbit coupling, dissociation mechanisms of CH{sub 3}Br{sup +} ion in A{sup 2}A{sub 1} and B{sup 2}E states along C–Br rupture are revealed. For CH{sub 3}Br{sup +}(A{sup 2}A{sub 1}) ion, the CH{sub 3}{sup +} + Br({sup 2}P{sub 1/2}) channel is occurred via an adiabatic dissociation by vibration, while the Br({sup 2}P{sub 3/2}) formation is through vibronic coupling to the high vibrational level of X{sup 2}E state followed by rapid dissociation. C–Br bond breaking of CH{sub 3}Br{sup +}(B{sup 2}E) ion can occur via slow internal conversion to the excited vibrational level of the lower electronic states and then dissociation.

  10. 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.

  11. Biomedical imaging of colorectal cancer by near infrared fluorescent nanoparticles.

    Science.gov (United States)

    Tivony, Ran; Larush, Liraz; Sela-Tavor, Osnat; Magdassi, Shlomo

    2014-06-01

    In this paper we describe the preparation of novel Near Infrared (NIR) fluorescent nanoparticles for application in medical imaging of colorectal tumors. The nanoparticles are prepared by using only non-covalent binding processes of molecules which are approved for clinical use. The preparation process is based on the precipitation of a polycation, Eudragit-RS, followed by sequential adsorption of a blocking protein, sodium caseinate, NIR fluorescent dye, Indocyanine Green (ICG) and optionally, a targeting molecule, anti-CEA antibody. Fluorescence measurements have shown that these nanoparticles have higher resistance to photobleaching and higher quantum yield relatively to free ICG. Imaging experiments in orthotopic colorectal cancer mice models have shown that these fluorescent nanoparticles are capable of binding to LS174T human colon tumors in vivo with high specificity, even without the targeting molecule. These nanoparticles, composed of all FDA approved materials, open the way to clinical bioimaging and diagnostics of colon cancer.

  12. Near-Infrared Fluorescent Nanoprobes for in Vivo Optical Imaging

    Directory of Open Access Journals (Sweden)

    Chai-Hoon Quek

    2012-03-01

    Full Text Available Near-infrared (NIR fluorescent probes offer advantages of high photon penetration, reduced light scattering and minimal autofluorescence from living tissues, rendering them valuable for noninvasive mapping of molecular events, assessment of therapeutic efficacy, and monitoring of disease progression in animal models. This review provides an overview of the recent development of the design and optical property of the different classes of NIR fluorescent nanoprobes associated with in vivo imaging applications.

  13. Near-Infrared Fluorescent Nanoprobes for in Vivo Optical Imaging

    Science.gov (United States)

    Quek, Chai-Hoon; Leong, Kam W.

    2012-01-01

    Near-infrared (NIR) fluorescent probes offer advantages of high photon penetration, reduced light scattering and minimal autofluorescence from living tissues, rendering them valuable for noninvasive mapping of molecular events, assessment of therapeutic efficacy, and monitoring of disease progression in animal models. This review provides an overview of the recent development of the design and optical property of the different classes of NIR fluorescent nanoprobes associated with in vivo imaging applications. PMID:28348298

  14. Modulated electron-multiplied fluorescence lifetime imaging microscope : All-solid-state camera for fluorescence lifetime imaging

    NARCIS (Netherlands)

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

    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

  15. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

    OpenAIRE

    Icha, Jaroslav; Schmied, Christopher; Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized data...

  16. 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.

  17. Ion-coincidence momentum imaging of three-body Coulomb explosion of formaldehyde in ultrashort intense laser fields

    Energy Technology Data Exchange (ETDEWEB)

    Fushitani, M.; Matsuda, A.; Hishikawa, A., E-mail: hishi@chem.nagoya-u.ac.jp [Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602 (Japan); Tseng, C.-M. [Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Aichi 444-8585 (Japan)

    2015-12-31

    Three-body Coulomb explosion of formaldehyde (H{sub 2}CO) in intense 7- and 35-fs laser fields (1.3 × 10{sup 15} W/cm{sup 2}) has been investigated by using ion-coincidence momentum imaging technique. Two types of explosion pathways from the triply charged state, H{sub 2}CO{sup 3+} → (i) H{sup +} + H{sup +} + CO{sup +} and (ii) H{sup +} + CH{sup +} + O{sup +}, have been identified. It is shown from the momentum correlation of the fragment ions of pathway (i), that the geometrical structure of the molecule is essentially frozen along the H-C-H bending coordinate for the 7-fs case. On the other hand, for a longer pulse duration (35 fs), structural deformation along the C-H stretching and H-C-H bending coordinates is identified, which is ascribed to the nuclear dynamics in the dication states populated within the laser pulse duration.

  18. 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

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

    Science.gov (United States)

    Warren, Sean C; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda; Dunsby, Chris; French, Paul M W

    2013-01-01

    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 of live cell

  20. A new electron-ion coincidence 3D momentum-imaging method and its application in probing strong field dynamics of 2-phenylethyl-N, N-dimethylamine

    Science.gov (United States)

    Fan, Lin; Lee, Suk Kyoung; Tu, Yi-Jung; Mignolet, Benoît; Couch, David; Dorney, Kevin; Nguyen, Quynh; Wooldridge, Laura; Murnane, Margaret; Remacle, Françoise; Schlegel, H. Bernhard; Li, Wen

    2017-07-01

    We report the development of a new three-dimensional (3D) momentum-imaging setup based on conventional velocity map imaging to achieve the coincidence measurement of photoelectrons and photo-ions. This setup uses only one imaging detector (microchannel plates (MCP)/phosphor screen) but the voltages on electrodes are pulsed to push both electrons and ions toward the same detector. The ion-electron coincidence is achieved using two cameras to capture images of ions and electrons separately. The time-of-flight of ions and electrons are read out from MCP using a digitizer. We demonstrate this new system by studying the dissociative single and double ionization of PENNA (2-phenylethyl-N,N-dimethylamine). We further show that the camera-based 3D imaging system can operate at 10 kHz repetition rate.

  1. Adaptive fluorescence microscopy by online feedback image analysis.

    Science.gov (United States)

    Tischer, Christian; Hilsenstein, Volker; Hanson, Kirsten; Pepperkok, Rainer

    2014-01-01

    Obtaining sufficient statistics in quantitative fluorescence microscopy is often hampered by the tedious and time-consuming task of manually locating comparable specimen and repeatedly launching the same acquisition protocol. Recent advances in combining fluorescence microscopy with online image analysis tackle this problem by fully integrating the task of identifying and locating the specimen of interest in an automated acquisition workflow. Here, we describe the general requirements and specific microscope control and image analysis software solutions for implementing such automated online feedback microscopy. We demonstrate the power of the method by two selected applications addressing high-throughput 3D imaging of sparsely parasite-infected tissue culture cells and automated fluorescence recovery after photobleaching experiments to quantify the turnover of vesicular coat proteins at ER exit sites. © 2014 Elsevier Inc. All rights reserved.

  2. 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.

  3. Led induced chlorophyll fluorescence transient imager for measurements of health and stress status of whole plants

    NARCIS (Netherlands)

    Jalink, H.; Schoor, van der R.

    2011-01-01

    We have developed LED (light emitting diode) induced fluorescence transient imaging instrumentation to image the plant health/stress status by calculation of two images: Fv/Fm (variable fluorescence over saturation level of fluorescence) and the time response, tTR, of the fluorescence time curve.

  4. APPLICATION OF MODULATED CHLOROPHYLL FLUORESCENCE AND MODULATED CHLOROPHYLL FLUORESCENCE IMAGING IN STUDYING ENVIRONMENTAL STRESSES EFFECT

    Directory of Open Access Journals (Sweden)

    L. Guidi

    2016-03-01

    Full Text Available Chlorophyll (Chl a fluorescence is a widely used tool to monitor the photosynthetic process in plants subjected to environmental stresses.this review reports the theoretical bases of Chl fluorescence, and the significance of the most important Chl fluorescence parameters. it also reportshow these parameters can be utilised to estimate changes in photosystem ii (PSII photochemistry, linear electron flux and energy dissipationmechanisms. the relation between actual PSII photochemistry and CO2 assimilation is discussed, as is the role of photochemical andnon-photochemical quenching in inducing changes in PSII activity. the application of Chl fluorescence imaging to study heterogeneity on leaflamina is also considered. this review summarises only some of the results obtained by this methodology to study the effects of differentenvironmental stresses, namely water and nutrients availability, pollutants, temperature and salinity.

  5. 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.

  6. Refractive index sensing using Fluorescence Lifetime Imaging (FLIM)

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Carolyn; Suhling, Klaus [Department of Physics, King' s College London, Strand, London, WC2R 2LS (United Kingdom)

    2006-07-15

    The fluorescence lifetime is a function of the refractive index of the fluorophore's environment, for example in the case of the biologically important green fluorescent protein (GFP). In order to address the question whether this effect can be exploited to image the local environment of specific proteins in cell biology, we need to determine the distance over which the fluorophore's lifetime is sensitive to the refractive index. To this end, we employ Fluorescence Lifetime Imaging (FLIM) of fluorescein in NaOH buffer at an interface. This approach allows us to map the fluorescence lifetime as a function of distance from a buffer/air and buffer/oil interface. Preliminary data show that the fluorescence lifetime of fluorescein increases near a buffer/air interface and decreases near a buffer/oil interface. The range over which this fluorescence lifetime change occurs is found to be of the order several {mu}m which is consistent with a theoretical model based on the full width at half maximum of the emission spectrum proposed by Toptygin.

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

    Science.gov (United States)

    Ranjan Gartia, Manas; Hsiao, Austin; Sivaguru, Mayandi; Chen, Yi; Logan Liu, G.

    2011-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-07

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

  9. Optofluidic fluorescent imaging cytometry on a cell phone.

    Science.gov (United States)

    Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

    2011-09-01

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in

  10. Towards whole-body fluorescence imaging in humans.

    Directory of Open Access Journals (Sweden)

    Sophie K Piper

    Full Text Available Dynamic near-infrared fluorescence (DNIF whole-body imaging of small animals has become a popular tool in experimental biomedical research. In humans, however, the field of view has been limited to body parts, such as rheumatoid hands, diabetic feet or sentinel lymph nodes. Here we present a new whole-body DNIF-system suitable for adult subjects. We explored whether this system (i allows dynamic whole-body fluorescence imaging and (ii can detect modulations in skin perfusion. The non-specific fluorescent probe indocyanine green (ICG was injected intravenously into two subjects, and fluorescence images were obtained at 5 Hz. The in- and out-flow kinetics of ICG have been shown to correlate with tissue perfusion. To validate the system, skin perfusion was modulated by warming and cooling distinct areas on the chest and the abdomen. Movies of fluorescence images show a bolus passage first in the face, then in the chest, abdomen and finally in the periphery (~10, 15, 20 and 30 seconds, respectively. When skin perfusion is augmented by warming, bolus arrives about 5 seconds earlier than when the skin is cooled and perfusion decreased. Calculating bolus arrival times and spatial fitting of basis time courses extracted from different regions of interest allowed a mapping of local differences in subcutaneous skin perfusion. This experiment is the first to demonstrate the feasibility of whole-body dynamic fluorescence imaging in humans. Since the whole-body approach demonstrates sensitivity to circumscribed alterations in skinperfusion, it may be used to target autonomous changes in polyneuropathy and to screen for peripheral vascular diseases.

  11. Simultaneous single molecule atomic force and fluorescence lifetime imaging

    Science.gov (United States)

    Schulz, Olaf; Koberling, Felix; Walters, Deron; Koenig, Marcelle; Viani, Jacob; Ros, Robert

    2010-02-01

    The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

  12. Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging.

    Science.gov (United States)

    Yamada, Arisa; Hiruta, Yuki; Wang, Jian; Ayano, Eri; Kanazawa, Hideko

    2015-08-10

    We report the development of environmentally responsive fluorescent polymers. The reversible temperature-induced phase transition of copolymers composed of N-isopropylacrylamide and a fluorescent monomer based on the fluorescein (FL), coumarin (CO), rhodamine (RH), or dansyl (DA) skeleton was used as a molecular switch to control the fluorescence intensity. The poly(N-isopropylacrylamide) (PNIPAAm) chain showed an expanded coil conformation below the lower critical solution temperature (LCST) due to hydration, but it changed to a globular form above the LCST due to dehydration. Through the combination of a polarity-sensitive fluorophore with PNIPAAm, the synthetic fluorescent polymer displayed a response to external temperature, with the fluorescence strength dramatically changing close to the LCST. Additionally, the P(NIPAAm-co-FL) and P(NIPAAm-co-CO) polymers, containing fluorescein and coumarin groups, respectively, exhibited pH responsiveness. The environmental responsiveness of the reported polymers is derived directly from the PNIPAAm and fluorophore structures, thus allowing for the cellular uptake of the fluorescence copolymer by RAW264.7 cells to be temperature-controlled. Cellular uptake was suppressed below the LCST but enhanced above the LCST. Furthermore, the cellular uptake of both P(NIPAAm-co-CO) and P(NIPAAm-co-RH) conjugated with a fusogenic lipid, namely, l-α-phosphatidylethanolamine, dioleoyl (DOPE), was enhanced. Such lipid-conjugated fluorescence probes are expected to be useful as physiological indicators for intracellular imaging.

  13. PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides

    Science.gov (United States)

    De Angelis, Dino A.; Miesenböck, Gero; Zemelman, Boris V.; Rothman, James E.

    1998-01-01

    We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces. PMID:9770483

  14. Fluorescence microscope by using computational ghost imaging

    Directory of Open Access Journals (Sweden)

    Mizutani Yasuhiro

    2015-01-01

    Full Text Available We propose a fluorescence microscope by using the computational Ghost imaging (CGI for observing a living cell for a long duration over an hour. There is a problem for observing a cell about light-induced bleaching fora ling-term observation.Toover come the problem, we focused on an advantageof sensitivityof the CGI as second order colleration for an imaging with weak intensity excitation light. Setting for the CGI, a DMD projector was installed at an eye-piece part of a microscope and fluorescent light was detected using a bucket detectorofa photo-multiplier tube.Asaresults,wehaveshownthe imagingadvantageoftheCGI under weak light intensity, in addition, we have demonstrated to detect fluorescence images of biological samples for one day.

  15. The cyan fluorescent protein (CFP) transgenic mouse as a model for imaging pancreatic exocrine cells.

    Science.gov (United States)

    Tran Cao, Hop S; Kimura, Hiroaki; Kaushal, Sharmeela; Snyder, Cynthia S; Reynoso, Jose; Hoffman, Robert M; Bouvet, Michael

    2009-03-09

    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. To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. 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). 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. Determination of fluorescence intensity of different organs. 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. The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

  16. Coincident Aerosol and H2O Retrievals versus HSI Imager Field Campaign ReportH2O Retrievals versus HSI Imager Field Campaign Report

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Gail P. [National Oceanic and Atmospheric Administration (NOAA), Washington, DC (United States); Cipar, John [Air Force Research Lab. (AFRL), Wright-Patterson AFB, OH (United States); Armstrong, Peter S. [Air Force Research Lab. (AFRL), Wright-Patterson AFB, OH (United States); van den Bosch, J. [Air Force Research Lab. (AFRL), Wright-Patterson AFB, OH (United States)

    2016-05-01

    Two spectrally calibrated tarpaulins (tarps) were co-located at a fixed Global Positioning System (GPS) position on the gravel antenna field at the U.S. Department of Energy (DOE) Atmospheric Radiation Measurement (ARM) Climate Research Facility’s Southern Great Plains (SGP) site. Their placement was timed to coincide with the overflight of a new hyperspectral imaging satellite. The intention was to provide an analysis of the data obtained, including the measured and retrieved spectral albedos for the calibration tarps. Subsequently, a full suite of retrieved values of H2O column, and the aerosol overburden, were to be compared to those determined by alternate SGP ground truth assets. To the extent possible, the down-looking cloud images would be assessed against the all-sky images. Because cloud contamination above a certain level precludes the inversion processing of the satellite data, coupled with infrequent targeting opportunities, clear-sky conditions were imposed. The SGP site was chosen not only as a target of opportunity for satellite validation, but as perhaps the best coincident field measurement site, as established by DOE’s ARM Facility. The satellite team had every expectation of using the information obtained from the SGP to improve the inversion products for all subsequent satellite images, including the cloud and radiative models and parameterizations and, thereby, the performance assessment for subsequent and historic image collections. Coordinating with the SGP onsite team, four visits, all in 2009, to the Central Facility occurred: • June 6-8 (successful exploratory visit to plan tarp placements, etc.) • July 18-24 (canceled because of forecast for heavy clouds) • Sep 9-12 (ground tarps placed, onset of clouds) • Nov 7-9 (visit ultimately canceled because of weather predictions). As noted, in each instance, any significant overcast prediction precluded image collection from the satellite. Given the long task-scheduling procedures

  17. Applications of fluorescence lifetime imaging in clinical medicine

    Directory of Open Access Journals (Sweden)

    Zhanwen Wang

    2018-01-01

    Full Text Available Fluorescence lifetime is not only associated with the molecular structure of fluorophores, but also strongly depends on the environment around them, which allows fluorescence lifetime imaging microscopy (FLIM to be used as a tool for precise measurement of the cell or tissue microenvironment. This review introduces the basic principle of fluorescence lifetime imaging technology and its application in clinical medicine, including research and diagnosis of diseases in skin, brain, eyes, mouth, bone, blood vessels and cavity organs, and drug evaluation. As a noninvasive, nontoxic and nonionizing radiation technique, FLIM demonstrates excellent performance with high sensitivity and specificity, which allows to determine precise position of the lesion and, thus, has good potential for application in biomedical research and clinical diagnosis.

  18. Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

    Science.gov (United States)

    Storrie-Lombardi, M. C.; Douglas, S.; Sun, H.; McDonald, G. D.; Bhartia, R.; Nealson, K. H.; Hug, W. F.

    2001-01-01

    An interdisciplinary team at the Jet Propulsion Laboratory Center for Life Detection has embarked on a project to provide in situ chemical and morphological characterization of Antarctic cryptoendolithic microbial communities. We present here in situ deep ultraviolet (UV) native fluorescence and environmental scanning electron microscopy images transiting 8.5 mm into a sandstone sample from the Antarctic Dry Valleys. The deep ultraviolet imaging system employs 224.3, 248.6, and 325 nm lasers to elicit differential fluorescence and resonance Raman responses from biomolecules and minerals. The 224.3 and 248.6 nm lasers elicit a fluorescence response from the aromatic amino and nucleic acids. Excitation at 325 nm may elicit activity from a variety of biomolecules, but is more likely to elicit mineral fluorescence. The resultant fluorescence images provide in situ chemical and morphological maps of microorganisms and the associated organic matrix. Visible broadband reflectance images provide orientation against the mineral background. Environmental scanning electron micrographs provided detailed morphological information. The technique has made possible the construction of detailed fluorescent maps extending from the surface of an Antarctic sandstone sample to a depth of 8.5 mm. The images detect no evidence of microbial life in the superficial 0.2 mm crustal layer. The black lichen component between 0.3 and 0.5 mm deep absorbs all wavelengths of both laser and broadband illumination. Filamentous deep ultraviolet native fluorescent activity dominates in the white layer between 0.6 mm and 5.0 mm from the surface. These filamentous forms are fungi that continue into the red (iron-rich) region of the sample extending from 5.0 to 8.5 mm. Using differential image subtraction techniques it is possible to identify fungal nuclei. The ultraviolet response is markedly attenuated in this region, apparently from the absorption of ultraviolet light by iron-rich particles coating

  19. Polyester Fabric's Fluorescent Dyeing in Supercritical Carbon Dioxide and its Fluorescence Imaging.

    Science.gov (United States)

    Xiong, Xiaoqing; Xu, Yanyan; Zheng, Laijiu; Yan, Jun; Zhao, Hongjuan; Zhang, Juan; Sun, Yanfeng

    2017-03-01

    As one of the most important coumarin-like dyes, disperse fluorescent Yellow 82 exhibits exceptionally large two-photon effects. Here, it was firstly introduced into the supercritical CO2 dyeing polyester fabrics in this work. Results of the present work showed that the dyeing parameters such as the dyeing time, pressure and temperature had remarkable influences on the color strength of fabrics. The optimized dyeing condition in supercritical CO2 dyeing has been proposed that the dyeing time was 60 min; the pressure was 25 MPa and the temperature was 120 °C. As a result, acceptable products were obtained with the wash and rub fastness rating at 5 or 4-5. The polyester fabrics dyed with fluorescent dyes can be satisfied for the requirement of manufacturing warning clothing. Importantly, the confocal microscopy imaging technology was successfully introduced into textile fields to observe the distribution and fluorescence intensity of disperse fluorescent Yellow 82 on polyester fabrics. As far as we know, this is the first report about supercritical CO2 dyeing polyester fabrics based on disperse fluorescent dyes. It will be very helpful for the further design of new fluorescent functional dyes suitable for supercritical CO2 dyeing technique.

  20. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    Science.gov (United States)

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  1. Plant response to destruxins visualized by imaging of chlorophyll fluorescence

    Czech Academy of Sciences Publication Activity Database

    Soukupová, Julie; Smatanová, Sylvie; Nedbal, Ladislav; Jegorov, A.

    2003-01-01

    Roč. 118, č. 118 (2003), s. 399-405 ISSN 0031-9317 Institutional research plan: CEZ:MSM 123100001; CEZ:AV0Z6087904 Keywords : fungal infection, destruxins * fluorescence imaging Subject RIV: GF - Plant Pathology, Vermin, Weed, Plant Protection Impact factor: 1.767, year: 2003

  2. A Review of Indocyanine Green Fluorescent Imaging in Surgery

    Directory of Open Access Journals (Sweden)

    Jarmo T. Alander

    2012-01-01

    Full Text Available The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluorescence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in clinical setting are reviewed. In addition to the surgical applications, other recent medical applications of ICG are briefly examined.

  3. Synchrotron-based X-ray fluorescence, imaging and elemental ...

    Indian Academy of Sciences (India)

    Analysis of the small areas will be better suited to establish the physiology of metals in specific structures like small animal shell and the distribution of other elements. Keywords. X-ray fluorescence; synchrotron-based excitation; imaging; elemental mapping; biological samples. PACS Nos 78.70.En; 29.20.dk; 87.59.

  4. Colorful protein-based fluorescent probes for collagen imaging.

    Directory of Open Access Journals (Sweden)

    Stijn J A Aper

    Full Text Available Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488. The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

  5. Imaging Ca2+ with a Fluorescent Rhodol.

    Science.gov (United States)

    Contractor, Alisha A; Miller, Evan W

    2018-01-16

    Ca2+ mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca2+-sensitive fluorophores has played an instrumental role in our understanding of the temporal and spatial dynamics of Ca2+. Coupling Ca2+-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca2+ affinities suited to a variety of cellular contexts. One underdeveloped area is the use of hybrid rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca2+ sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σTPA), making them excellent candidates for incorporation into Ca2+-sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca2+ sensor 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca2+ binding constant of 240 nM and a 10-fold turn response to Ca2+. RCS-1 effectively absorbs infrared light and has a σTPA of 76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca2+ fluctuations and cultured neurons using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca2+ sensing using two-photon illumination in neurons.

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

    Science.gov (United States)

    Kaifosh, Patrick; Zaremba, Jeffrey D.; Danielson, Nathan B.; Losonczy, Attila

    2014-01-01

    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/. PMID:25295002

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

    Science.gov (United States)

    Kaifosh, Patrick; Zaremba, Jeffrey D; Danielson, Nathan B; Losonczy, Attila

    2014-01-01

    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/.

  8. Fluorescence Lifetime Imaging System for in Vivo Studies

    Directory of Open Access Journals (Sweden)

    Moinuddin Hassan

    2007-07-01

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

  9. Linear and non-linear fluorescence imaging of neuronal activity

    Science.gov (United States)

    Fisher, Jonathan A. N.

    Optical imaging of neuronal activity offers new possibilities for understanding brain physiology. The predominant methods in neuroscience for measuring electrical activity require electrodes inserted into the tissue. Such methods, however, provide limited spatial information and are invasive. Optical methods are less physically invasive and offer the possibility for simultaneously imaging the activity of many neurons. In this thesis one- and two-photon fluorescence microscopy techniques were applied to several in vivo and in vitro mammalian preparations. Using one-photon absorption fluorescence microscopy and gradient index (GRIN) lens optics, cortical electrical activity in response to electric stimulation was resolved in three-dimensions at high-speed in the primary somatosensory cortex of the mouse in vivo using voltage-sensitive dyes. Imaging at depths up to 150 mum below the cortex surface, it was possible to resolve depth-dependent patterns of neuronal activity in response to cortical and thalamic electric stimulation. The patterns of activity were consistent with known cortical cellular architecture. In a qualitatively different set of experiments, one-photon fluorescence microscopy via voltage-sensitive dyes was successfully employed to image an in vitro preparation of the perfused rat brainstem during the process of respiratory rhythmogenesis. Imaging results yielded insights into the spatial organization of the central respiratory rhythm generation region in the ventrolateral medulla. A multifocal two-photon scanning microscope was constructed, and design and operation principles are described. Utilizing the novel device, anatomical and functional two-photon imaging via potentiometric dyes and calcium dyes is described, and the results of in vivo versus in vitro imaging are compared. Anatomical imaging results used either functional probe background fluorescence or green fluorescent protein (GFP) expression. Spectroscopic experiments measuring the two

  10. Near infrared fluorescence imaging of rabbit thyroid and parathyroid glands.

    Science.gov (United States)

    Antakia, Ramez; Gayet, Pascal; Guillermet, Stephanie; Stephenson, Tim J; Brown, Nicola J; Harrison, Barney J; Balasubramanian, Saba P

    2014-12-01

    Near infrared fluorescence imaging using intravenous methylene blue (MB) is a novel technique that has potential to aid the parathyroid gland (PG) localization during thyroid and parathyroid surgery. The aim of this study was to examine MB fluorescence in the rabbit neck and determine the influence of MB dose and time following administration on fluorescence from thyroid and PGs. Thyroid and external PGs were exposed in six New Zealand white rabbits under anesthesia. Varying doses of MB (0.025-3 mg/kg) were injected through the marginal ear vein. Near infrared fluorescence from exposed tissues was recorded at different time intervals (10-74 min) using Fluobeam 700. Specimens of identified glands were then resected for histologic assessment. Histology confirmed accurate identification of all excised thyroid and PGs; these were the only neck structures to demonstrate significant fluorescence. The parathyroid demonstrated lower fluorescence intensities and reduced washout times at all MB doses compared with the thyroid gland. A dose of 0.1 mg/kg MB was adequate to identify fluorescence; this also delineated the blood supply of the external PGs. The study demonstrates that near infrared fluorescence with intravenous MB helps differentiate between thyroid and PGs in the rabbit. This has potential to improve outcomes in thyroid and parathyroid surgery by increasing the accuracy of parathyroid identification; however, the findings require replication in human surgery. The use of low doses of MB may also avoid the side effects associated with currently used doses in humans (3-7 mg/kg). Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Multiresolution Multiscale Active Mask Segmentation of Fluorescence Microscope Images.

    Science.gov (United States)

    Srinivasa, Gowri; Fickus, Matthew; Kovačević, Jelena

    2009-01-01

    We propose an active mask segmentation framework that combines the advantages of statistical modeling, smoothing, speed and flexibility offered by the traditional methods of region-growing, multiscale, multiresolution and active contours respectively. At the crux of this framework is a paradigm shift from evolving contours in the continuous domain to evolving multiple masks in the discrete domain. Thus, the active mask framework is particularly suited to segment digital images. We demonstrate the use of the framework in practice through the segmentation of punctate patterns in fluorescence microscope images. Experiments reveal that statistical modeling helps the multiple masks converge from a random initial configuration to a meaningful one. This obviates the need for an involved initialization procedure germane to most of the traditional methods used to segment fluorescence microscope images. While we provide the mathematical details of the functions used to segment fluorescence microscope images, this is only an instantiation of the active mask framework. We suggest some other instantiations of the framework to segment different types of images.

  12. 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.

  13. Compressive fluorescence microscopy for biological and hyperspectral imaging.

    Science.gov (United States)

    Studer, Vincent; Bobin, Jérome; Chahid, Makhlad; Mousavi, Hamed Shams; Candes, Emmanuel; Dahan, Maxime

    2012-06-26

    The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy.

  14. Registering plant dysfunction in artificial biosystems through fluorescence imaging technique

    Science.gov (United States)

    Nikolova, Alexandra; Krumov, Alexandar; Vassilev, Vesselin

    Humanity ambitions in space exploration and long-term men-operated space missions evoke an increasing interest to artificial ecosystem researches. Advanced studies of plant biosystems provoke development of new innovative technologies for plant cultivation in man-made environment. Closed ecosystems of different types and structure are now used for space horticulture, cultivation of genetically modified species, bio-products for pharmacies and industry etc. New technologies are required to monitor and control basic parameters of future bioregenerative life support system, especially of plants photosynthetic activity as the most fundamental biological process. Authors propose a conception for a non-invasive control of plant physiological status in closed biosystem through spatial registration of chlorophyll fluorescence. This approach allows an early detection of stress impact on plants, reveal the dynamic and direction of the negative influence and the level of plant stress. Technical requirements for obtaining plant fluorescence images are examined in close relation with plant illumination conditions. Problems related with optimised plant illumination are discussed. Examples of fluorescence images of healthy and stressed plants demonstrate the sensibility and rapidity of signal changes caused by plant dysfunction. Proposed conception could be used for developing new technical solutions in autocontrolled bio-support systems, based on real time analysis of fluorescence images.

  15. Fast automatic quantitative cell replication with fluorescent live cell imaging

    Directory of Open Access Journals (Sweden)

    Wang Ching-Wei

    2012-01-01

    Full Text Available Abstract Background live cell imaging is a useful tool to monitor cellular activities in living systems. It is often necessary in cancer research or experimental research to quantify the dividing capabilities of cells or the cell proliferation level when investigating manipulations of the cells or their environment. Manual quantification of fluorescence microscopic image is difficult because human is neither sensitive to fine differences in color intensity nor effective to count and average fluorescence level among cells. However, auto-quantification is not a straightforward problem to solve. As the sampling location of the microscopy changes, the amount of cells in individual microscopic images varies, which makes simple measurement methods such as the sum of stain intensity values or the total number of positive stain within each image inapplicable. Thus, automated quantification with robust cell segmentation techniques is required. Results An automated quantification system with robust cell segmentation technique are presented. The experimental results in application to monitor cellular replication activities show that the quantitative score is promising to represent the cell replication level, and scores for images from different cell replication groups are demonstrated to be statistically significantly different using ANOVA, LSD and Tukey HSD tests (p-value Conclusion A robust automated quantification method of live cell imaging is built to measure the cell replication level, providing a robust quantitative analysis system in fluorescent live cell imaging. In addition, the presented unsupervised entropy based cell segmentation for live cell images is demonstrated to be also applicable for nuclear segmentation of IHC tissue images.

  16. Quantum dots for fluorescent biosensing and bio-imaging applications.

    Science.gov (United States)

    Li, Jingjing; Zhu, Jun-Jie

    2013-05-07

    Quantum dots (QDs) have been facilitating the development of sensitive fluorescence biosensors over the past two decades due to their high quantum yield, narrow and tunable emission spectrum and good photostability. The new emerging QDs with improved biocompatibility further promote their biological applications. In this review, we first briefly introduce the prevalently used QDs and their preparation and bioconjugation approaches. Then we summarize QDs-based fluorescent biosensing for proteins and nucleic acids, and QDs-based applications in cellular and in vivo targeting and imaging. Last but not the least, we envision the potential QDs-based applications in future perspectives.

  17. In Vivo Fluorescence Imaging in the Second Near-Infrared Window Using Carbon Nanotubes.

    Science.gov (United States)

    Hong, Guosong; Dai, Hongjie

    2016-01-01

    In vivo fluorescence imaging in the second near-infrared window (NIR-II window, 1000-1700 nm) is a powerful imaging technique that emerged in recent years. This imaging tool allows for noninvasive, deep-tissue visualization and interrogation of anatomical features and functions with improved imaging resolution and contrast at greater tissue penetration depths than traditional fluorescence imaging. Here, we present the detailed protocol for conducting NIR-II fluorescence imaging in live animals, including the procedures for preparation of biocompatible and NIR-II fluorescent carbon nanotube solution, live animal administration and NIR-II fluorescence image acquisition.

  18. FLUORESCENT X-RAY IMAGING WITH PINHOLE CAMERA

    Directory of Open Access Journals (Sweden)

    Yu. I. Dudchik

    2016-01-01

    Full Text Available Pinhole camera is one of X-ray optics devices. The pinhole camera is used for obtaining the image of synchrotron and laboratory sources, and also as a lens in a method of X-ray fluorescent microscopy. This method allows to obtain information on a spatial distribution of various chemical elements to the areas of several square centimeters with spatial resolution at the level of 50–100 μm. As a rule energy-dispersive two-dimensional CCD cameras are used for imaging. Such cameras are expensive devices and have low sensitivity for X-rays with energy of photons higher than 8 keV. Therefore it is perspective to use for X-ray fluorescent microscopy more effective CCD cameras with a scintillator layer. The purpose of this work consists in development of the device for imaging with secondary fluorescent X-rays by using pinhole as a lens and CCD camera for registration of the X-ray image. The device for obtaining the image of objects in secondary X-rays is developed. The device consists of an X-ray tube, pinhole and CCD camera. The object of research was irradiated with radiation from the X-ray tube and emits secondary X-rays. 100-microns pinhole was used for formation of the image of the object at an entrance window of the CCD camera. Images of a number of the iron springs differing in the sizes are received. It is established that the spatial resolution of the device is about 200 μm at an exposition of 60 s. It is possible to improve permission of the device by increasing in an exposition, optimization imaging conditions and reduction of the pinhole size. 

  19. Clinical results of fluorescence lifetime imaging in ophthalmology

    Science.gov (United States)

    Schweitzer, D.; Quick, S.; Klemm, M.; Hammer, M.; Jentsch, S.; Dawczynski, J.; Becker, W.

    2009-07-01

    A laser scanner ophthalmoscope was developed for in vivo fluorescence lifetime measurements at the human retina. Measurements were performed in 30 degree fundus images. The fundus was excited by pulses of 75 ps (FWHM). The dynamic fluorescence was detected in two spectral channels K1(490-560nm), K2(560-700 nm) by time-correlated single photon counting. The decay of fluorescence was three-exponentially. Local and global alterations in lifetimes were found between healthy subjects and patients suffering from age-related macular degeneration, diabetic retinopathy, and vessel occlusion. The lifetimes T1, T2, and T3 in both channels are changed to longer values in AMD and diabetic retinopathy in comparison with healthy subjects. The lifetime T2 in K1 is most sensitive to metabolic alterations in branch arterial vessel occlusion.

  20. Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe.

    Science.gov (United States)

    Ali, Haydar; Bhunia, Susanta Kumar; Dalal, Chumki; Jana, Nikhil R

    2016-04-13

    Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes.

  1. Fluorescence lifetime imaging (FLIM) of membrane markers in living cells

    Science.gov (United States)

    Schneckenburger, Herbert; Wagner, Michael; Kretzschmar, Martina; Strauss, Wolfgang S.; Sailer, Reinhard

    2003-10-01

    An experimental setup for fluorescence lifetime imaging (FLIM) has been combined with total internal reflection fluorescence microscopy (TIRFM) in order to detect various membrane markers within living cells. The method is established using T47D human breast cancer cells transfected by a plasmid encoding for a membrane associated yellow fluorescent protein (EYFPmem). For further measurements the mitochondrial marker rhodamine 123 (R123) as well as the membrane marker laurdan are used. With increasing concentration R123 is accumulated outside the mitochondria, in particular within the plasma membrane, whereas mitochondrial fluorescence is quenched. Fluorescence lifetime of laurdan can be used to probe membrane dynamics, in particular the phase of membrane lipids. These lipids are in a rigid gel phase at temperatures around 24°C, whereas the gel phases and a liquid crystalline phase coexist at T >= 30°C. This phase pattern also depends on the age and the growth phase of the cells and may play a role in the uptake of pharmaceutical agents.

  2. Prostate cancer diagnosis with fluorescence lifetime imaging (Conference Presentation)

    Science.gov (United States)

    Sridharan, Shamira; Gandour-Edwards, Regina F.; Dall'Era, Marc; Marcu, Laura

    2017-02-01

    More than 1 million men in the United States undergo a prostate biopsy procedure annually and approximately 200,000 men receive a diagnosis of prostate cancer. 5-10% of these men have to undergo a repeat biopsy due to insufficient tissue sampling. We are studying the utility of a multi-spectral time resolved fluorescence spectroscopy (MS-TRFS) technique for real-time prostate cancer diagnosis. The MS-TRFS imaging setup, which includes a fiberoptic set-up with a 355nm excitation light source coupled with a blue (450nm) aiming beam, was used to image ex-vivo prostatectomy specimen. The prostate tissue from 11 patients was sectioned at 2mm thickness and the fluorescence lifetime information was overlaid spatially for histology and thus, diagnostic co-registration. Initial results show that fluorescence lifetime in the 390±40nm channel, which measures collagen and elastin signatures, is longer for glandular regions than in the stromal regions. Additionally, lifetime in the 452±45nm channel, corresponding to NAD redox state, is longer in the cancerous glandular region in comparison with the normal glandular regions. Current work is focused on developing real-time quantitative algorithms to combine the fluorescence signatures from the two channels for performing prostate cancer diagnosis on biopsies.

  3. Fluorescence imaging of dendritic spines of Golgi-Cox-stained neurons using brightening background

    Science.gov (United States)

    Ai, Min; Xiong, Hanqing; Yang, Tao; Shang, Zhenhua; Chen, Muqing; Liu, Xiuli; Zeng, Shaoqun

    2015-01-01

    We report a novel fluorescence imaging approach to imaging nonfluorescence-labeled biological tissue samples. The method was demonstrated by imaging neurons in Golgi-Cox-stained and epoxy-resin-embedded samples through the excitation of the background fluorescence of the specimens. The dark neurons stood out clearly against background fluorescence in the images, enabling the tracing of a single dendritic spine using both confocal and wide-field fluorescence microscopy. The results suggest that the reported fluorescence imaging method would provide an effective alternative solution to image nonfluorescence-labeled samples, and it allows tracing the dendritic spine structure of neurons.

  4. Proton-induced x-ray fluorescence CT imaging.

    Science.gov (United States)

    Bazalova-Carter, Magdalena; Ahmad, Moiz; Matsuura, Taeko; Takao, Seishin; Matsuo, Yuto; Fahrig, Rebecca; Shirato, Hiroki; Umegaki, Kikuo; Xing, Lei

    2015-02-01

    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. 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(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. A linear relationship between gold pXRF and gold concentration was observed in both experimental and MC simulation data (R(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. Proton-induced x-ray fluorescence CT imaging of 3%-5% gold solutions in a small animal sized water phantom has been demonstrated

  5. Directional bilateral filters for smoothing fluorescence microscopy images

    Science.gov (United States)

    Venkatesh, Manasij; Mohan, Kavya; Seelamantula, Chandra Sekhar

    2015-08-01

    Images obtained through fluorescence microscopy at low numerical aperture (NA) are noisy and have poor resolution. Images of specimens such as F-actin filaments obtained using confocal or widefield fluorescence microscopes contain directional information and it is important that an image smoothing or filtering technique preserve the directionality. F-actin filaments are widely studied in pathology because the abnormalities in actin dynamics play a key role in diagnosis of cancer, cardiac diseases, vascular diseases, myofibrillar myopathies, neurological disorders, etc. We develop the directional bilateral filter as a means of filtering out the noise in the image without significantly altering the directionality of the F-actin filaments. The bilateral filter is anisotropic to start with, but we add an additional degree of anisotropy by employing an oriented domain kernel for smoothing. The orientation is locally adapted using a structure tensor and the parameters of the bilateral filter are optimized for within the framework of statistical risk minimization. We show that the directional bilateral filter has better denoising performance than the traditional Gaussian bilateral filter and other denoising techniques such as SURE-LET, non-local means, and guided image filtering at various noise levels in terms of peak signal-to-noise ratio (PSNR). We also show quantitative improvements in low NA images of F-actin filaments.

  6. Directional bilateral filters for smoothing fluorescence microscopy images

    Directory of Open Access Journals (Sweden)

    Manasij Venkatesh

    2015-08-01

    Full Text Available Images obtained through fluorescence microscopy at low numerical aperture (NA are noisy and have poor resolution. Images of specimens such as F-actin filaments obtained using confocal or widefield fluorescence microscopes contain directional information and it is important that an image smoothing or filtering technique preserve the directionality. F-actin filaments are widely studied in pathology because the abnormalities in actin dynamics play a key role in diagnosis of cancer, cardiac diseases, vascular diseases, myofibrillar myopathies, neurological disorders, etc. We develop the directional bilateral filter as a means of filtering out the noise in the image without significantly altering the directionality of the F-actin filaments. The bilateral filter is anisotropic to start with, but we add an additional degree of anisotropy by employing an oriented domain kernel for smoothing. The orientation is locally adapted using a structure tensor and the parameters of the bilateral filter are optimized for within the framework of statistical risk minimization. We show that the directional bilateral filter has better denoising performance than the traditional Gaussian bilateral filter and other denoising techniques such as SURE-LET, non-local means, and guided image filtering at various noise levels in terms of peak signal-to-noise ratio (PSNR. We also show quantitative improvements in low NA images of F-actin filaments.

  7. Novel fluorescent carbonic nanomaterials for sensing and imaging

    Science.gov (United States)

    Demchenko, Alexander P.; Dekaliuk, Mariia O.

    2013-12-01

    Small brightly fluorescent carbon nanoparticles have emerged as a new class of materials important for sensing and imaging applications. We analyze comparatively the properties of nanodiamonds, graphene and graphene oxide ‘dots’, of modified carbon nanotubes and of diverse carbon nanoparticles known as ‘C-dots’ obtained by different methods. The mechanisms of their light absorption and luminescence emission are still unresolved and the arguments are presented for their common origin. Regarding present and potential applications, we provide critical comparison with the other types of fluorescence reporters, such as organic dyes and semiconductor quantum dots. Their most prospective applications in sensing (based on the changes of intensity, FRET and lifetime) and in imaging technologies on the level of living cells and whole bodies are overviewed. The possibilities for design on their basis of multifunctional nanocomposites on a broader scale of theranostics are outlined.

  8. Fluorescence microscopy imaging of electroperturbation in mammalian cells.

    Science.gov (United States)

    Sun, Yinghua; Vernier, P Thomas; Behrend, Matthew; Wang, Jingjing; Thu, Mya Mya; Gundersen, Martin; Marcu, Laura

    2006-01-01

    We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation--the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.

  9. Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors

    Science.gov (United States)

    Hosny, Neveen A.; Mohamedi, Graciela; Rademeyer, Paul; Owen, Joshua; Wu, Yilei; Tang, Meng-Xing; Eckersley, Robert J.; Stride, Eleanor; Kuimova, Marina K.

    2013-01-01

    Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a “molecular rotor” embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component. PMID:23690599

  10. Fluorescence resonance energy transfer imaging of CFP/YFP labeled NDH in cyanobacterium cell

    Energy Technology Data Exchange (ETDEWEB)

    Ji Dongmei [State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100080 (China); Lv Wei [State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100080 (China); Huang Zhengxi [State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100080 (China); Xia Andong [State Key Laboratory of Molecular Reaction Dynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100080 (China)]. E-mail: andong@iccas.ac.cn; Xu Min [Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai, 200031 (China); Ma Weimin [Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai, 200031 (China); Mi Hualing [Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai, 200031 (China)]. E-mail: mihl@iris.sipp.ac.cn; Ogawa Teruo [Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai, 200031 (China)]. E-mail: ogawater@xd6.so-net.ne.jp

    2007-01-15

    The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell.

  11. Development and integration of Raman imaging capabilities to Sandia National Laboratories hyperspectral fluorescence imaging instrument.

    Energy Technology Data Exchange (ETDEWEB)

    Timlin, Jerilyn Ann; Nieman, Linda T.

    2005-11-01

    Raman spectroscopic imaging is a powerful technique for visualizing chemical differences within a variety of samples based on the interaction of a substance's molecular vibrations with laser light. While Raman imaging can provide a unique view of samples such as residual stress within silicon devices, chemical degradation, material aging, and sample heterogeneity, the Raman scattering process is often weak and thus requires very sensitive collection optics and detectors. Many commercial instruments (including ones owned here at Sandia National Laboratories) generate Raman images by raster scanning a point focused laser beam across a sample--a process which can expose a sample to extreme levels of laser light and requires lengthy acquisition times. Our previous research efforts have led to the development of a state-of-the-art two-dimensional hyperspectral imager for fluorescence imaging applications such as microarray scanning. This report details the design, integration, and characterization of a line-scan Raman imaging module added to this efficient hyperspectral fluorescence microscope. The original hyperspectral fluorescence instrument serves as the framework for excitation and sample manipulation for the Raman imaging system, while a more appropriate axial transmissive Raman imaging spectrometer and detector are utilized for collection of the Raman scatter. The result is a unique and flexible dual-modality fluorescence and Raman imaging system capable of high-speed imaging at high spatial and spectral resolutions. Care was taken throughout the design and integration process not to hinder any of the fluorescence imaging capabilities. For example, an operator can switch between the fluorescence and Raman modalities without need for extensive optical realignment. The instrument performance has been characterized and sample data is presented.

  12. Fluorescent antitumor titanium(iv) salen complexes for cell imaging.

    Science.gov (United States)

    Tzubery, Avia; Melamed-Book, Naomi; Tshuva, Edit Y

    2018-02-16

    Two differently substituted fluorescent salen Ti(iv) complexes were developed. One was inactive on human cancer cells, whereas the other showed high cytotoxicity. Based on live cell imaging, both complexes penetrated the cell, but were not detected in the nuclei. Moreover, the inactive complex was trapped in endocytic vesicles, whereas the active complex accumulated in the perinuclear region and inflected phototoxicity upon continuous irradiation.

  13. Kinetic Requirements for Spatiotemporal Chemical Imaging with Fluorescent Nanosensors.

    Science.gov (United States)

    Meyer, Daniel; Hagemann, Annika; Kruss, Sebastian

    2017-04-25

    Fluorescent nanosensors are powerful tools for basic research and bioanalytical applications. Individual nanosensors are able to detect single molecules, while ensembles of nanosensors can be used to measure the bulk concentration of an analyte. Collective imaging of multiple nanosensors could provide both spatial and temporal chemical information from the nano- to the microscale. This type of chemical imaging with nanosensors would be very attractive to study processes such as chemical signaling between cells (e.g., neurons). So far, it is not understood what processes are resolvable (concentration, time, space) and how optimal sensors should be designed. Here, we develop a theoretical framework to simulate the fluorescence image of arrays of nanosensors in response to a concentration gradient. For that purpose, binding and unbinding of the analyte is simulated for each single nanosensor by using a Monte Carlo simulation and varying rate constants (kon, koff). Multiple nanosensors are arranged on a surface and exposed to a concentration pattern cA(x,y,t) of an analyte. We account for the resolution limit of light microscopy (Abbe limit) and the acquisition speed and resolution of optical setups and determine the resulting response images ΔI(x,y,t). Consequently, we introduce terms for the spatial and temporal resolution and simulate phase diagrams for different rate constants that allow us to predict how a sensor should be designed to provide a desired spatial and temporal resolution. Our results show, for example, that imaging of neurotransmitter release requires rate constants of kon = 106 M-1 s-1and koff = 102 s-1 in many scenarios, which corresponds to high dissociation constants of Kd > 100 μM. This work predicts if a given fluorescent nanosensor array (rate constants, size, shape, geometry, density) is able to resolve fast concentration changes such as neurotransmitter release from cells. Additionally, we provide rational design principles to engineer

  14. [Development of a Fluorescence Probe for Live Cell Imaging].

    Science.gov (United States)

    Shibata, Aya

    2017-01-01

     Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

  15. Fluorescent quantum dots: synthesis, biomedical optical imaging, and biosafety assessment.

    Science.gov (United States)

    Ji, Xiaoyuan; Peng, Fei; Zhong, Yiling; Su, Yuanyuan; He, Yao

    2014-12-01

    The marriage of nanomaterials with biology has significantly promoted advancement of biological techniques, profoundly facilitating basic research and practical applications in biological and biomedical fields. Taking advantages of unique optical properties (e.g., strong fluorescence, robust photostability, size-tunable emission wavelengths, etc.), fluorescent quantum dots (QDs), appearing as high-performance biological fluorescent nanoprobes, have been extensively explored for a variety of biomedical optical imaging applications. In this review, we present representative synthetic strategies for preparation of QDs and their applications in biomedical optical imaging, as well as risk assessments in vitro and in vivo. Briefly, we first summarize recent progress in fabrication of QDs via two rudimentary approaches, i.e., organometallic route and aqueous synthesis. Next we present representative achievement in QDs-based in vitro and in vivo biomedical optical imaging applications. We further discuss the toxicity assessment of QDs, ranging from cell studies to animal models. In the final section, we discuss challenges and perspectives for the QDs-relative bioapplications in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2013-01-01

    Full Text Available Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM can provide high resolution and high imaging frame during mature FLIM methods. An abstract time-gated FLIM model was given, and important temporal parameters are shown as well. Aiming at different applications of steady and transient fluorescence processes, two different operation modes, timing and lifetime computing algorithm are designed. High resolution and high frame can be achieved by one-excitation one-sampling mode and least square algorithm for steady imaging applications. Correspondingly, one-excitation two-sampling mode and rapid lifetime determination algorithm contribute to transient fluorescence situations.

  17. Normalized fluorescence lifetime imaging for tumor identification and margin delineation

    Science.gov (United States)

    Sherman, Adria J.; Papour, Asael; Bhargava, Siddharth; Taylor, Zach; Grundfest, Warren S.; Stafsudd, Oscar M.

    2013-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is a technique that has been proven to produce quantitative and qualitative differentiation and identification of substances with good specificity and sensitivity based on lifetime extracted information. This technique has shown the ability to also differentiate between a wide range of tissue types to identify malignant from benign tissue in vivo and ex vivo. However, the complexity, long duration and effort required to generate this information has limited the adoption of these techniques in a clinical setting. Our group has developed a time-resolved imaging system (patent pending) that does not require the extraction of lifetimes or use of complex curve fitting algorithms to display the needed information. The technique, entitled Lifetime Fluorescence Imaging (LFI, or NoFYI), converts fluorescence lifetime decay information directly into visual contrast. Initial studies using Fluorescein and Rhodamine-B demonstrated the feasibility of this approach. Subsequent studies demonstrated the ability to separate collagen and elastin powders. The technique uses nanosecond pulsed UV LEDs at 375 nm for average illumination intensities of ~4.5 μW on the tissue surface with detection by a gated CCD camera. To date, we have imaged 11 surgical head and neck squamous cell carcinoma and brain cancer biopsy specimens including 5 normal and 6 malignant samples. Images at multiple wavelengths clearly demonstrate differentiation between benign and malignant tissue, which was later confirmed by histology. Contrast was obtained between fluorophores with 35 μm spatial resolution and an SNR of ~30 dB allowing us to clearly define tumor margins in these highly invasive cancers. This method is capable of providing both anatomical and chemical information for the pathologist and the surgeon. These results suggest that this technology has a possible role in identifying tumors in tissue specimens and detecting tumor margins during procedures.

  18. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy.

    Science.gov (United States)

    Min, Wei; Lu, Sijia; Chong, Shasha; Roy, Rahul; Holtom, Gary R; Xie, X Sunney

    2009-10-22

    Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.

  19. 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.

  20. 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.

  1. Coincidence Planar Imaging for Dynamic [18F]FDG Uptake in Nude Mice with Tumors and Inflammation: Correlated With Histopathology and Micro-autoradiography

    Directory of Open Access Journals (Sweden)

    Ying-Ling Sun

    2005-06-01

    Full Text Available The Institute of Nuclear Energy Research of Taiwan has developed a dynamic coincidence detection device for positron emitted radiotracer pharmacodynamic study in small mice models. In this study, we set up an experimental paradigm by determining [fluorine-18]-2-deoxy-2-fluoro-D-glucose ([18F]FDG dynamic uptake in tumors and inflammations in nude mice as the foundation for future applications in therapy development. Histopathology and micro-autoradiography of these tumors and inflammations were obtained for confirmation. Dynamic coincidence planar images of six tumors and two inflammations in nude mice were acquired over 4 hours immediately after injection of 25.9 MBq of [18F]FDG into the right thigh of each animal. After image reconstruction, the lesion-to-background ratios were calculated in regions of interest over the lesion and contralateral thigh to determine the equilibrium status of the radiotracer. All mice were sacrificed for histopathologic examination and six of the mice were examined with micro-autoradiography. [18F]FDG uptake in tumors and inflammations both reached equilibrium about 3 hours after injection. At equilibrium, [18F]FDG uptake into tumors was two to four times higher than the background. Uptake into the 4-day and 8-day inflammations was 2.3 and 5.5 times higher than the background, respectively. Histopathology showed macrophage and neutrophil infiltration around the tumors and in the inflammations. Micro-autoradiography showed dense silver grains in the granulation tissue surrounding the tumors and inflammations. The preliminary results suggested that dynamic [18F]FDG coincidence planar imaging can help in determining the suitable time for static [18F]FDG imaging in nude mice models. The optimal time for static [18F]FDG positron emission tomography imaging was around 3 hours after injection. The paradigm for determining a dynamic [18F]FDG uptake pattern was demonstrated for future new therapeutic drug experimental use.

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

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-01

    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.

  3. Single camera imaging system for color and near-infrared fluorescence image guided surgery.

    Science.gov (United States)

    Chen, Zhenyue; Zhu, Nan; Pacheco, Shaun; Wang, Xia; Liang, Rongguang

    2014-08-01

    Near-infrared (NIR) fluorescence imaging systems have been developed for image guided surgery in recent years. However, current systems are typically bulky and work only when surgical light in the operating room (OR) is off. We propose a single camera imaging system that is capable of capturing NIR fluorescence and color images under normal surgical lighting illumination. Using a new RGB-NIR sensor and synchronized NIR excitation illumination, we have demonstrated that the system can acquire both color information and fluorescence signal with high sensitivity under normal surgical lighting illumination. The experimental results show that ICG sample with concentration of 0.13 μM can be detected when the excitation irradiance is 3.92 mW/cm(2) at an exposure time of 10 ms.

  4. Fluorescence lifetime imaging of endogenous biomarker of oxidative stress

    Science.gov (United States)

    Datta, Rupsa; Alfonso-García, Alba; Cinco, Rachel; Gratton, Enrico

    2015-01-01

    Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets. PMID:25993434

  5. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

    Science.gov (United States)

    Hayashi, Shinichi; Okada, Yasushi

    2015-05-01

    Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  6. 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.

  7. Small portable interchangeable imager of fluorescence for fluorescence guided surgery and research.

    Science.gov (United States)

    Okusanya, Olugbenga T; Madajewski, Brian; Segal, Erin; Judy, Brendan F; Venegas, Ollin G; Judy, Ryan P; Quatromoni, Jon G; Wang, May D; Nie, Shuming; Singhal, Sunil

    2015-04-01

    Fluorescence guided surgery (FGS) is a developing field of surgical and oncologic research. Practically, FGS has shown useful applications in urologic surgery, benign biliary surgery, colorectal cancer liver metastasis resection, and ovarian cancer debulking. Most notably in in cancer surgery, FGS allows for the clear delineation of cancerous tissue from benign tissue. FGS requires the utilization of a fluorescent contrast agent and an intraoperative fluorescence imaging device (IFID). Currently available IFIDs are expensive, unable to work with multiple fluorophores, and can be cumbersome. This study aims to describe the development and utility of a small, cost-efficient, and interchangeable IFID made from commercially available components. Extensive research was done to design and construct a light-weight, portable, and cost-effective IFID. We researched the capabilities, size, and cost of several camera types and eventually decided on a near-infrared (NIR) charged couple device (CCD) camera for its overall profile. The small portable interchangeable imager of fluorescence (SPIIF) is a "scout" IFID system for FGS. The main components of the SPIIF are a NIR CCD camera with an articulating light filter. These components and a LED light source with an attached heat sink are mounted on a small metal platform. The system is connected to a laptop by a USB 2.0 cable. Pixielink © software on the laptop runs the system by controlling exposure time, gain, and image capture. After developing the system, we evaluated its utility as an IFID. The system weighs less than two pounds and can cover a large area. Due to its small size, it is easily made sterile by covering it with any sterile plastic sheet. To determine the system's ability to detect fluorescent signal, we used the SPIIF to detect indocyanine green under ex and in-vivo conditions and fluorescein under ex-vivo conditions. We found the SPIIF was able to detect both ICG and fluorescein under different depths of a

  8. A simple protocol for attenuating the auto-fluorescence of cyanobacteria for optimized fluorescence in situ hybridization (FISH) imaging.

    Science.gov (United States)

    Zeller, Perrine; Ploux, Olivier; Méjean, Annick

    2016-03-01

    Cyanobacteria contain pigments, which generate auto-fluorescence that interferes with fluorescence in situ hybridization (FISH) imaging of cyanobacteria. We describe simple chemical treatments using CuSO4 or H2O2 that significantly reduce the auto-fluorescence of Microcystis strains. These protocols were successfully applied in FISH experiments using 16S rRNA specific probes and filamentous cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

    Directory of Open Access Journals (Sweden)

    Svitlana M. Levchenko

    2018-01-01

    Full Text Available Fluorescence lifetime imaging (FLIM is an effective noninvasive bioanalytical tool based on measuring fluorescent lifetime of fluorophores. A growing number of FLIM studies utilizes genetically engineered fluorescent proteins targeted to specific subcellular structures to probe local molecular environment, which opens new directions in cell science. This paper highlights the unconventional applications of FLIM for studies of molecular processes in diverse organelles of live cultured cells.

  10. Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

    OpenAIRE

    Svitlana M. Levchenko; Artem Pliss; Junle Qu

    2018-01-01

    Fluorescence lifetime imaging (FLIM) is an effective noninvasive bioanalytical tool based on measuring fluorescent lifetime of fluorophores. A growing number of FLIM studies utilizes genetically engineered fluorescent proteins targeted to specific subcellular structures to probe local molecular environment, which opens new directions in cell science. This paper highlights the unconventional applications of FLIM for studies of molecular processes in diverse organelles of live cultured cells.

  11. Snapshot spectrally encoded fluorescence imaging through a fiber bundle

    Science.gov (United States)

    Bedard, Noah; Tkaczyk, Tomasz S.

    2012-08-01

    Fiber optic endomicroscopy is a valuable tool for clinical diagnostics and animal studies because it can capture images of tissue in vivo with subcellular resolution. Current configurations for endomicroscopes have either limited spatial resolution or require a scanning mechanism at the distal end of the fiber, which can slow imaging speed and increase the probe size. We present a novel configuration that provides high contrast 350×350 pixel images at 7.2 frames per second, without the need for mechanical scanning at the proximal or distal end of the fiber. The proof-of-concept benchtop system is tested in fluorescence mode and can resolve 1.5 μm features of a high resolution 1951 USAF target.

  12. In situ Analysis of Coral Recruits Using Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Adi Zweifler

    2017-09-01

    Full Text Available Recruitment is a fundamental process that influences coral population dynamics as well as reef community structure. To date, coral recruitment success rates are poorly quantified because survey methods are labor-intensive and require manual interpretation. Thus, they are prone to human errors and have low repeatability—a gap we aim to bridge in this research. Since both corals and their symbiotic algae contain fluorescent pigments (chlorophyll and fluorescent proteins, we used the non-invasive Fluorescence Imaging System (FluorIS and developed a methodology to acquire daytime fluorescent photographs and identify coral recruits in them. We tested our method by monitoring 20 random quadrats at two sites in the Gulf of Aqaba, Israel. The quadrats were surveyed once a month for 8 months in order to track the settlement, mortality and survival rates of new coral recruits. We demonstrate daytime imaging using our method and identification of coral recruits as small as 1 mm in diameter, in a 20 × 20 cm quadrat. Our results show that this photographic method reduces surveyor errors and improves precision. The surveys revealed that on average, there are ~2 new coral recruit settlements (<2 cm for a quadrat (40 cm2 per month and that 83% of them survive the first month. Our study suggests a relative stability in the Gulf of Aqaba coral population during the survey period. The ability to survey recruits during the day using low-cost, easy-to-use photographic equipment has the potential to contribute significantly to the standardization of coral reef monitoring and management tools, at a time when the world's coral reefs are declining due to local and global stressors.

  13. Defining a superlens operating regime for imaging fluorescent molecules.

    Directory of Open Access Journals (Sweden)

    Kareem Elsayad

    Full Text Available It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such "lenses" is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close (>/ approximately 10 nm or too far (>/approximately 30 nm from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub

  14. Defining a superlens operating regime for imaging fluorescent molecules.

    Science.gov (United States)

    Elsayad, Kareem; Heinze, Katrin G

    2009-12-01

    It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such "lenses" is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix) calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close (>/ approximately 10 nm) or too far (>/approximately 30 nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating

  15. Fluorescence Intensity and Lifetime Cell Imaging with Luminescent Gold Nanoclusters.

    Science.gov (United States)

    Zhang, Jian; Fu, Yi; Conroy, Cecil V; Tang, Zhenghua; Li, Ge; Zhao, Richard Y; Wang, Gangli

    2012-12-20

    In this article, luminescent properties of gold nanoclusters (AuNCs) were studied at the single nanoparticle level and also used as novel imaging agents in cell media. Two types of water-soluble AuNCs which were stabilized with a monolayer composed of either mercaptosuccinic acid (MSA) or tiopronin thiolate ligands were synthesized by a chemical reduction reaction. These AuNCs were determined to have an average core diameter of less than 2 nm. On a time-resolved confocal microscope, the emission signals from the single AuNCs were distinctly recordable. The quantum yields of these AuNCs were measured to be ca. 5%. The lifetime of these AuNCs is also much longer than the lifetime of cellular autofluorescence in lifetime cell imaging as well as the lifetime of organic dye Alexa Fluor 488. After being derivatized with polyethylene glycol (PEG) moieties, the AuNCs were uploaded efficiently in the HeLa cells. Fluorescence intensity and lifetime cell images were recorded on the time-resolved confocal microscope in which the emission from the AuNCs was readily differentiated from the cellular autofluorescence background because of their relatively stronger emission intensities and longer lifetimes. These loaded nanoclusters in the cells were observed to widely distribute throughout the cells and especially densely loaded near the cell nucleuses. The AuNCs in the cells were also tested to have a better photostability relative to the organic fluorophores under the same conditions. We thus conclude that the AuNCs have a great potential as novel nanoparticle imaging agents, especially as lifetime imaging agents, in fluorescence imaging applications. We also prospect much broader applications of these AuNCs after further improvements of their luminescence quantum yields.

  16. 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

    2011-06-16

    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.

  17. Intraoperative detection of liver tumors aided by a fluorescence goggle system and multimodal imaging.

    Science.gov (United States)

    Liu, Yang; Akers, Walter J; Bauer, Adam Q; Mondal, Suman; Gullicksrud, Kyle; Sudlow, Gail P; Culver, Joseph P; Achilefu, Samuel

    2013-04-21

    Real-time image guidance in the operating room is needed to improve instantaneous surgical decisions. Toward this goal, we utilized a new fluorescence goggle system and a near-infrared fluorescent dye approved for human use, indocyanine green, to demonstrate the feasibility of detecting liver tumors intraoperatively. The fluorescence goggle provided successful imaging of multifocal breast cancer metastases in mouse liver. Diffused tumor deposits as small as 0.8 mm in diameter were detected, which were not obvious without the fluorescence goggle. A combination of surface-weighted fluorescence imaging and deep tissue-sensitive ultrasound imaging allowed comprehensive image guidance with the fluorescence goggle system for tumor resection in a rabbit VX2 liver metastasis model. This multimodal detection and guided surgical intervention strategy using ultrasonic imaging and real-time intraoperative fluorescence guidance is a promising and innovative technology platform for improving surgical outcome of human patients with primary or metastatic liver cancer.

  18. High resolution fluorescence bio-imaging upconversion nanoparticles in insects.

    Science.gov (United States)

    Alkahtani, Masfer; Chen, Yunyun; Pedraza, Julie J; González, Jorge M; Parkinson, Dilworth Y; Hemmer, Philip R; Liang, Hong

    2017-01-23

    Imaging fluorescent markers with brightness, photostability, and continuous emission with auto fluorescence background suppression in biological samples has always been challenging due to limitations of available and economical techniques. Here we report a new approach, to achieve high contrast imaging inside small and difficult biological systems with special geometry such as fire ants, an important agricultural pest, using a homemade cost-effective optical system. Unlike the commonly used rare-earth doped fluoride nanoparticles, we utilized nanoparticles with a high upconversion efficiency in water. Specifically Y2O3:Er+3,Yb+3 nanoparticles (40-50 nm diameter) were fed to fire ants as food and then a simple illuminating experiment was conducted at 980 nm wavelength at relatively low pump intensity8 kW.cm-2. The locations were further confirmed by X-ray tomography, where most particles aggregated inside the ant's mouth. High resolution, fast, and economical optical imaging system opens the door for studying more complex biological systems.

  19. 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/.

  20. 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.

  1. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.

    NARCIS (Netherlands)

    Hoebe, R.A.; van Oven, C.H.; Gadella, Th.W.J.; Dhonukshe, P.B.; van Noorden, C.J.F.; Manders, E.M.M.

    2007-01-01

    Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy

  2. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging

    NARCIS (Netherlands)

    Hoebe, R. A.; van Oven, C. H.; Gadella, T. W. J.; Dhonukshe, P. B.; van Noorden, C. J. F.; Manders, E. M. M.

    2007-01-01

    Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy (

  3. Image processing in standing-wave fluorescence microscopy

    Science.gov (United States)

    Krishnamurthi, Vijaykumar

    Fluorescence microscopes are valuable tools in determining the structure of the objects they image, typically, fixed and living cells and their components. However, the axial resolution of such microscopes is much worse than their transverse resolution. In addition, due to the finite aperture of the lenses used in all microscopes, not all the light emitted from the object is collected by the microscope. Every microscope acts like a low-pass filter and is therefore characterized by a transfer function, known as the optical transfer function (OTF). The bandpass region of OTF that determines which frequency components of the object will be transmitted has a characteristic shape with a cone shaped region in the middle in addition to bandlimits. Any object frequency components that fall in this cone shaped region, or outside the bandlimits are lost during imaging. The standing-wave fluorescence microscope (SWIM) uses interference of two beams, resulting in a non-uniform, planar excitation pattern in the specimen. The optical transfer function of this microscope (SWOTF) has three distinct bands, a central band that is identical to the optical transfer function (OTF) in a conventional fluorescence microscope, and two additional sidebands that are offset from the central band. Therefore, the SWOTF has gaps between the central band and the sidebands, and information about the object that falls in the gaps or outside the three bands is lost when imaging. The principal questions answered by this thesis are: (1) Do the data in the sidebands of the SWOTF contribute additional information about the object? (2) Can the information about the object lost in the gaps in the SWOTF be recovered using computational means? In SWFM, three images per plane of focus are required to capture all the information about the object. In the case of multiple plane data sets, there are three stacks of such triplets. In order to answer the questions posed above, the three SWFM images (or stacks

  4. Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

    Science.gov (United States)

    Jabbour, Joey M.; Cheng, Shuna; Malik, Bilal H.; Cuenca, Rodrigo; Jo, Javier A.; Wright, John; Cheng, Yi-Shing Lisa; Maitland, Kristen C.

    2013-04-01

    Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

  5. 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 (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.

  6. Intraoperative real-time localization of parathyroid gland with near infrared fluorescence imaging.

    Science.gov (United States)

    Kim, Sung Won; Lee, Hyoung Shin; Lee, Kang Dae

    2017-10-01

    Surgeons have cited difficulties in identifying the parathyroid glands (PG) during thyroidectomy. To overcome the limitation of naked eye, many studies on near-infrared fluorescence imaging of PGs have been introduced and suggested that fluorescence imaging is useful for both localizing PGs and evaluating their function. This imaging technique has been reported in two ways: (I) imaging using a fluorescent material called indocyanine green (ICG); and (II) autofluorescence using intrinsic fluorophores. These innovative and novel techniques are expected to have a significant impact on performing thyroid or parathyroid surgery. In this article, current papers that describe ICG fluorescence and autofluorescence imaging of PG during thyroid and parathyroid surgery are reviewed.

  7. Zebrafish as a good vertebrate model for molecular imaging using fluorescent probes.

    Science.gov (United States)

    Ko, Sung-Kyun; Chen, Xiaoqiang; Yoon, Juyoung; Shin, Injae

    2011-05-01

    Fluorescent probes have been used extensively to monitor biomolecules and biologically relevant species in vitro and in vivo. A new trend in this area that has been stimulated by the desire to obtain more detailed information about the biological effects of analytes is the change from live cell to whole animal fluorescent imaging. Zebrafish has received great attention for live vertebrate imaging due to several noticeable advantages. In this tutorial review, recent advances in live zebrafish imaging using fluorescent probes, such as fluorescent proteins, synthetic fluorescent dyes and quantum dots, are highlighted.

  8. Near-Infrared Fluorescence Lymphatic Imaging in Lymphangiomatosis.

    Science.gov (United States)

    Rasmussen, John C; Fife, Caroline E; Sevick-Muraca, Eva M

    2015-09-01

    Lymphangiomatosis is a rare disorder of the lymphatic system that can impact the dermis, soft tissue, bone, and viscera and can be characterized by lymphangiomas, swelling, and chylous discharge. Whether disordered lymphangiogenesis in lymphangiomatosis affects the function and anatomy of the entire systemic lymphatic circulation or is localized to specific sites is not fully known. A 35-year-old Caucasian female diagnosed with whole-body lymphangiomatosis at 2 months of age and who continues to present with progressive disease was imaged with near-infrared fluorescence lymphatic imaging. While the peripheral lymphatics in the extremities appeared largely normal compared to prior studies, we observed tortuous lymphatic vessels, fluorescence drainage from the peripheral lymphatics into lymphangiomas, and extensive dermal lymphatics in the left thigh and inguinal regions where the subject had previously had surgical assaults, potentially indicating defective systemic lymphangiogenesis. Further research into anatomical and functional lymphatic changes associated with the progression and treatment of lymphangiomatosis could aid in understanding the pathophysiology of the disease as well as point to treatment strategies.

  9. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.

    Science.gov (United States)

    Icha, Jaroslav; Schmied, Christopher; Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-04-10

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

  10. phiFLIM: a new method to avoid aliasing in frequency domain fluorescence lifetime imaging microscopy.

    NARCIS (Netherlands)

    van Munster, E.B.; Gadella, Th.W.J.

    2004-01-01

    In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded

  11. Hyperspectral fluorescence imaging coupled with multivariate image analysis techniques for contaminant screening of leafy greens

    Science.gov (United States)

    Everard, Colm D.; Kim, Moon S.; Lee, Hoyoung

    2014-05-01

    The production of contaminant free fresh fruit and vegetables is needed to reduce foodborne illnesses and related costs. Leafy greens grown in the field can be susceptible to fecal matter contamination from uncontrolled livestock and wild animals entering the field. Pathogenic bacteria can be transferred via fecal matter and several outbreaks of E.coli O157:H7 have been associated with the consumption of leafy greens. This study examines the use of hyperspectral fluorescence imaging coupled with multivariate image analysis to detect fecal contamination on Spinach leaves (Spinacia oleracea). Hyperspectral fluorescence images from 464 to 800 nm were captured; ultraviolet excitation was supplied by two LED-based line light sources at 370 nm. Key wavelengths and algorithms useful for a contaminant screening optical imaging device were identified and developed, respectively. A non-invasive screening device has the potential to reduce the harmful consequences of foodborne illnesses.

  12. Fluorescence lifetime images of different green fluorescent proteins in fly brain

    Science.gov (United States)

    Lai, Sih-Yu; Lin, Y. Y.; Chiang, A. S.; Huang, Y. C.

    2009-02-01

    The mechanisms of learning and memory are the most important functions in an animal brain. Investigating neuron circuits and network maps in a brain is the first step toward understanding memory and learning behavior. Since Drosophila brain is the major model for understanding brain functions, we measure the florescence lifetimes of different GFP-based reporters expressed in a fly brain. In this work, two Gal4 drivers, OK 107 and MZ 19 were used. Intracellular calcium ([Ca2+]) concentration is an importation indicator of neuronal activity. Therefore, several groups have developed GFP-based calcium sensors, among which G-CaMP is the most popular and reliable. The fluorescence intensity of G-CaMP will increase when it binds to calcium ion; however, individual variation from different animals prevents quantitative research. In this work, we found that the florescence lifetime of G-CaMP will shrink from 1.8 ns to 1.0 ns when binding to Ca2+. This finding can potentially help us to understand the neuron circuits by fluorescence lifetime imaging microscopy (FLIM). Channelrhodopsin-2 (ChR2) is a light-activated ion-channel protein on a neuron cell membrane. In this work, we express ChR2 and G-CaMP in a fly brain. Using a pulsed 470-nm laser to activate the neurons, we can also record the fluorescence lifetime changes in the structure. Hence, we can trace and manipulate a specific circuit in this animal. This method provides more flexibility in brain research.

  13. Fluorescence imaging of lymphatic outflow of cerebrospinal fluid in mice.

    Science.gov (United States)

    Kwon, Sunkuk; Janssen, Christopher F; Velasquez, Fred Christian; Sevick-Muraca, Eva M

    2017-10-01

    Cerebrospinal fluid (CSF) is known to be reabsorbed by the lymphatic vessels and drain into the lymph nodes (LNs) through peripheral lymphatic vessels. In the peripheral lymphatics, the contractile pumping action of lymphangions mediates lymph drainage; yet it is unknown whether lymphatic vessels draining cranial and spinal CSF show similar function. Herein, we used non-invasive near-infrared fluorescence imaging (NIRFI) to image (i) indocyanine green (ICG) distribution along the neuraxis and (ii) routes of ICG-laden CSF outflow into the lymphatics following intrathecal lumbar administration. We demonstrate lymphatic contractile function in peripheral lymphatics draining from the nasal lymphatics to the mandibular LNs. In addition, we observed afferent sciatic lymphatic vessels, which also show contractile activity and transport spinal CSF into the sciatic LNs. This drainage pattern was also visualized by NIRFI following intrathecal thoracic injection. In situ intravital imaging following intrathecal lumbar injection of blue dye shows similar distributions to that seen in vivo with ICG. NIRFI could be used as a tool to probe CSF pathology including neurological disorders by imaging CSF outflow dynamics to lymphatics. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Detecting fluorescence hot-spots using mosaic maps generated from multimodal endoscope imaging

    Science.gov (United States)

    Yang, Chenying; Soper, Timothy D.; Seibel, Eric J.

    2013-03-01

    Fluorescence labeled biomarkers can be detected during endoscopy to guide early cancer biopsies, such as high-grade dysplasia in Barrett's Esophagus. To enhance intraoperative visualization of the fluorescence hot-spots, a mosaicking technique was developed to create full anatomical maps of the lower esophagus and associated fluorescent hot-spots. The resultant mosaic map contains overlaid reflectance and fluorescence images. It can be used to assist biopsy and document findings. The mosaicking algorithm uses reflectance images to calculate image registration between successive frames, and apply this registration to simultaneously acquired fluorescence images. During this mosaicking process, the fluorescence signal is enhanced through multi-frame averaging. Preliminary results showed that the technique promises to enhance the detectability of the hot-spots due to enhanced fluorescence signal.

  15. Intraoperative near-infrared fluorescent imaging during robotic operations.

    Science.gov (United States)

    Macedo, Antonio Luiz de Vasconcellos; Schraibman, Vladimir

    2016-01-01

    The intraoperative identification of certain anatomical structures because they are small or visually occult may be challenging. The development of minimally invasive surgery brought additional difficulties to identify these structures due to the lack of complete tactile sensitivity. A number of different forms of intraoperative mapping have been tried. Recently, the near-infrared fluorescence imaging technology with indocyanine green has been added to robotic platforms. In addition, this technology has been tested in several types of operations, and has advantages such as safety, low cost and good results. Disadvantages are linked to contrast distribution in certain clinical scenarios. The intraoperative near-infrared fluorescent imaging is new and promising addition to robotic surgery. Several reports show the utility of this technology in several different procedures. The ideal dose, time and site for dye injection are not well defined. No high quality evidence-based comparative studies and long-term follow-up outcomes have been published so far. Initial results, however, are good and safe. RESUMO A identificação intraoperatória de certas estruturas anatômicas, por seu tamanho ou por elas serem ocultas à visão, pode ser desafiadora. O desenvolvimento da cirurgia minimamente invasiva trouxe dificuldades adicionais, pela falta da sensibilidade tátil completa. Diversas formas de detecção intraoperatória destas estruturas têm sido tentadas. Recentemente, a tecnologia de fluorescência infravermelha com verde de indocianina foi associada às plataformas robóticas. Além disso, essa tecnologia tem sido testada em uma variedade de cirurgias, e suas vantagens parecem estar ligadas a baixo custo, segurança e bons resultados. As desvantagens estão associadas à má distribuição do contraste em determinados cenários. A imagem intraoperatória por fluorescência infravermelha é uma nova e promissora adição à cirurgia robótica. Diversas séries mostram

  16. Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

    Science.gov (United States)

    Feeks, James A; Hunter, Jennifer J

    2017-05-01

    In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

  17. Concurrent fluorescence macro-imaging across multiple spectral regions in the visible and the near infrared

    Science.gov (United States)

    Kazemzadeh, Farnoud; Haider, Shahid; Jin, Chao; Clausi, David A.; Wong, Alexander

    2015-09-01

    Fluorescent imaging, often synonymous with microscopic imaging, is an imaging modality whereby various features of a target are observed based on assignment of chemical labels. These labels are in most cases indirect tracers of specific structures or chemical compounds which cannot be otherwise identified. The tracers are excited by an illuminating source and they in turn emit light at specific wavelengths. This light is then captured by an imaging device and represented as an indirect observation of the specific feature in the sample. The process of excitation and imaging of the emitted light is performed sequentially and is proportional to the number of tracers or fluorescence species present in the sample. We present an imaging system that can image fluorescent tracers, in the visible and the near Infra-red, simultaneously. This system is capable of illuminating the target with different excitation light sources and capture the corresponding fluorescence images in one snapshot using a series of mirrors to capture different views of the sample. The simultaneously captured image are fused using a computational reconstruction process to present a coherent multispectral fluorescence image. The system is proposed for use in applications where the rapid enumeration of fluorescent species in a large field of view is paramount as opposed to their microscopic image in a narrow field of view. The system was tested using a controlled cocktail solution of four different types fluorescent microspheres and was able to enumerate the microspheres based on their different fluorescent signatures as captured by the system.

  18. FLEX: an imaging spectrometer for measurement of vegetation fluorescence

    Science.gov (United States)

    Smorenburg, Kees; Visser, Huib; Court, Andrew; Stoll, Marc Ph.

    2017-11-01

    Detection of vegetation fluorescence gives information about plant functioning, stress and vitality. During the past decades several ground based laser fluorosensors have been developed to investigate these processes and to demonstrate the value of this technique. FLEX (= FLuorescense EXplorer) is a space mission to measure the fluorescence of vegetation on earth over large areas from space. Such a mission would greatly improve the understanding and enhance the capability to quantify e.g. the role of terrestrial vegetation in global carbon sequestration. Because the fluorescence signal, which is excited by solar irradiation is low with respect to the reflected sunlight the signal from a satellite is proposed to be measured in the solar Fraunhofer lines, where the reflection signal is much reduced. The heart of FLEX is a high resolution imaging spectrometer with 2 channels: channel 1 around the Fraunhofer lines at ‡ = 397 nm, ‡= 423 nm and/or ‡ = 434 nm and channel 2 around the Fraunhofer line at ‡ = 656 nm. The required spectral resolution will depend on the linewidth (0.02-0.3 nm). A first definition of the field of view is 8.4 degrees, leading from an 800 km satellite altitude to a swath of about 120 km. For detection a 1024x1024 pixel frame transfer CCD detector is proposed, with a pixel dimension of 13 x 13 ‡ mm2. The maximum footprint is about 500x500m2. The optical configuration contains a scan mirror for solar calibration, for pointing the FOV in swath direction and for freezing the observed ground scene up to a few seconds to increase the signal to noise performance. At this moment the concept of FLEX is elaborated in a feasibility study. Both the scientific and instrument requirements are updated and the concept is studied in detail. Besides a development plan for FLEX is made. In this paper the idea and the headlines of FLEX are described.

  19. Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

    NARCIS (Netherlands)

    Broess, K.; Borst, J.W.; Amerongen, van H.

    2009-01-01

    This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of

  20. Video Histories, Memories, and Coincidences

    DEFF Research Database (Denmark)

    Kacunko, Slavko

    2012-01-01

    Looping images allows us to notice things that we have never noticed before. Looping a small but exquisite selection of the video tapes of Marcel Odenbach, Dieter Kiessling and Matthias Neuenhofer may allow the discovering of Histories, Coincidences, and Infinitesimal Aesthetics inscribed...

  1. Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging

    Science.gov (United States)

    Peter, Sébastien; Elgass, Kirstin; Sackrow, Marcus; Caesar, Katharina; Born, Anne-Kathrin; Maniura, Katharina; Harter, Klaus; Meixner, Alfred J.; Schleifenbaum, Frank

    2010-02-01

    Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

  2. 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.

  3. 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).

  4. 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}.

  5. Enhanced imaging resolution in dynamic fluorescence molecular tomography by multispectral excitation method (Conference Presentation)

    Science.gov (United States)

    Chen, Maomao; Zhou, Yuan; Su, Han; Zhang, Dong; Luo, Jianwen

    2017-03-01

    Dynamic fluorescence molecular tomography (DFMT) is a promising method for the quantitative evaluation of the metabolic process of fluorescent agents in body. However, the resolution is limited due to the ill-posed nature of fluorescence molecular tomography (FMT) and the high absorption and scattering of the fluorescent light in biological tissues. In this paper, the resolution of DFMT is improved by multispectral excitation method. Firstly, the imaged object with varied fluorescent concentrations at different time points is excited by several excitation lights with different wavelengths, and the fluorescent images are collected. Secondly, the individual FMT images at different time points are respectively reconstructed, and independent component analysis (ICA) is employed to decompose the fluorescent targets. The independent components (ICs) and corresponding spectrum courses (SCs) which obtained from ICA represent the spatial structures and spectral variations of the fluorescent targets, respectively. Thirdly, the ICs and SCs are combined to quantitatively recover the concentrations of individual fluorescent targets. Finally, the metabolic parameters and DFMT images are obtained by fitting the FMT images of each fluorescent targets at different time points into a two compartment model. Numerical simulations are carried out to validate the feasibility of the proposed method. The results demonstrate that the resolution of DFMT is significantly improved. The metabolic curves can be correctly recovered even when the edge-edge-distance of the fluorescent targets is less than 0.1 cm.

  6. 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.

  7. Tumor Endothelial Marker Imaging in Melanomas Using Dual-Tracer Fluorescence Molecular Imaging

    Science.gov (United States)

    Tichauer, Kenneth M.; Deharvengt, Sophie J.; Samkoe, Kimberley S.; Gunn, Jason R.; Bosenberg, Marcus W.; Turk, Mary-Jo; Hasan, Tayyaba; Stan, Radu V.; Pogue, Brian W.

    2014-01-01

    Purpose Cancer-specific endothelial markers available for intravascular binding are promising targets for new molecular therapies. In this study, a molecular imaging approach of quantifying endothelial marker concentrations (EMCI) is developed and tested in highly light-absorbing melanomas. The approach involves injection of targeted imaging tracer in conjunction with an untargeted tracer, which is used to account for nonspecific uptake and tissue optical property effects on measured targeted tracer concentrations. Procedures Theoretical simulations and a mouse melanoma model experiment were used to test out the EMCI approach. The tracers used in the melanoma experiments were fluorescently labeled anti-Plvap/PV1 antibody (plasmalemma vesicle associated protein Plvap/PV1 is a transmembrane protein marker exposed on the luminal surface of endothelial cells in tumor vasculature) and a fluorescent isotype control antibody, the uptakes of which were measured on a planar fluorescence imaging system. Results The EMCI model was found to be robust to experimental noise under reversible and irreversible binding conditions and was capable of predicting expected overexpression of PV1 in melanomas compared to healthy skin despite a 5-time higher measured fluorescence in healthy skin compared to melanoma: attributable to substantial light attenuation from melanin in the tumors. Conclusions This study demonstrates the potential of EMCI to quantify endothelial marker concentrations in vivo, an accomplishment that is currently unavailable through any other methods, either in vivo or ex vivo. PMID:24217944

  8. Results of imaging infection with [{sup 18}F]FDG and coincidence cameras; Ergebnisse der Entzuendungsdiagnostik mit [{sup 18}F]Fluor-Deoxyglukose (FDG) und Koinzidenz-Gammakameras

    Energy Technology Data Exchange (ETDEWEB)

    Meller, J.; Becker, W. [Goettingen Univ. (Germany). Abt. fuer Nuklearmedizin

    2001-11-01

    During the last years several reports about the role of FDG-PET in imaging inflammatory diseases either with dedicated systems or coincidence gamma cameras have been published. Based on these studies the value of FDG-imaging can now be determined for several entities. Most important data come from prospective studies which compared FDG-PET with established nuclear medicine procedures. Studies with coincidence cameras demonstrated that FDG may be the tracer of choice in the diagnostic work-up in patients with fever of unknown origin (FUO) and chronic bacterial osteomyelitis. The method also proved to be sensitive and specific in imaging infection outside the surgical wound in patients with postoperative fever, but the specificity of the method of detecting the focus of fever within the surgical wound was poor. It can be expected that FDG will be of further diagnostic value in other inflammatory diseases, especially in autoimmune disorders. (orig.) [German] Erst in den letzten Jahren wurden systematische klinische Arbeiten zum Einsatz von FDG, vorwiegend mit dedizierten PET-Systemen, aber auch mit Koinzidenz-Gammakameras bei entzuendlichen Erkrankungen publiziert, die es ermoeglichen, zu einzelnen Eintitaeten Aussagen ueber den Stellenwert der Methode machen zu koennen. Am aussagekraeftigsten sind hier vor allem Studien, die die FDG-PET mit etablierten nuklearmedizinischen Verfahren prospektiv verglichen haben. Bisherige Untersuchungen zeigen, dass eine Entzuendungsdiagnostik mittels Koinzidenz-Gammakamerasystemen und [{sup 18}F]FDG bei der Abklaerung von Fieber unklarer Genese (FUO) und der chronischen Osteomyelitis herkoemmlichen und bisher als Referenzmethoden angesehenen szintigraphischen Methoden ueberlegen ist. Bei postoperativen Fieberzustaenden kann die Methode aufgrund ihrer Unspezifitaet bei Verdacht auf eine Infektion im Operationsgebiet nicht empfohlen werden, waehrend sie ausserhalb des Operationsgebietes eine hohe diagnostische Genauigkeit aufweist. Es

  9. 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.

  10. Aptamer-assembled nanomaterials for fluorescent sensing and imaging

    Science.gov (United States)

    Lu, Danqing; He, Lei; Zhang, Ge; Lv, Aiping; Wang, Ruowen; Zhang, Xiaobing; Tan, Weihong

    2017-01-01

    Aptamers, which are selected in vitro by a technology known as the systematic evolution of ligands by exponential enrichment (SELEX), represent a crucial recognition element in molecular sensing. With advantages such as good biocompatibility, facile functionalization, and special optical and physical properties, various nanomaterials can protect aptamers from enzymatic degradation and nonspecific binding in living systems and thus provide a preeminent platform for biochemical applications. Coupling aptamers with various nanomaterials offers many opportunities for developing highly sensitive and selective sensing systems. Here, we focus on the recent applications of aptamer-assembled nanomaterials in fluorescent sensing and imaging. Different types of nanomaterials are examined along with their advantages and disadvantages. Finally, we look toward the future of aptamer-assembled nanomaterials.

  11. Inspection of fecal contamination on strawberries using fluorescence imaging

    Science.gov (United States)

    Chuang, Yung-Kun; Yang, Chun-Chieh; Kim, Moon S.; Delwiche, Stephen R.; Lo, Y. Martin; Chen, Suming; Chan, Diane E.

    2013-05-01

    Fecal contamination of produce is a food safety issue associated with pathogens such as Escherichia coli that can easily pollute agricultural products via animal and human fecal matters. Outbreaks of foodborne illnesses associated with consuming raw fruits and vegetables have occurred more frequently in recent years in the United States. Among fruits, strawberry is one high-potential vector of fecal contamination and foodborne illnesses since the fruit is often consumed raw and with minimal processing. In the present study, line-scan LED-induced fluorescence imaging techniques were applied for inspection of fecal material on strawberries, and the spectral characteristics and specific wavebands of strawberries were determined by detection algorithms. The results would improve the safety and quality of produce consumed by the public.

  12. GPU acceleration of time-domain fluorescence lifetime imaging

    Science.gov (United States)

    Wu, Gang; Nowotny, Thomas; Chen, Yu; Li, David Day-Uei

    2016-01-01

    Fluorescence lifetime imaging microscopy (FLIM) plays a significant role in biological sciences, chemistry, and medical research. We propose a graphic processing unit (GPU) based FLIM analysis tool suitable for high-speed, flexible time-domain FLIM applications. With a large number of parallel processors, GPUs can significantly speed up lifetime calculations compared to CPU-OpenMP (parallel computing with multiple CPU cores) based analysis. We demonstrate how to implement and optimize FLIM algorithms on GPUs for both iterative and noniterative FLIM analysis algorithms. The implemented algorithms have been tested on both synthesized and experimental FLIM data. The results show that at the same precision, the GPU analysis can be up to 24-fold faster than its CPU-OpenMP counterpart. This means that even for high-precision but time-consuming iterative FLIM algorithms, GPUs enable fast or even real-time analysis.

  13. 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

    2014-11-07

    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.

  14. The Yeast Resource Center Public Image Repository: A large database of fluorescence microscopy images

    Directory of Open Access Journals (Sweden)

    Riffle Michael

    2010-05-01

    Full Text Available Abstract Background There is increasing interest in the development of computational methods to analyze fluorescent microscopy images and enable automated large-scale analysis of the subcellular localization of proteins. Determining the subcellular localization is an integral part of identifying a protein's function, and the application of bioinformatics to this problem provides a valuable tool for the annotation of proteomes. Training and validating algorithms used in image analysis research typically rely on large sets of image data, and would benefit from a large, well-annotated and highly-available database of images and associated metadata. Description The Yeast Resource Center Public Image Repository (YRC PIR is a large database of images depicting the subcellular localization and colocalization of proteins. Designed especially for computational biologists who need large numbers of images, the YRC PIR contains 532,182 TIFF images from nearly 85,000 separate experiments and their associated experimental data. All images and associated data are searchable, and the results browsable, through an intuitive web interface. Search results, experiments, individual images or the entire dataset may be downloaded as standards-compliant OME-TIFF data. Conclusions The YRC PIR is a powerful resource for researchers to find, view, and download many images and associated metadata depicting the subcellular localization and colocalization of proteins, or classes of proteins, in a standards-compliant format. The YRC PIR is freely available at http://images.yeastrc.org/.

  15. The Yeast Resource Center Public Image Repository: A large database of fluorescence microscopy images.

    Science.gov (United States)

    Riffle, Michael; Davis, Trisha N

    2010-05-19

    There is increasing interest in the development of computational methods to analyze fluorescent microscopy images and enable automated large-scale analysis of the subcellular localization of proteins. Determining the subcellular localization is an integral part of identifying a protein's function, and the application of bioinformatics to this problem provides a valuable tool for the annotation of proteomes. Training and validating algorithms used in image analysis research typically rely on large sets of image data, and would benefit from a large, well-annotated and highly-available database of images and associated metadata. The Yeast Resource Center Public Image Repository (YRC PIR) is a large database of images depicting the subcellular localization and colocalization of proteins. Designed especially for computational biologists who need large numbers of images, the YRC PIR contains 532,182 TIFF images from nearly 85,000 separate experiments and their associated experimental data. All images and associated data are searchable, and the results browsable, through an intuitive web interface. Search results, experiments, individual images or the entire dataset may be downloaded as standards-compliant OME-TIFF data. The YRC PIR is a powerful resource for researchers to find, view, and download many images and associated metadata depicting the subcellular localization and colocalization of proteins, or classes of proteins, in a standards-compliant format. The YRC PIR is freely available at http://images.yeastrc.org/.

  16. Clearing up the signal: spectral imaging and linear unmixing in fluorescence microscopy.

    Science.gov (United States)

    Zimmermann, Timo; Marrison, Joanne; Hogg, Karen; O'Toole, Peter

    2014-01-01

    The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluorophores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given.

  17. Multi-pinhole fluorescent x-ray computed tomography for molecular imaging

    National Research Council Canada - National Science Library

    Tenta Sasaya; Naoki Sunaguchi; Kazuyuki Hyodo; Tsutomu Zeniya; Tetsuya Yuasa

    2017-01-01

    We propose a multi-pinhole fluorescent x-ray computed tomography (mp-FXCT) technique for preclinical molecular imaging that can provide the complete data necessary to produce 3-D tomographic images during anaesthesia...

  18. Imaging of surfaces by concurrent surface plasmon resonance and surface plasmon resonance-enhanced fluorescence.

    Directory of Open Access Journals (Sweden)

    Rahber Thariani

    Full Text Available Surface plasmon resonance imaging and surface plasmon induced fluorescent are sensitive tools for surface analysis. However, existing instruments in this area have provided limited capability for concurrent detection, and may be large and expensive. We demonstrate a highly cost-effective system capable of concurrent surface plasmon resonance microscopy (SPRM and surface plasmon resonance-enhanced fluorescence (SPRF imaging, allowing for simultaneous monitoring of reflectivity and fluorescence from discrete spatial regions. The instrument allows for high performance imaging and quantitative measurements with surface plasmon resonance, and surface plasmon induced fluorescence, with inexpensive off-the-shelf components.

  19. Application of Fluorescence Lifetime Imaging (FLIM) to Measure Intracellular Environments in a Single Cell.

    Science.gov (United States)

    Nakabayashi, Takakazu; Awasthi, Kamlesh; Ohta, Nobuhiro

    2017-01-01

    Fluorescence lifetime imaging (FLIM) has now been used in many bioscience fields, which comes from the quantification of fluorescence lifetime. The procedure for obtaining lifetime images is very similar to that used in fluorescence microscopy. However, obtaining reliable lifetime images requires an understanding of the theory of fluorescence lifetime, principle of FLIM systems, and evaluation procedure of intracellular environments. In this chapter, the materials, methods, and notes on FLIM measurements have been described, in conjunction with a brief explanation of the background of FLIM.

  20. Model for the brightness uniformity of fluorescence screen of image intensifier

    Science.gov (United States)

    Qiu, YaFeng; Chang, BenKang; Qian, YunSheng; Fu, RongGuo; Gao, Youtang; Si, Tian

    2007-01-01

    The three elements of photoelectrical cathode, microchannel plate and fluorescence screen are important parts to imaging quality of low light and ultraviolet Image intensifier. To do research and analysis work on the Fluorescence screen parameter testing have practical significance to the understanding of the performance of fluorescence screen and then can help to know where improvement should be made and then achieve a best performance entire tube, This article mainly introduce the testing theory of the brightness uniformity of fluorescence screen of Image Intensifier and how to build a mathematic model.

  1. Quantitative analysis of fluorescence lifetime measurements of the macula using the fluorescence lifetime imaging ophthalmoscope in healthy subjects.

    Science.gov (United States)

    Dysli, Chantal; Quellec, Gwénolé; Abegg, Mathias; Menke, Marcel N; Wolf-Schnurrbusch, Ute; Kowal, Jens; Blatz, Johannes; La Schiazza, Olivier; Leichtle, Alexander B; Wolf, Sebastian; Zinkernagel, Martin S

    2014-04-03

    Fundus autofluorescence (FAF) cannot only be characterized by the intensity or the emission spectrum, but also by its lifetime. As the lifetime of a fluorescent molecule is sensitive to its local microenvironment, this technique may provide more information than fundus autofluorescence imaging. We report here the characteristics and repeatability of FAF lifetime measurements of the human macula using a new fluorescence lifetime imaging ophthalmoscope (FLIO). A total of 31 healthy phakic subjects were included in this study with an age range from 22 to 61 years. For image acquisition, a fluorescence lifetime ophthalmoscope based on a Heidelberg Engineering Spectralis system was used. Fluorescence lifetime maps of the retina were recorded in a short- (498-560 nm) and a long- (560-720 nm) spectral channel. For quantification of fluorescence lifetimes a standard ETDRS grid was used. Mean fluorescence lifetimes were shortest in the fovea, with 208 picoseconds for the short-spectral channel and 239 picoseconds for the long-spectral channel, respectively. Fluorescence lifetimes increased from the central area to the outer ring of the ETDRS grid. The test-retest reliability of FLIO was very high for all ETDRS areas (Spearman's ρ = 0.80 for the short- and 0.97 for the long-spectral channel, P macula in healthy subjects. By using a custom-built software, we were able to quantify fluorescence lifetimes within the ETDRS grid. Establishing a clinically accessible standard against which to measure FAF lifetimes within the retina is a prerequisite for future studies in retinal disease.

  2. Digital aberration correction of fluorescent images with coherent holographic image reconstruction by phase transfer (CHIRPT)

    Science.gov (United States)

    Field, Jeffrey J.; Bartels, Randy A.

    2016-03-01

    Coherent holographic image reconstruction by phase transfer (CHIRPT) is an imaging method that permits digital holographic propagation of fluorescent light. The image formation process in CHIRPT is based on illuminating the specimen with a precisely controlled spatio-temporally varying intensity pattern. This pattern is formed by focusing a spatially coherent illumination beam to a line focus on a spinning modulation mask, and image relaying the mask plane to the focal plane of an objective lens. Deviations from the designed spatio-temporal illumination pattern due to imperfect mounting of the circular modulation mask onto the rotation motor induce aberrations in the recovered image. Here we show that these aberrations can be measured and removed non-iteratively by measuring the disk aberration phase externally. We also demonstrate measurement and correction of systematic optical aberrations in the CHIRPT microscope.

  3. Improved in Vivo Whole-Animal Detection Limits of Green Fluorescent Protein–Expressing Tumor Lines by Spectral Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Jenny M. Tam

    2007-07-01

    Full Text Available Green fluorescent protein (GFP has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI. To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 × 103 cells for spectral imaging versus 1 × 106 cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.

  4. Coincidence measurements on detectors for microPET II: A 1 mm3 resolution PET scanner for small animal imaging

    CERN Document Server

    Chatziioannou, A; Shao, Y; Doshi, N K; Silverman, B; Meadors, K; Cherry, SR

    2000-01-01

    We are currently developing a small animal PET scanner with a design goal of 1 mm3 image resolution. We have built three pairs of detectors and tested performance in terms of crystal identification, spatial, energy and timing resolution. The detectors consisted of 12 multiplied by 12 arrays of 1 multiplied by 1 multiplied by 10mm LSO crystals (1.15 mm pitch) coupled to Hamamatsu H7546 64 channel PMTs via 5cm long coherent glass fiber bundles. Optical fiber connection is necessary to allow high packing fraction in a ring geometry scanner. Fiber bundles with and without extramural absorber (EMA) were tested. The results demonstrated an intrinsic spatial resolution of 1.12 mm (direct coupled LSO array), 1.23 mm (bundle without EMA) and 1.27 mm (bundle with EMA) using a similar to 500 micron diameter Na-22 source. Using a 330 micron line source filled with F-18, intrinsic resolution for the EMA bundle improved to 1.05 mm. The respective timing and energy resolution values were 1.96 ns, 21% (direct coupled), 2.20 ...

  5. Multimodality Imaging Probe for Positron Emission Tomography and Fluorescence Imaging Studies

    Directory of Open Access Journals (Sweden)

    Suresh K. Pandey

    2014-05-01

    Full Text Available Our goal is to develop multimodality imaging agents for use in cell tracking studies by positron emission tomography (PET and optical imaging (OI. For this purpose, bovine serum albumin (BSA was complexed with biotin (histologic studies, 5(6- carboxyfluorescein, succinimidyl ester (FAM SE (OI studies, and diethylenetriamine pentaacetic acid (DTPA for chelating gallium 68 (PET studies. For synthesis of BSA-biotin-FAM-DTPA, BSA was coupled to (+-biotin N-hydroxysuccinimide ester (biotin-NHSI. BSA- biotin was treated with DTPA-anhydride and biotin-BSA-DTPA was reacted with FAM. The biotin-BSA-DTPA-FAM was reacted with gallium chloride 3 to 5 mCi eluted from the generator using 0.1 N HCl and was passed through basic resin (AG 11 A8 and 150 mCi (100 μL, pH 7–8 was incubated with 0.1 mg of FAM conjugate (100 μL at room temperature for 15 minutes to give 66Ga-BSA-biotin-DTPA-FAM. A shaved C57 black mouse was injected with FAM conjugate (50 μL at one flank and FAM-68Ga (50 μL, 30 mCi at the other. Immediately after injection, the mouse was placed in a fluorescence imaging system (Kodak In-Vivo F, Bruker Biospin Co., Woodbridge, CT and imaged (Λex: 465 nm, Λem: 535 nm, time: 8 seconds, Xenon Light Source, Kodak. The same mouse was then placed under an Inveon microPET scanner (Siemens Medical Solutions, Knoxville, TN injected (intravenously with 25 μCi of 18F and after a half-hour (to allow sufficient bone uptake was imaged for 30 minutes. Molecular weight determined using matrix-associated laser desorption ionization (MALDI for the BSA sample was 66,485 Da and for biotin-BSA was 67,116 Da, indicating two biotin moieties per BSA molecule; for biotin-BSA-DTPA was 81,584 Da, indicating an average of 30 DTPA moieties per BSA molecule; and for FAM conjugate was 82,383 Da, indicating an average of 1.7 fluorescent moieties per BSA molecule. Fluorescence imaging clearly showed localization of FAM conjugate and FAM-68Ga at respective flanks of the mouse

  6. Hyperspectral instrumentation to image and characterize the fluorescence of materials

    Science.gov (United States)

    Bourcier, Frédéric; Walter, Philippe; Pedetti, Silvia; Faye, Delphine; Spezzigu, Piero; Infante, Fulvio; Le Nouy, Patrice; Zedda, Edoardo

    2016-09-01

    Optical instruments for space applications with improved performances (smaller pixels and spectral range extension) are becoming more and more sensitive to chemical contamination and particle sedimentation. Outgassing under vacuum conditions causes dramatic flux losses, especially in the UV bandwidth. Furthermore, it is difficult to perform physicochemical analyses of contaminated surfaces on flight models, in a clean room. Conventional analytical techniques such as FTIR (Fourier Transform Infrared interferometer) need the tool to be in contact with the studied area, which is forbidden when working on satellites. In addition, it does not give any information about the distribution of the contaminants in the field of view. The probed area is large, mono-pixel, and the sensitivity of the instrument is too low for hundred nanometer thin film deposits. A first study has shown that we could benefit from using the UV/visible fluorescence spectra to partially identify contaminants and polymer materials. The shape of the fluorescence spectra of adhesives, paints and varnishes have specific signatures that could be recorded into a designated reference database. The location of the presence of these contaminants on such sensitive optics is also relevant. To acquire both these parameters, we designed a specific compact hyperspectral instrument to remotely acquire cube images (500x500 pixels) in a 5 degree field of view, and on a wide range of continuous wavelengths from UV at 320 nm up to the near infrared at 1000 nm. This paper will present the chosen trade-off between different critical optics for a new portable version of this instrument. It is dedicated to space and cultural heritage applications and the first results on an engineering prototype will be shown.

  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.......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. Time-resolved imaging system for fluorescence-guided surgery with lifetime imaging capability

    Science.gov (United States)

    Powolny, F.; Homicsko, K.; Sinisi, R.; Bruschini, Claudio E.; Grigoriev, E.; Homulle, H.; Prior, John O.; Hanahan, D.; Dubikovskaya, E.; Charbon, E.

    2014-05-01

    We present a single-photon camera for fluorescence imaging, with a time resolution better than 100ps, capable of providing both intensity and lifetime images. the camera was fabricated in standard CMOS technology. With this FluoCam we show the possibility to study sub-nanosecond fluorescence mechanisms. The FluoCam was used to characterize a near-infrared probe, indocyanine green, conjugated with multimeric cyclic pentapeptide (cRGD). The fluorescent probe-conjugated was used to target and mark tumors with better specificity, in particular aiming at targeting the integrins αvβ3 and αvβ5. As a first step towards clinical studies, preliminary results obtained in-vivo are presented. The first envisioned clinical application would be image-guided surgical oncology to help the surgeon to remove tumor tissue by a better discrimination from normal tissues and also to improve the detection of metastatic lymph nodes. A further application could be the in-vivo determination of the αvβ3 and αvβ5 targets to select patients for therapy with RGD chemotherapy conjugates.

  10. Singlet oxygen phosphorescence lifetime imaging based on a fluorescence lifetime imaging microscope.

    Science.gov (United States)

    Tian, Wenming; Deng, Liezheng; Jin, Shengye; Yang, Heping; Cui, Rongrong; Zhang, Qing; Shi, Wenbo; Zhang, Chunlei; Yuan, Xiaolin; Sha, Guohe

    2015-04-09

    The feasibility of singlet oxygen phosphorescence (SOP) lifetime imaging microscope was studied on a modified fluorescence lifetime imaging microscope (FLIM). SOP results from the infrared radiative transition of O2(a(1)Δg → X(3)Σg(-)) and O2(a(1)Δg) was produced in a C60 powder sample via photosensitization process. To capture the very weak SOP signal, a dichroic mirror was placed between the objective and tube lens of the FLIM and used to divide the luminescence returning from the sample into two beams: the reflected SOP beam and the transmitted photoluminescence of C60 (C60-PL) beam. The C60-PL beam entered the scanner of the FLIM and followed the normal optical path of the FLIM, while the SOP steered clear of the scanner and directly entered a finely designed SOP detection channel. Confocal C60-PL images and nonconfocal SOP images were then simultaneously obtained by using laser-scanning mode. Experimental results show that (1) under laser-scanning mode, the obstacle to confocal SOP imaging is the infrared-incompatible scanner, which can be solved by using an infrared-compatible scanner. Confocal SOP imaging is also expected to be realized under stage-scanning mode when the laser beam is parked and meanwhile a pinhole is added into the SOP detection channel. (2) A great challenge to SOP imaging is its extraordinarily long imaging time, and selecting only a few interesting points from fluorescence images to measure their SOP time-dependent traces may be a correct compromise.

  11. Development of a handheld fluorescence imaging device to investigate the characteristics of protoporphyrin IX fluorescence in healthy and diseased skin.

    Science.gov (United States)

    Kulyk, Olena; Ibbotson, Sally H; Moseley, Harry; Valentine, Ronan M; Samuel, Ifor D W

    2015-12-01

    Topical Photodynamic therapy (PDT) is an effective treatment for superficial non-melanoma skin cancers (NMSC) and dysplasia. During PDT light activates the photosensitiser (PpIX), metabolised from a topical pro-drug. A combination of PpIX, light and molecular oxygen results in inflammation and cell death. However, the outcomes of the treatment could be better. Insufficient biosynthesis of PpIX may be one of the causes of incomplete response or recurrence. Measuring surface fluorescence is usually employed as a means of studying PpIX formation. The aim of this work was to develop a device and a method for convenient fluorescence imaging in clinical settings to gather information on PpIX metabolism in healthy skin and NMSC with a view to improving PDT regimes. A handheld fluorescence camera and a time course imaging method was developed and used in healthy volunteers and patients diagnosed with basal cell carcinoma (BCC) and actinic keratosis (AK). The photosensitiser (precursor) creams used were 5-aminolaevulinic acid (ALA; Ameluz(®)) and methyl aminolevulinate (MAL; Metvix(®)). Pain was assessed using a visual analogue score immediately after the PDT. Fluorescence due to PpIX increases over three hours incubation in healthy skin and in lesional BCC and AK. Distribution of PpIX fluorescence varies between the lesion types and between subjects. There was no significant correlation between PpIX fluorescence characteristics and pro-drug, diagnosis or pain experienced. However, there was a clear dependence on body site. The device and the method developed can be used to assess the characteristics of PpIX fluorescence, quantitative analysis and time course. Our findings show that body site influences PpIX fluorescence which we suggest may be due to the difference in skin temperature at different body sites. Copyright © 2015. Published by Elsevier B.V.

  12. 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

  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.

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

    Science.gov (United States)

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

    2016-07-01

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

  15. 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.

  16. Live Cell Imaging Using Photoswitchable Diarylethene-Doped Fluorescent Polymer Dots.

    Science.gov (United States)

    Osakada, Yasuko; Fukaminato, Tuyoshi; Ichinose, Yuma; Fujitsuka, Mamoru; Harada, Yoshie; Majima, Tetsuro

    2017-10-18

    Fluorescence photoswitching using nanomaterials has recently emerged as a promising approach for the imaging of biological targets. However, despite intensive research efforts during the last decade, practical microscopy of biological targets using photoswitchable nanoparticles in real time remains challenging. To address this problem, we have developed live macrophage cell imaging and single particle imaging methods, using photoswitchable fluorescent diarylethene-doped polymer nanoparticles (P-dots) under Xe lamp irradiation. We established a 34-times prolonged "off-state", using P-dots doped with a diarylethene-containing methoxy substituent, upon visible-light irradiation using a Xe lamp and a green fluorescent protein filter cube. To demonstrate the practicality of doped P-dots imaging, we imaged lysosomes in macrophage cells, and observed 11-times slower recovery of the fluorescence from the "off-state" to the "on-state", indicating their potential for cellular imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Deep-brain imaging via epi-fluorescence Computational Cannula Microscopy

    Science.gov (United States)

    Kim, Ganghun; Nagarajan, Naveen; Pastuzyn, Elissa; Jenks, Kyle; Capecchi, Mario; Shepherd, Jason; Menon, Rajesh

    2017-03-01

    Here we demonstrate widefield (field diameter = 200 μm) fluorescence microscopy and video imaging inside the rodent brain at a depth of 2 mm using a simple surgical glass needle (cannula) of diameter 0.22 mm as the primary optical element. The cannula guides excitation light into the brain and the fluorescence signal out of the brain. Concomitant image-processing algorithms are utilized to convert the spatially scrambled images into fluorescent images and video. The small size of the cannula enables minimally invasive imaging, while the long length (>2 mm) allow for deep-brain imaging with no additional complexity in the optical system. Since no scanning is involved, widefield fluorescence video at the native frame rate of the camera can be achieved.

  18. Line scan--structured illumination microscopy super-resolution imaging in thick fluorescent samples.

    Science.gov (United States)

    Mandula, Ondrej; Kielhorn, Martin; Wicker, Kai; Krampert, Gerhard; Kleppe, Ingo; Heintzmann, Rainer

    2012-10-22

    Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen's inner structure.

  19. Optical imaging of non-fluorescent nanoparticle probes in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Gufeng; Stender, Anthony S.; Sun, Wei; and Fang, Ning

    2009-12-17

    Precise imaging of cellular and subcellular structures and dynamic processes in live cells is crucial for fundamental research in life sciences and in medical applications. Non-fluorescent nanoparticles are an important type of optical probe used in live-cell imaging due to their photostability, large optical cross-sections, and low toxicity. Here, we provide an overview of recent developments in the optical imaging of non-fluorescent nanoparticle probes in live cells.

  20. A fast image registration approach of neural activities in light-sheet fluorescence microscopy images

    Science.gov (United States)

    Meng, Hui; Hui, Hui; Hu, Chaoen; Yang, Xin; Tian, Jie

    2017-03-01

    The ability of fast and single-neuron resolution imaging of neural activities enables light-sheet fluorescence microscopy (LSFM) as a powerful imaging technique in functional neural connection applications. The state-of-art LSFM imaging system can record the neuronal activities of entire brain for small animal, such as zebrafish or C. elegans at single-neuron resolution. However, the stimulated and spontaneous movements in animal brain result in inconsistent neuron positions during recording process. It is time consuming to register the acquired large-scale images with conventional method. In this work, we address the problem of fast registration of neural positions in stacks of LSFM images. This is necessary to register brain structures and activities. To achieve fast registration of neural activities, we present a rigid registration architecture by implementation of Graphics Processing Unit (GPU). In this approach, the image stacks were preprocessed on GPU by mean stretching to reduce the computation effort. The present image was registered to the previous image stack that considered as reference. A fast Fourier transform (FFT) algorithm was used for calculating the shift of the image stack. The calculations for image registration were performed in different threads while the preparation functionality was refactored and called only once by the master thread. We implemented our registration algorithm on NVIDIA Quadro K4200 GPU under Compute Unified Device Architecture (CUDA) programming environment. The experimental results showed that the registration computation can speed-up to 550ms for a full high-resolution brain image. Our approach also has potential to be used for other dynamic image registrations in biomedical applications.

  1. Image-guided cancer surgery : the value of near-infrared fluorescence imaging during oncologic and gastrointestinal procedures

    NARCIS (Netherlands)

    Verbeek, Floris Paul Reinier

    2015-01-01

    Intraoperative imaging using near-infrared (NIR) fluorescence is a relatively new technique that can be used to visualize tumor tissue, sentinel nodes and vital anatomical structures. This thesis is divided in three parts. In part one the ability to visualize surgical margins using NIR fluorescence

  2. Development of a neutral embedding resin for optical imaging of fluorescently labeled biological tissue

    Science.gov (United States)

    Zhou, Hongfu; Gang, Yadong; Chen, Shenghua; Wang, Yu; Xiong, Yumiao; Li, Longhui; Yin, Fangfang; Liu, Yue; Liu, Xiuli; Zeng, Shaoqun

    2017-10-01

    Plastic embedding is widely applied in light microscopy analyses. Previous studies have shown that embedding agents and related techniques can greatly affect the quality of biological tissue embedding and fluorescent imaging. Specifically, it is difficult to preserve endogenous fluorescence using currently available acidic commercial embedding resins and related embedding techniques directly. Here, we developed a neutral embedding resin that improved the green fluorescent protein (GFP), yellow fluorescent protein (YFP), and DsRed fluorescent intensity without adjusting the pH value of monomers or reactivating fluorescence in lye. The embedding resin had a high degree of polymerization, and its fluorescence preservation ratios for GFP, YFP, and DsRed were 126.5%, 155.8%, and 218.4%, respectively.

  3. 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.

  4. Dual-modal nanoprobes for imaging of mesenchymal stem cell transplant by MRI and fluorescence imaging.

    Science.gov (United States)

    Sung, Chang Kyu; Hong, Kyung Ah; Lin, Shunmei; Lee, Yuwon; Cha, Jinmyung; Lee, Jin-Kyu; Hong, Cheol Pyo; Han, Bong Soo; Jung, Sung Il; Kim, Seung Hyup; Yoon, Kang Sup

    2009-01-01

    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(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(2)(RITC)-PEG showed both superparamagnetic and fluorescent properties. The r(1) and r(2) relaxivity values of the MNP@SiO(2)(RITC)-PEG were 0.33 and 398 mM(-1) s(-1) at 1.5T, respectively, and 0.29 and 453 mM(-1) s(-1) at 3T, respectively. The effective internalization of MNP@SiO(2)(RITC)-PEG into hMSCs was observed by confocal laser scanning fluorescence microscopy. The transmission electron microscopy images showed that MNP@SiO(2)(RITC)-PEG was internalized into the cells and mainly resided in the cytoplasm. The viability and proliferation of MNP@SiO(2)(RITC)-PEG-labeled hMSCs were not significantly different from the control cells. MNP@SiO(2)(RITC)-PEG-labeled hMSCs were observed in vitro and in vivo with optical and MR imaging. MNP@SiO(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. Time-domain imaging with quench-based fluorescent contrast agents

    Science.gov (United States)

    Akers, Walter J.; Solomon, Metasebya; Sudlow, Gail P.; Berezin, Mikhail; Achilefu, Samuel

    2012-03-01

    Quench-based probes utilize unique characteristics of fluorescence resonance energy transfer (FRET) to enhance contrast upon de-quenching. This mechanism has been used in a variety of molecular probes for imaging of cancer related enzyme activity such as matrix metalloproteinases, cathepsins and caspases. While non-fluorescent upon administration, fluorescence can be restored by separation of donor and acceptor, resulting in higher intensity in the presence of activator. Along with decreased quantum yield, FRET also results in altered fluorescence lifetime. Time-domain imaging can further enhance contrast and information yield from quench-based probes. We present in vivo time-domain imaging for detecting activation of quench-based probes. Quench-based probes utilize unique characteristics of fluorescence resonance energy transfer (FRET) to enhance contrast upon de-quenching. This mechanism has been used in a variety of molecular probes for imaging of cancer related enzyme activity such as matrix metalloproteinases, cathepsins and caspases. While non-fluorescent upon administration, fluorescence can be restored by separation of donor and acceptor, resulting in higher intensity in the presence of activator. Along with decreased quantum yield, FRET also results in altered fluorescence lifetime. Time-domain imaging can further enhance contrast and information yield from quench-based probes. We present in vivo time-domain imaging for detecting activation of quench-based probes. Time-domain diffuse optical imaging was performed to assess the FRET and quenching in living mice with orthotopic breast cancer. Tumor contrast enhancement was accompanied by increased fluorescence lifetime after administration of quenched probes selective for matrix metalloproteinases while no significant change was observed for non-quenched probes for integrin receptors. These results demonstrate the utility of timedomain imaging for detection of cancer-related enzyme activity in vivo.

  6. Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for "Technicolor" cancer imaging.

    Science.gov (United States)

    Tran Cao, Hop S; Reynoso, Jose; Yang, Meng; Kimura, Hiroaki; Kaushal, Sharmeela; Snyder, Cynthia S; Hoffman, Robert M; Bouvet, Michael

    2009-05-15

    A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non-transgenic nude mice with the transgenic CK/ECFP mouse in which the beta-actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA-1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color-coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color-coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment. 2009 Wiley-Liss, Inc.

  7. Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

    Science.gov (United States)

    Sun, Yen; Lo, Wen; Lin, Sung-Jan; Lin, Wei-Chou; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover, increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area. In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.

  8. 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...

  9. Application of fluorescence spectroscopy and imaging in the detection of a photosensitizer in photodynamic therapy

    Science.gov (United States)

    Zang, Lixin; Zhao, Huimin; Zhang, Zhiguo; Cao, Wenwu

    2017-02-01

    Photodynamic therapy (PDT) is currently an advanced optical technology in medical applications. However, the application of PDT is limited by the detection of photosensitizers. This work focuses on the application of fluorescence spectroscopy and imaging in the detection of an effective photosenzitizer, hematoporphyrin monomethyl ether (HMME). Optical properties of HMME were measured and analyzed based on its absorption and fluorescence spectra. The production mechanism of its fluorescence emission was analyzed. The detection device for HMME based on fluorescence spectroscopy was designed. Ratiometric method was applied to eliminate the influence of intensity change of excitation sources, fluctuates of excitation sources and photo detectors, and background emissions. The detection limit of this device is 6 μg/L, and it was successfully applied to the diagnosis of the metabolism of HMME in the esophageal cancer cells. To overcome the limitation of the point measurement using fluorescence spectroscopy, a two-dimensional (2D) fluorescence imaging system was established. The algorithm of the 2D fluorescence imaging system is deduced according to the fluorescence ratiometric method using bandpass filters. The method of multiple pixel point addition (MPPA) was used to eliminate fluctuates of signals. Using the method of MPPA, SNR was improved by about 30 times. The detection limit of this imaging system is 1.9 μg/L. Our systems can be used in the detection of porphyrins to improve the PDT effect.

  10. Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies

    NARCIS (Netherlands)

    van Staveren, HJ; Speelman, OC; Witjes, MJH; Cincotta, L

    Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing

  11. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

    Science.gov (United States)

    Boreham, Alexander; Brodwolf, Robert; Walker, Karolina; Haag, Rainer; Alexiev, Ulrike

    2016-12-24

    The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

  12. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine

    Directory of Open Access Journals (Sweden)

    Alexander Boreham

    2016-12-01

    Full Text Available The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

  13. Soft-x-ray-induced ionization and fragmentation dynamics of Sc3N @C80 investigated using an ion-ion-coincidence momentum-imaging technique

    Science.gov (United States)

    Xiong, Hui; Obaid, Razib; Fang, Li; Bomme, Cédric; Kling, Nora G.; Ablikim, Utuq; Petrovic, Vladimir; Liekhus-Schmaltz, Chelsea E.; Li, Heng; Bilodeau, Rene C.; Wolf, Thomas; Osipov, Timur; Rolles, Daniel; Berrah, Nora

    2017-09-01

    The fragmentation dynamics of an endohedral fullerene, S c3N @C80 , after absorption of a soft-x-ray photon, has been studied with an ion-ion-coincidence momentum-imaging technique. Molecular inner-shell ionization at 406.5 eV, targeting the Sc (2 p ) shell of the encapsulated S c3N moiety and the C (1 s ) shell of the C80 cage, leads to the cage fragmentation through evaporation of C2, emission of small molecular carbon ions (Cn+ , n ≤24 ), and release of Sc and Sc-containing ions associated with the carbon cage opening or fragmentation. The predominant charge states of Sc and Sc-containing ionic fragments are +1 despite an effective Sc valence of 2.4, indicating that charge transfer or redistribution plays an important role in the fragmentation of the encaged S c3N . Sequential emission of two out of the three Sc atoms of the encaged moiety, via Coulomb explosion in the form of S c+ or Sc-containing ions, is significant. We also find that the resonant excitation of the Sc (2 p ) shell electrons significantly increased the yield of the parent S c3N @C80 and its fragment ions, partially attributed to the collision of the energetic Auger electrons from the Sc site with the carbon cage.

  14. Impact of indocyanine green fluorescent image-guided surgery for parapharyngeal space tumours.

    Science.gov (United States)

    Yokoyama, Junkichi; Ooba, Shinichi; Fujimaki, Mitsuhisa; Anzai, Takashi; Yoshii, Ryota; Kojima, Masataka; Ikeda, Katsuhisa

    2014-09-01

    In parapharyngeal space dissection, significant complications such as dysphagia and carotid artery rupture have been reported. In order to resect tumours safely in narrow parapharyngeal space, we propose indocyanine green (ICG) florescence image for navigation surgery. To evaluate the usefulness of ICG fluorescent image-guided surgery for parapharyngeal space tumours. 0.5 mg/kg of ICG was injected via the cephalic vein. Observation of the fluorescent image was performed with HEMS (HyperEye Medical System) at 10-30 min after injection. At first, the position of the tumour was marked over pharyngeal mucosa according to ICG fluorescence imaging with HEMS. We also confirmed submucosal tumours hidden under fascia using HEMS imaging again and resected them. All tumours displayed bright fluorescence emissions which clearly contrasted with the normal structures. Even with the submucosal tumour covered with and obscured by fasciae, we could observe the tumour clearly under HEMS imaging. Tumours behind the carotid artery and lower cranial nerves also were displayed bright fluorescence emissions and were clearly detected. As a result, we could completely remove the tumour safely and noninvasively to preserve pharyngeal functions. ICG fluorescence imaging is effective for the detection and resection of the parapharyngeal space tumours with preserving functions. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  15. Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images

    NARCIS (Netherlands)

    Fereidouni, F.|info:eu-repo/dai/nl/372641431; Bader, A.N.|info:eu-repo/dai/nl/291137334; Gerritsen, H.C.|info:eu-repo/dai/nl/071548777

    2012-01-01

    A new global analysis algorithm to analyse (hyper-) spectral images is presented. It is based on the phasor representation that has been demonstrated to be very powerful for the analysis of lifetime imaging data. In spectral phasor analysis the fluorescence spectrum of each pixel in the image is

  16. Molecular imaging of small animals with fluorescent proteins: from projection to multimodality.

    Science.gov (United States)

    Yang, Xiaoquan; Gong, Hui; Fu, Jianwei; Quan, Guotao; Huang, Chuan; Luo, Qingming

    2012-06-01

    Fluorescent proteins (FPs) have been widely adopted in cell research for protein trafficking and reporter gene expression studies, as well as to study other biological processes. However, biological tissue has high light scattering and high absorption coefficients of visible light; hence, using FPs in small animal imaging remains a challenge, especially when the FPs are located deep in the tissue. In small animals, fluorescence molecular imaging could potentially address this difficulty. We constructed fluorescence molecular imaging systems that have two modes: a planner mode (projection imaging) and a multimodality mode (fluorescence molecular tomography and micro-CT). The planner mode can provide projection images of a fluorophore in the whole body of a small animal, whereas three-dimensional information can be offered by multimodality mode. The planner imaging system works in the reflection mode and is designed to provide fast imaging. The multimodality imaging system is designed to allow quantification and three-dimensional localization of fluorophores. A nude mouse with a tumour targeted with a far-red FP, which is appropriate for in vivo imaging, was adopted to validate the two systems. The results indicate that the planner imaging system is probably suitable for high throughput molecular imaging, whereas the multimodality imaging system is fit for quantitative research. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Multifunctional nanoprobes for both fluorescence and 19F magnetic resonance imaging.

    Science.gov (United States)

    Guo, Chang; Xu, Minmin; Xu, Suying; Wang, Leyu

    2017-06-01

    Fluorescence is widely used for cell imaging due to its high sensitivity and rich color choice but limited for in vivo imaging because of its low light penetration. Meanwhile, magnetic resonance imaging (MRI) is widely applied for in vivo diagnosis but not suitable for cell imaging because of its low resolution. Compared to 1H-MRI, 19F-MRI is more suitable for clinical application due to its high sensitivity but fabricating 19F-MRI probes is a great challenge. Therefore, it is highly desirable to develop a dual-modal imaging probe for both cell fluorescence imaging and in vivo19F-MRI with high sensitivity and deep penetration. In this study, 19F moiety loaded nanocomposites with an organic fluorescent core were successfully prepared via a facile strategy by encapsulating organic dyes with oleylamine-functionalized polysuccinimide and 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PDTES). The aggregation of organic fluorescent dyes in the core results in significant fluorescence for optical imaging, while the 19F moieties on PDTES allow for simultaneous 19F MRI. Moreover, the nanocomposites exhibited high water dispersibility and excellent biocompatibility. These properties make them promising for both cell imaging and in vivo imaging applications.

  18. Evaluating the use of fluorescent imaging for the quantification of dental fluorosis

    Science.gov (United States)

    2012-01-01

    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 pfluorosis or at low levels of fluorosis severity had an adverse impact on tooth fluorescence and hence the outcome variable. This in conjunction with an uneven distribution of subjects across the range of fluorosis presentations may have resulted in the lower than anticipated correlations between the fluorescent imaging metrics and the photographic fluorosis scores. Nevertheless, the fluorescence imaging technique was able to discriminate between a fluoridated and non-fluoridated population (pfluorosis when used adjunctively with photographic scoring. PMID:23116324

  19. Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

    Science.gov (United States)

    Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

    2015-10-07

    Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

  20. Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes

    National Research Council Canada - National Science Library

    Mark Bates; Bo Huang; Graham T. Dempsey; Xiaowei Zhuang

    2007-01-01

    .... Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demostrate multicolor stochastic optical reconstruction microscopy (STORM...

  1. QUANTITATIVE IMAGING AND STATISTICAL ANALYSIS OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) OF AUREOBASIDIUM PULLULANS. (R823845)

    Science.gov (United States)

    AbstractImage and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal o...

  2. Multiphoton Laser Microscopy and Fluorescence Lifetime Imaging for the Evaluation of the Skin

    Directory of Open Access Journals (Sweden)

    Stefania Seidenari

    2012-01-01

    Full Text Available Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM, is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.

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

    Energy Technology Data Exchange (ETDEWEB)

    Autiero, Maddalena [Dipartimento di Scienze Fisiche, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Celentano, Luigi [Dipartimento di Scienze Biomorfologiche e Funzionali, Universita di Napoli Federico II, Via Pansini 5, I-80126 Naples (Italy); Cozzolino, Rosanna [Dipartimento di Biologia Strutturale e Funzionale, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Laccetti, Paolo [Dipartimento di Biologia Strutturale e Funzionale, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Marotta, Marcello [Dipartimento di Medicina Clinica e Sperimentale, Universita di Napoli Federico II, Via Pansini 5, I-80131 Naples (Italy); Mettivier, Giovanni [Dipartimento di Scienze Fisiche, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Istituto Nazionale di Fisica Nucleare (INFN), Via Cinthia, I-80126 Naples (Italy); Cristina Montesi, Maria [Dipartimento di Scienze Fisiche, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Istituto Nazionale di Fisica Nucleare (INFN), Via Cinthia, I-80126 Naples (Italy); Quarto, Maria [Dipartimento di Scienze Fisiche, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Riccio, Patrizia [Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita di Napoli Federico II, Via Pansini 5, I-80131 Naples (Italy); Roberti, Giuseppe [Dipartimento di Scienze Fisiche, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy)]. E-mail: roberti@unina.it; Russo, Paolo [Dipartimento di Scienze Fisiche, Universita di Napoli Federico II, Via Cinthia, I-80126 Naples (Italy); Istituto Nazionale di Fisica Nucleare (INFN), Via Cinthia, I-80126 Naples (Italy)

    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.

  4. B-Spline potential function for maximum a-posteriori image reconstruction in fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Shilpa Dilipkumar

    2015-03-01

    Full Text Available An iterative image reconstruction technique employing B-Spline potential function in a Bayesian framework is proposed for fluorescence microscopy images. B-splines are piecewise polynomials with smooth transition, compact support and are the shortest polynomial splines. Incorporation of the B-spline potential function in the maximum-a-posteriori reconstruction technique resulted in improved contrast, enhanced resolution and substantial background reduction. The proposed technique is validated on simulated data as well as on the images acquired from fluorescence microscopes (widefield, confocal laser scanning fluorescence and super-resolution 4Pi microscopy. A comparative study of the proposed technique with the state-of-art maximum likelihood (ML and maximum-a-posteriori (MAP with quadratic potential function shows its superiority over the others. B-Spline MAP technique can find applications in several imaging modalities of fluorescence microscopy like selective plane illumination microscopy, localization microscopy and STED.

  5. 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

  6. Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells

    OpenAIRE

    Oida, T; Sako, Y.; Kusumi, A.

    1993-01-01

    A new method of fluorescence microscopy for cell imaging has been developed that takes advantage of the spatial variations of fluorescence lifetimes in single cells as a source of image contrast, and thus it is named "fluorescence lifetime imaging microscopy (flimscopy)". Since time-resolved fluorescence measurements are sensitive to molecular dynamics and interactions, flimscopy allows the molecular information to be visualized in single cells. In flimscopy measurements, several (nanosecond)...

  7. 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.

  8. LED-Induced fluorescence and image analysis to detect stink bug damage in cotton bolls.

    Science.gov (United States)

    Mustafic, Adnan; Roberts, Erin E; Toews, Michael D; Haidekker, Mark A

    2013-02-20

    Stink bugs represent a major agricultural pest complex attacking more than 200 wild and cultivated plants, including cotton in the southeastern US. Stink bug feeding on developing cotton bolls will cause boll abortion or lint staining and thus reduced yield and lint value. Current methods for stink bug detection involve manual harvesting and cracking open of a sizable number of immature cotton bolls for visual inspection. This process is cumbersome, time consuming, and requires a moderate level of experience to obtain accurate estimates. To improve detection of stink bug feeding, we present here a method based on fluorescent imaging and subsequent image analyses to determine the likelihood of stink bug damage in cotton bolls. Damage to different structures of cotton bolls including lint and carpal wall can be observed under blue LED-induced fluorescence. Generally speaking, damaged regions fluoresce green, whereas non-damaged regions with chlorophyll fluoresce red. However, similar fluorescence emission is also observable on cotton bolls that have not been fed upon by stink bugs. Criteria based on fluorescent intensity and the size of the fluorescent spot allow to differentiate between true positives (fluorescent regions associated with stink bug feeding) and false positives (fluorescent regions due to other causes). We found a detection rates with two combined criteria of 87% for true-positive marks and of 8% for false-positive marks. The imaging technique presented herein gives rise to a possible detection apparatus where a cotton boll is imaged in the field and images processed by software. The unique fluorescent signature left by stink bugs can be used to determine with high probability if a cotton boll has been punctured by a stink bug. We believe this technique, when integrated in a suitable device, could be used for more accurate detection in the field and allow for more optimized application of pest control.

  9. A Concept for a Sensitive Micro Total Analysis System for High Throughput Fluorescence Imaging

    OpenAIRE

    Rabner, Arthur; Shacham, Yosi

    2006-01-01

    This paper discusses possible methods for on-chip fluorescent imaging for integrated bio-sensors. The integration of optical and electro-optical accessories, according to suggested methods, can improve the performance of fluorescence imaging. It can boost the signal to background ratio by a few orders of magnitudes in comparison to conventional discrete setups. The methods that are present in this paper are oriented towards building reproducible arrays for high-throughput micro total analysis...

  10. The use of near-infrared fluorescence imaging in endocrine surgical procedures.

    Science.gov (United States)

    Kahramangil, Bora; Berber, Eren

    2017-06-01

    Near-infrared fluorescence imaging in endocrine surgery is a new, yet highly investigated area. It involves indocyanine green use as well as parathyroid autofluorescence. Several groups have described their technique and reported on the observed utility. However, there is no consensus on technical details. Furthermore, the correlation between intraoperative findings and postoperative outcomes is unclear. With this study, we aim to review the current literature on fluorescence imaging and share our insights on technical details. © 2017 Wiley Periodicals, Inc.

  11. Whole-field fluorescence microscope with digital micromirror device: imaging of biological samples.

    Science.gov (United States)

    Fukano, Takashi; Miyawaki, Atsushi

    2003-07-01

    We have developed a whole-field fluorescence microscope equipped with a Digital Micromirror Device to acquire optically sectioned images by using the fringe-projection technique and the phase-shift method. This system allows free control of optical sectioning strength through computer-controlled alteration of the fringe period projected onto a sample. We have employed this system to image viable cells expressing fluorescent proteins and discussed its biological applications.

  12. Lifetime Fluorescence and Raman Imaging for Detection of Wound Failure and Heterotopic Ossification

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-12-2-0075 TITLE: Lifetime Fluorescence and Raman Imaging for Detection of Wound Failure and Heterotopic Ossification ...15Sep2012 - 14Sep2015 4. TITLE AND SUBTITLE Lifetime Fluorescence and Raman Imaging for Detection of Wound Failure and Heterotopic Ossification 5a...with the presence of inflammatory cytokines [1]. An additional wound failure mechanism is the formation of Heterotopic Ossification (HO) [3, 7]. In

  13. Carbon Dots as Nontoxic and High-Performance Fluorescence Imaging Agents

    OpenAIRE

    Yang, Sheng-Tao; Wang, Xin; Wang, Haifang; Lu, Fushen; Luo, Pengju G.; Cao, Li; Meziani, Mohammed J.; Liu, Jia-Hui; Liu, Yuanfang; Chen, Min; Huang, Yipu; Sun, Ya-Ping

    2009-01-01

    Fluorescent carbon dots (small carbon nanoparticles with the surface passivated by oligomeric PEG molecules) were evaluated for their cytotoxicity and in vivo toxicity and also for their optical imaging performance in reference to that of the commercially supplied CdSe/ZnS quantum dots. The results suggested that the carbon dots were biocompatible, and their performance as fluorescence imaging agents was competitive. The implication to the use of carbon dots for in vitro and in vivo applicati...

  14. Carbon Dots as Nontoxic and High-Performance Fluorescence Imaging Agents.

    Science.gov (United States)

    Yang, Sheng-Tao; Wang, Xin; Wang, Haifang; Lu, Fushen; Luo, Pengju G; Cao, Li; Meziani, Mohammed J; Liu, Jia-Hui; Liu, Yuanfang; Chen, Min; Huang, Yipu; Sun, Ya-Ping

    2009-09-28

    Fluorescent carbon dots (small carbon nanoparticles with the surface passivated by oligomeric PEG molecules) were evaluated for their cytotoxicity and in vivo toxicity and also for their optical imaging performance in reference to that of the commercially supplied CdSe/ZnS quantum dots. The results suggested that the carbon dots were biocompatible, and their performance as fluorescence imaging agents was competitive. The implication to the use of carbon dots for in vitro and in vivo applications is discussed.

  15. Green Synthesis of Bifunctional Fluorescent Carbon Dots from Garlic for Cellular Imaging and Free Radical Scavenging.

    Science.gov (United States)

    Zhao, Shaojing; Lan, Minhuan; Zhu, Xiaoyue; Xue, Hongtao; Ng, Tsz-Wai; Meng, Xiangmin; Lee, Chun-Sing; Wang, Pengfei; Zhang, Wenjun

    2015-08-12

    Nitrogen and sulfur codoped carbon dots (CDs) were prepared from garlic by a hydrothermal method. The as-prepared CDs possess good water dispersibility, strong blue fluorescence emission with a fluorescent quantum yield of 17.5%, and excellent photo and pH stabilities. It is also demonstrated that the fluorescence of CDs are resistant to the interference of metal ions, biomolecules, and high ionic strength environments. Combining with low cytotoxicity properties, CDs could be used as an excellent fluorescent probe for cellular multicolor imaging. Moreover, the CDs were also demonstrated to exhibit favorable radical scavenging activity.

  16. Fiber optic probe for region of interest (ROI) selective time averaged multi-fluorescence imaging

    Science.gov (United States)

    Shinde, Anant; Perinchery, Sandeep M.; Murukeshan, V. M.

    2017-06-01

    Time averaged imaging is one of the widely used methods to achieve improved image quality, used in different types of microscopic methods. Time averaged imaging refers to adjusting the exposure time of the imaging system to obtain optimal images. In state of the art microscopes, the region of interest (ROI) of illumination beam for time averaged imaging can be selected to be of regular shapes such as circle or rectangle. This forces smallest possible ROI to be larger than the actual sample's ROI which can be of a specific shape with complex contours. In this context, we present a flexible fiber bundle based illumination probe capable of illuminating samples of irregular shapes for time averaged imaging. Further, this probe is capable of multi-wavelength illumination, hence can be used for multi-fluorescence imaging. The fiber probe with features such as region selective and multi- wavelength illumination allows it to be used for optimal imaging of multi-fluorescence sample.

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

    Science.gov (United States)

    2009-01-01

    Background Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. Methods Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM) and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. Results Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. Conclusion Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer. PMID:19133127

  18. Automated analysis of fluorescence lifetime imaging microscopy (FLIM) data based on the Laguerre deconvolution method.

    Science.gov (United States)

    Pande, Paritosh; Jo, Javier A

    2011-01-01

    In fluorescence lifetime imaging microscopy (FLIM), fluorescence time decay at each pixel of the imaged sample are measured. Every recorded fluorescence decay corresponds to the time convolution of the instrument response with the intrinsic fluorescence impulse response function (IRF), from which the sample fluorescence lifetime is determined. To estimate the IRF, the instrument response thus needs to be deconvolved from the recorded fluorescence decay. We have recently introduced a novel FLIM time-deconvolution method based on the linear expansion of the fluorescence decays on an orthonormal Laguerre basis. Since this method allows simultaneous estimation of the IRFs at all pixels, it performs at least two orders of magnitude faster than standard algorithms. In its original implementation, however, the Laguerre basis, determined by the Laguerre parameter α, is selected using a heuristic approach. Here, we present an automated implementation, whereby the Laguerre parameter α is treated as a free parameter within a nonlinear least squares optimization scheme. The new implementation combines the unmatched inherent computational speed of the Laguerre deconvolution method with a systematic model selection approach. This method will thus facilitate applications of FLIM requiring automatic estimation of the spatial distribution of fluorescence lifetimes, such as in in vivo tissue FLIM imaging.

  19. 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. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  20. Optically sectioned wide-field fluorescence lifetime imaging microscopy enabled by structured illumination

    Science.gov (United States)

    Hinsdale, Taylor; Olsovsky, Cory; Rico-Jimenez, Jose J.; Maitland, Kristen C.; Jo, Javier A.; Malik, Bilal H.

    2017-01-01

    In this paper, we demonstrate the ability of structured illumination microscopy to enhance the ability of fluorescence lifetime imaging to resolve fluorescence lifetimes in relatively thick samples that possess distinct but spectrally overlapping fluorescent layers. Structured illumination fluorescent lifetime imaging microscopy (SI-FLIM) is shown to be able to accurately reconstruct lifetime values in homogenous fluorophore samples (POPOP, NADH, and FAD) as well as accurately measure fluorescent lifetime in two layer models that are layered with NADH/FAD over POPOP, where NADH/FAD and POPOP have spectral overlap. Finally, the ability of SI-FLIM was demonstrated in a hamster cheek pouch ex vivo to show that more accurate lifetimes could be measured for each layer of interest in the oral mucosa (epithelium and submucosa). PMID:28663841

  1. Non-fused Phospholes as Fluorescent Probes for Imaging of Lipid Droplets in Living Cells

    Directory of Open Access Journals (Sweden)

    K. Peter R. Nilsson

    2017-04-01

    Full Text Available Molecular tools for fluorescent imaging of specific compartments in cells are essential for understanding the function and activity of cells. Here, we report the synthesis of a series of pyridyl- and thienyl-substituted phospholes and the evaluation of these dyes for fluorescent imaging of cells. The thienyl-substituted phospholes proved to be successful for staining of cultured normal and malignant cells due to their fluorescent properties and low toxicity. Co-staining experiments demonstrated that these probes target lipid droplets, which are, lipid-storage organelles found in the cytosol of nearly all cell types. Our findings confirm that thienyl-substituted phospholes can be utilized as fluorescent tools for vital staining of cells, and we foresee that these fluorescent dyes might be used in studies to unravel the roles that lipid droplets play in cellular physiology and in diseases.

  2. Development of Micro-Structured Fluorescent Plates for High-resolution Imaging

    Science.gov (United States)

    Sakai, Takuro; Yasuda, Ryo; Iikura, Hiroshi; Matsubayashi, Masahito

    We have developed novel fluorescent plates for high-resolution imaging. The devices consist of capillary plates and fine phosphor grains; specifically, each capillary is filled with the grains. The fabricated fluorescent plates were characterized by optical microscopy and scanning electron microscopy. The observation revealed that all capillaries were well filled with grains. Imaging experiments were performed using a small X-ray source. Results show that the fluorescent plates are expected to be compatible with both high spatial resolution and good detection efficiency.

  3. Low power super resolution fluorescence microscopy by lifetime modification and image reconstruction.

    Science.gov (United States)

    Marsh, Richard J; Culley, Siân; Bain, Angus J

    2014-05-19

    We demonstrate a new method for obtaining sub-diffraction resolution in fluorescence microscopy. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured (fundamental Gaussian) continuous wave stimulated emission depletion. A reduced point spread functions (PSF) is obtained by the recombination of time segments of the evolving image. A significant reduction in the PSF for 20 nm fluorescent beads (ca. 240 nm to 125 nm) is obtained with an on-sample power of 7.5 mW (17 MW/cm2) - substantially lower than that required for spatially structured stimulated emission depletion microscopy.

  4. 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 effic...... 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. An endoscopic fluorescence imaging system for simultaneous visual examination and photodetection of cancers

    Science.gov (United States)

    Wagnières, Georges A.; Studzinski, André P.; van den Bergh, Hubert E.

    1997-01-01

    We describe the design and performance tested during six years of clinical trials of a fluorescence endoscope for the detection and delineation of cancers in several hollow organs. The apparatus is based on the imaging of the laser-induced fluorescence that differs between a tumor and its surrounding normal tissue. The tests are carried out in the upper aerodigestive tract, the tracheobronchial tree, the esophagus, and the colon. In the three former cases an exogenous dye is used (Photofrin II), whereas in the latter case fluorescein molecules conjugated with monoclonal antibodies directed against carcinoembryonic antigen are injected. The decrease of native tissue autofluorescence observed in early cancers is also used for detecting lesions in the tracheobronchial tree. The fluorescence contrast between the tumor and surrounding normal tissue is enhanced by real time image processing. This is done by simultaneously recording the fluorescence image in two spectral domains, after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Moreover, the device that is described below allows for an immediate observation of the endoscopic area under white light illumination during fluorescence detection in order to localize the origin of the "positive" fluorescence signals. Typical results obtained in the tracheobronchial tree and in the colon are presented and the sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.

  6. Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

    Science.gov (United States)

    Heppert, Jennifer K; Dickinson, Daniel J; Pani, Ariel M; Higgins, Christopher D; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R; Goldstein, Bob

    2016-11-07

    Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. © 2016 Heppert et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  7. Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

    Science.gov (United States)

    Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

    2013-02-01

    The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

  8. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques

    Directory of Open Access Journals (Sweden)

    Biliana Todorova

    2017-01-01

    Full Text Available Purpose. We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Procedures. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM. Results. The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. Conclusions. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

  9. Energy dispersive detector for white beam synchrotron x-ray fluorescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Matthew D., E-mail: Matt.Wilson@stfc.ac.uk; Seller, Paul; Veale, Matthew C. [Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus,UK (United Kingdom); Connolley, Thomas [Diamond Light Source, I12 Beamline, Harwell Campus, Didcot, Oxfordshire (United Kingdom); Dolbnya, Igor P.; Malandain, Andrew; Sawhney, Kawal [Diamond Light Source, B16 Beamline, Harwell Campus, Didcot, Oxfordshire (United Kingdom); Grant, Patrick S.; Liotti, Enzo; Lui, Andrew [Department of Materials, University of Oxford Parks Road, Oxford (United Kingdom)

    2016-07-27

    A novel, “single-shot” fluorescence imaging technique has been demonstrated on the B16 beamline at the Diamond Light Source synchrotron using the HEXITEC energy dispersive imaging detector. A custom made furnace with 200µm thick metal alloy samples was positioned in a white X-ray beam with a hole made in the furnace walls to allow the transmitted beam to be imaged with a conventional X-ray imaging camera consisting of a 500 µm thick single crystal LYSO scintillator, mirror and lens coupled to an AVT Manta G125B CCD sensor. The samples were positioned 45° to the incident beam to enable simultaneous transmission and fluorescence imaging. The HEXITEC detector was positioned at 90° to the sample with a 50 µm pinhole 13 cm from the sample and the detector positioned 2.3m from pinhole. The geometric magnification provided a field of view of 1.1×1.1mm{sup 2} with one of the 80×80 pixels imaging an area equivalent to 13µm{sup 2}. Al-Cu alloys doped with Zr, Ag and Mo were imaged in transmission and fluorescence mode. The fluorescence images showed that the dopant metals could be simultaneously imaged with sufficient counts on all 80x80 pixels within 60 s, with the X-ray flux limiting the fluorescence imaging rate. This technique demonstrated that it is possible to simultaneously image and identify multiple elements on a spatial resolution scale ~10µm or higher without the time consuming need to scan monochromatic energies or raster scan a focused beam of X-rays. Moving to high flux beamlines and using an array of detectors could improve the imaging speed of the technique with element specific imaging estimated to be on a 1 s timescale.

  10. Breast Cancer Imaging Using the Near-Infrared Fluorescent Agent, CLR1502

    Directory of Open Access Journals (Sweden)

    Melissa L. Korb

    2015-01-01

    Full Text Available Positive margins after breast conservation surgery represent a significant problem in the treatment of breast cancer. The near-infrared fluorescence agent CLR1502 (Cellectar Biosciences, Madison, WI was studied in a preclinical breast cancer model to determine imaging properties and ability to detect small islands of malignancy. Nude mice bearing human breast cancer flank xenografts were given a systemic injection of CLR1502, and imaging was performed using LUNA (Novadaq Technologies Inc., Richmond, BC and Pearl Impulse (LI-COR Biosciences, Lincoln, NE devices. Normal tissues were examined for fluorescence signal, and conventional and fluorescence histology was performed using the Odyssey scanner. Peak tumor to background ratio occurred 2 days after injection with CLR1502. The smallest amount of tumor that was imaged and detected using these devices was 1.9 mg, equivalent to 1.9 × 106 cells. The highest fluorescence signal was seen in tumor and normal lymph node tissue, and the lowest fluorescence signal was seen in muscle and plasma. Human breast cancer tumors can be imaged in vivo with multiple optical imaging platforms using CLR1502. This pilot study supports further investigations of this fluorescent agent for improving surgical resection of malignancies, with the goal of eventual clinical translation.

  11. Breast Cancer Imaging Using the Near-Infrared Fluorescent Agent, CLR1502.

    Science.gov (United States)

    Korb, Melissa L; Warram, Jason M; Grudzinski, Joseph; Weichert, Jamey; Jeffery, Justin; Rosenthal, Eben L

    2015-01-01

    Positive margins after breast conservation surgery represent a significant problem in the treatment of breast cancer. The near-infrared fluorescence agent CLR1502 (Cellectar Biosciences, Madison, WI) was studied in a preclinical breast cancer model to determine imaging properties and ability to detect small islands of malignancy. Nude mice bearing human breast cancer flank xenografts were given a systemic injection of CLR1502, and imaging was performed using LUNA (Novadaq Technologies Inc., Richmond, BC) and Pearl Impulse (LI-COR Biosciences, Lincoln, NE) devices. Normal tissues were examined for fluorescence signal, and conventional and fluorescence histology was performed using the Odyssey scanner. Peak tumor to background ratio occurred 2 days after injection with CLR1502. The smallest amount of tumor that was imaged and detected using these devices was 1.9 mg, equivalent to 1.9 × 106 cells. The highest fluorescence signal was seen in tumor and normal lymph node tissue, and the lowest fluorescence signal was seen in muscle and plasma. Human breast cancer tumors can be imaged in vivo with multiple optical imaging platforms using CLR1502. This pilot study supports further investigations of this fluorescent agent for improving surgical resection of malignancies, with the goal of eventual clinical translation.

  12. Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging

    Science.gov (United States)

    Silva, Susana F.; Batista, Ana; Castejón, Olga C.; Quadrado, Maria João.; Domingues, José Paulo; Morgado, Miguel

    2015-07-01

    Metabolic imaging can be a valuable tool in the early diagnosis of corneal diseases. Cell metabolic changes can be assessed through non-invasive optical methods due to the autofluorescence of metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Both molecules exhibit double exponential fluorescence decays, with well-separated short and long lifetime components, which are related to their protein-bound and free states. Corneal metabolism can be monitored by measuring the relative contribution of these two components. Here we report on the development of a fluorescence lifetime imaging microscope for in vivo measurement of FAD fluorescence lifetimes in corneal cells. The microscope is based on one-photon fluorescence excitation, through a pulsed blue diode laser. Fluorescence lifetime imaging is achieved using the Time-Gated technique. Structured illumination is used to improve the low axial resolution of wide-field time-gated FLIM. A Digital Micromirror Device (DMD) is used to produce the sinusoidal patterns required by structural illumination. The DMD control is integrated with the acquisition software of the imaging system which is based on an ultra-high speed gated image intensifier coupled to a CCD camera. We present preliminary results concerning optical and timing performance of the fluorescence lifetime microscope. Preliminary tests with ex-vivo bovine corneas are also described.

  13. Hyperspectral imaging of endogenous fluorescent metabolic molecules to identify pain states in central nervous system tissue

    Science.gov (United States)

    Staikopoulos, Vasiliki; Gosnell, Martin E.; Anwer, Ayad G.; Mustafa, Sanam; Hutchinson, Mark R.; Goldys, Ewa M.

    2016-12-01

    Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

  14. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P [Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Zhu, R; Mayer, B [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F [Agilent Technologies Austria GmbH, Aubrunnerweg 11, A-4040 Linz (Austria); Salio, M; Shepherd, D; Polzella, P; Cerundolo, V [Cancer Research UK Tumor Immunology Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS (United Kingdom); Dieudonne, M, E-mail: ferry_kienberger@agilent.com [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on {alpha}-galactosylceramide ({alpha}GalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from {approx} 25 to {approx} 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  15. Coincidence studies with antiprotons

    Energy Technology Data Exchange (ETDEWEB)

    McGovern, M; Walters, H R J [Department of Applied Mathematics and Theoretical Physics, Queen' s University, Belfast BT7 1NN (United Kingdom); Assafrao, D; Mohallem, J R [Laboratorio de Atomos e Moleculas Especiais, Departamento de Fisica, ICEx, Universidade Federal de Minas Gerais, P.O Box 702, 30123-970 Belo Horizonte, MG (Brazil); Whelan, Colm T, E-mail: mmcgovern06@qub.ac.u [Department of Physics, Old Dominion University, Norfolk, VA 23529-0116 (United States)

    2010-02-01

    We present a short overview of a new method for calculating fully differential cross sections that is able to describe any aspect of coincidence measurements involving heavy projectiles. The method is based upon impact parameter close coupling with pseudostates. Examples from antiproton impact ionization are shown.

  16. Detection of sentinel node in gastric cancer surgery by indocyanine green fluorescence imaging: comparison with infrared imaging.

    Science.gov (United States)

    Miyashiro, Isao; Miyoshi, Norikatsu; Hiratsuka, Masahiro; Kishi, Kentaro; Yamada, Terumasa; Ohue, Masayuki; Ohigashi, Hiroaki; Yano, Masahiko; Ishikawa, Osamu; Imaoka, Shingi

    2008-06-01

    Secure methods for clinical detection of the sentinel node (SN) are in great demand to avoid unnecessary resection. This was a clinical exploration/feasibility study of a novel detection system for SN biopsy using indocyanine green (ICG) fluorescence imaging in gastric cancer surgery. SN biopsy using ICG dye was performed in three patients who had gastric cancer. ICG fluorescence images were obtained using a detection system comprising a charge-coupled device (CCD) camera with a cut filter as the detector and light emitting diodes (LED) as the light source. The nodes were also examined simultaneously by an infrared (IR) imaging videoscope. Immediately after intraoperative ICG injection, the fluorescence imaging system allowed easy visualization of the lymphatic vessels draining from the primary gastric tumor toward the lymph nodes and tracing of the moving injected dye. Some lymph vessels and nodes were hardly recognized by ICG green color or IR imaging. The ICG fluorescence system also allowed visualization of the lymph node when ICG was injected the day before surgery, similar to the radio-guided method. Detection of SNs in gastric cancer surgery using the ICG fluorescence imaging system is a promising novel technique and may perhaps prove useful for laparoscopic surgery.

  17. Three-Dimensional Live Imaging of Filamentous Fungi with Light Sheet-Based Fluorescence Microscopy (LSFM).

    Science.gov (United States)

    Pampaloni, Francesco; Knuppertz, Laura; Hamann, Andrea; Osiewacz, Heinz D; Stelzer, Ernst H K

    2017-01-01

    We describe a method for the three-dimensional live imaging of filamentous fungi with light sheet-based fluorescence microscopy (LSFM). LSFM provides completely new opportunities to investigate the biology of fungal cells and other microorganisms with high spatial and temporal resolution. As an example, we study the established aging model Podospora anserina. The protocol explains the mounting of the live fungi for the light sheet imaging, the imaging procedure and illustrates basic image processing of data.

  18. 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).

  19. 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.

  20. Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis

    Science.gov (United States)

    Chen, Q. G.; Zhu, H. H.; Xu, Y.; Lin, B.; Chen, H.

    2015-08-01

    A quantitative method to discriminate caries lesions for a fluorescence imaging system is proposed in this paper. The autofluorescence spectral investigation of 39 teeth samples classified by the International Caries Detection and Assessment System levels was performed at 405 nm excitation. The major differences in the different caries lesions focused on the relative spectral intensity range of 565-750 nm. The spectral parameter, defined as the ratio of wavebands at 565-750 nm to the whole spectral range, was calculated. The image component ratio R/(G + B) of color components was statistically computed by considering the spectral parameters (e.g. autofluorescence, optical filter, and spectral sensitivity) in our fluorescence color imaging system. Results showed that the spectral parameter and image component ratio presented a linear relation. Therefore, the image component ratio was graded as 1.62 to quantitatively classify sound, early decay, established decay, and severe decay tissues, respectively. Finally, the fluorescence images of caries were experimentally obtained, and the corresponding image component ratio distribution was compared with the classification result. A method to determine the numerical grades of caries using a fluorescence imaging system was proposed. This method can be applied to similar imaging systems.

  1. Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

    Science.gov (United States)

    Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

    2018-01-01

    Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 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.

  3. Intraoperative fluorescence imaging for personalized brain tumor resection: Current state and future directions

    Directory of Open Access Journals (Sweden)

    Evgenii Belykh

    2016-10-01

    Full Text Available Introduction: Fluorescence-guided surgery is one of the rapidly emerging methods of surgical theranostics. In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients, as well as future applications of recent laboratory and translational studies.Methods: Review of the literature.Results: A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-ALA and ICG, less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine can be used for rapid tumor detection and pathological tissue examination. Other emerging agents such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed.Conclusion: We are standing on the doorstep of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.

  4. uPAR-targeted optical near-infrared (NIR) fluorescence imaging and PET for image-guided surgery in head and neck cancer

    DEFF Research Database (Denmark)

    Christensen, Anders; Juhl, Karina; Persson, Morten

    2017-01-01

    xenograft tongue cancer model. Experimental design and results: Tumor growth of tongue cancer was monitored by bioluminescence imaging (BLI) and MRI. Either ICG-Glu-Glu-AE105 (fluorescent agent) or 64Cu-DOTA-AE105 (PET agent) was injected systemically, and fluorescence imaging or PET/CT imaging...... was performed. Tissue was collected for micro-fluorescence imaging and histology. A clear fluorescent signal was detected in the primary tumor with a mean in vivo tumor-to-background ratio of 2.5. Real-time fluorescence-guided tumor resection was possible, and sub-millimeter tumor deposits could be localized...

  5. Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical Scaffolds.

    Science.gov (United States)

    Neto, Brenno A D; Carvalho, Pedro H P R; Correa, Jose R

    2015-06-16

    This Account describes the origins, features, importance, and trends of the use of fluorescent small-molecule 2,1,3-benzothiadiazole (BTD) derivatives as a new class of bioprobes applied to bioimaging analyses of several (live and fixed) cell types. BTDs have been successfully used as probes for a plethora of biological analyses for only a few years, and the impressive responses obtained by using this important class of heterocycle are fostering the development of new fluorescent BTDs and expanding the biological applications of such derivatives. The first use of a fluorescent small-molecule BTD derivative as a selective cellular probe dates back to 2010, and since then impressive advances have been described by us and others. The well-known limitations of classical scaffolds urged the development of new classes of bioprobes. Although great developments have been achieved by using classical scaffolds such as coumarins, BODIPYs, fluoresceins, rhodamines, cyanines, and phenoxazines, there is still much to be done, and BTDs aim to succeed where these dyes have shown their limitations. Important organelles and cell components such as nuclear DNA, mitochondria, lipid droplets, and others have already been successfully labeled by fluorescent small-molecule BTD derivatives. New technological systems that use BTDs as the fluorophores for bioimaging experiments have been described in recent scientific literature. The successful application of BTDs as selective bioprobes has led some groups to explore their potential for use in studying membrane pores or tumor cells under hypoxic conditions. Finally, BTDs have also been used as fluorescent tags to investigate the action mechanism of some antitumor compounds. The attractive photophysical data typically observed for π-extended BTD derivatives is fostering interest in the use of this new class of bioprobes. Large Stokes shifts, large molar extinction coefficients, high quantum yields, high stability when stored in solution or

  6. Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

    Science.gov (United States)

    Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

    2014-01-01

    Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Diagnostic potential of fluorescence of formalin-fixed paraffin-embedded malignant melanoma and pigmented skin lesions: quantitative study of fluorescence intensity using fluorescence microscope and digital imaging.

    Science.gov (United States)

    Chwirot, B W; Sypniewska, N; Swiatlak, J

    2001-12-01

    The background for this study was reports in the literature of stronger fluorescence observed visually for melanomas compared with benign naevi in formalin-fixed paraffin-embedded sections. Our objective was to carry out a quantitative study of the phenomenon and to investigate if such an approach could be used in the detection of melanomas. Microscopic digital imaging was used to measure quantitatively the fluorescence intensity in specimens from 50 malignant melanomas, four basal cell carcinomas and 58 benign lesions. The mean fluorescence intensity of the melanomas was considerably higher than of the other lesions. For melanomas, the intensity depended both on the distance from the skin surface and the distance from the centre of the lesion. A simple algorithm based on the intensity threshold correctly classified the melanomas with a sensitivity of 74% and a specificity of 59%. Quantitative measurements of the fluorescence of the pigmented skin lesions fixed with formalin and embedded in paraffin can be a useful auxiliary tool for differentiating melanoma from other pigmented lesions histopathologically.

  8. 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.

  9. Red fluorescence imaging for dental plaque detection and quantification: pilot study

    Science.gov (United States)

    Liu, Zhao; Gomez, Juliana; Khan, Soniya; Peru, Debbie; Ellwood, Roger

    2017-09-01

    The red fluorescence of dental plaque originating from porphyrins in oral bacteria may allow visualization, detection, and scoring of plaque without disclosing agents. Two studies were conducted. The first included 24 healthy participants who abstained from oral hygiene for 24 h. Dental plaque was collected from tooth surfaces, and a 10% solution was prepared. These were scanned by a molecular spectrometer to identify the optimum excitation and emission wavelengths of plaque for developing a red fluorescence imaging system. Fourteen healthy subjects completed the second study. After a washout period (1 week), participants had a prophylaxis at baseline and abstained from oral hygiene during the study. They were monitored using the fluorescence imaging system at baseline, 24 h, and 48 h. A dentist clinically assessed plaque after disclosing and on red fluorescence images. Three descriptors were extracted from images and a RUSBoost classifier derived computer fluorescence scores through cross-validation. Red fluorescence plaque levels increased during the 48-h accumulation. Plaque progression was identified by dentist assessment and computer analysis, presenting significant differences between visits at tooth and subject levels (pplaque and red fluorescence plaque (r=0.62 dentist, r=0.55 computer). The best agreement was observed when disclosing plaque threshold at level 2, for both dentist evaluation (sensitivity 71.1%, specificity 67.7%, accuracy 70.2%) and computer classification (sensitivity 68.4%, specificity 62.9%, accuracy 67.1%). Given the correlation with clinical diagnosis, red fluorescence imaging shows its potential for providing an objective and promising method for proper oral hygiene assessment.

  10. Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system

    NARCIS (Netherlands)

    Gadella, T.W.J.; van Hoek, A.; Visser, A.J.W.G.

    1997-01-01

    The construction of a homodyne frequency domain fluorescence lifetime imaging microscope is described. The system consists of (i) an intensity-modulated laser excitation source, (ii) an epifluorescence microscope, (iii) a gain-modulated microchannel plate (MCP) image intensifier, and (iv) a

  11. Shading correction and calibration in bacterial fluorescence measurement by image processing system

    NARCIS (Netherlands)

    Wilkinson, M.H.F.

    An image processing system with applications in bacterial (immuno-)fluorescence measurement has been developed. To reach quantitative results, correction for non-uniformities in system sensitivity, both as a function of time (calibration for drifts) and as a function of image coordinates (shading

  12. Time-domain whole-field fluorescence lifetime imaging with optical sectioning.

    Science.gov (United States)

    Cole, M J; Siegel, J; Webb, S E; Jones, R; Dowling, K; Dayel, M J; Parsons-Karavassilis, D; French, P M; Lever, M J; Sucharov, L O; Neil, M A; Juskaitis, R; Wilson, T

    2001-09-01

    A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.

  13. Fluorescence hyperspectral imaging technique for foreign substance detection on fresh-cut lettuce.

    Science.gov (United States)

    Mo, Changyeun; Kim, Giyoung; Kim, Moon S; Lim, Jongguk; Cho, Hyunjeong; Barnaby, Jinyoung Yang; Cho, Byoung-Kwan

    2017-09-01

    Non-destructive methods based on fluorescence hyperspectral imaging (HSI) techniques were developed to detect worms on fresh-cut lettuce. The optimal wavebands for detecting the worms were investigated using the one-way ANOVA and correlation analyses. The worm detection imaging algorithms, RSI-I(492-626)/492 , provided a prediction accuracy of 99.0%. The fluorescence HSI techniques indicated that the spectral images with a pixel size of 1 × 1 mm had the best classification accuracy for worms. The overall results demonstrate that fluorescence HSI techniques have the potential to detect worms on fresh-cut lettuce. In the future, we will focus on developing a multi-spectral imaging system to detect foreign substances such as worms, slugs and earthworms on fresh-cut lettuce. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. Experimental Design and Data Analysis of In Vivo Fluorescence Imaging Studies.

    Science.gov (United States)

    Ding, Ying; Lin, Hui-Min

    2016-01-01

    The objective of this chapter is to provide researchers who conduct in vivo fluorescence imaging studies with guidance in statistical aspects in the experimental design and data analysis of such studies. In the first half of this chapter, we introduce the key statistical components for designing a sound in vivo experiment. Particular emphasis is placed on the issues and designs that pertain to fluorescence imaging studies. Examples representing several popular types of fluorescence imaging experiments are provided as case studies to demonstrate how to appropriately design such studies. In the second half of this chapter, we explain the fundamental statistical concepts and methods used in the data analysis of typical in vivo experiments. We also provide specific examples in in vivo imaging studies to illustrate the key steps of analysis procedure.

  15. Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

    Science.gov (United States)

    Yilmaz, Hasan

    2016-03-01

    Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).

  16. 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.

  17. 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.

  18. Intraoperative Near-infrared Imaging for Parathyroid Gland Identification by Auto-fluorescence: A Feasibility Study.

    Science.gov (United States)

    De Leeuw, Frederic; Breuskin, Ingrid; Abbaci, Muriel; Casiraghi, Odile; Mirghani, Haïtham; Ben Lakhdar, Aïcha; Laplace-Builhé, Corinne; Hartl, Dana

    2016-09-01

    Parathyroid glands (PGs) can be particularly hard to distinguish from surrounding tissue and thus can be damaged or removed during thyroidectomy. Postoperative hypoparathyroidism is the most common complication after thyroidectomy. Very recently, it has been found that the parathyroid tissue shows near-infrared (NIR) auto-fluorescence which could be used for intraoperative detection, without any use of contrast agents. The work described here presents a histological validation ex vivo of the NIR imaging procedure and evaluates intraoperative PG detection by NIR auto-fluorescence using for the first time to our knowledge a commercially available clinical NIR imaging device. Ex vivo study on resected operative specimens combined with a prospective in vivo study of consecutive patients who underwent total or partial thyroid, or parathyroid surgery at a comprehensive cancer center. During surgery, any tissue suspected to be a potential PG by the surgeon was imaged with the Fluobeam 800 (®) system. NIR imaging was compared to conventional histology (ex vivo) and/or visual identification by the surgeon (in vivo). We have validated NIR auto-fluorescence with an ex vivo study including 28 specimens. Sensitivity and specificity were 94.1 and 80 %, respectively. Intraoperative NIR imaging was performed in 35 patients and 81 parathyroids were identified. In 80/81 cases, the fluorescence signal was subjectively obvious on real-time visualization. We determined that PG fluorescence is 2.93 ± 1.59 times greater than thyroid fluorescence in vivo. Real-time NIR imaging based on parathyroid auto-fluorescence is fast, safe, and non-invasive and shows very encouraging results, for intraoperative parathyroid identification.

  19. 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.

  20. Laser-induced fluorescence imaging of coronary arteries for open-heart surgery applications

    Science.gov (United States)

    Taylor, Roderick S.; Gladysz, D.; Brown, Derek W.; Higginson, Lyall A. J.

    1991-07-01

    A technique utilizing laser induced fluorescence has been developed to obtain direct real-time imaging of the coronary artery network for open heart surgery applications. Both excimer pumped dye and cw argon-ion laser radiation transmitted through a fused silica fiber were used as laser sources to irradiate swine, bovine, and human cadaver hearts whose coronary arteries had been injected with strongly fluorescent dyes. The laser induces fluorescence originating from within the coronary arteries and detected by the surgeon's eye, allows the entire coronary network to be directly viewed. A comparison between laser induced fluorescence and the use of direct visual inspection of arteries following injection of the dye Cardio-Green(R) as well as conventional thermal imaging is presented. The limitations imposed on each technique by layers of fat on top of the coronary arteries are also described. The possibility of using these techniques to detect mechanical or laser beam perforations during laser endarterectomy procedures is discussed.

  1. Deep-red emissive crescent-shaped fluorescent dyes: substituent effect on live cell imaging.

    Science.gov (United States)

    Liu, Weimin; Zhou, Bingjiang; Niu, Guangle; Ge, Jiechao; Wu, Jiasheng; Zhang, Hongyan; Xu, Haitao; Wang, Pengfei

    2015-04-08

    A series of crescent-shaped fluorescent dyes (CP1-CP6) were synthesized by hybridizing coumarin and pyronin moieties with different amino substituents at both ends. The molecular structures and photophysical properties of these fluorescent dyes were investigated through X-ray diffraction, absorption spectroscopy, and fluorescence spectroscopy. Results show that the fluorescent dyes exhibited crescent-shaped structures, deep-red emissions (approximately 650 nm), and significant Stokes shifts. In live-cell-imaging experiments, CP1 stains mitochondria, whereas CP3 and CP6 stain the lysosomes in a cytoplasm and the RNA in nucleoli. The relationships between different amino substituent groups and the imaging properties of CP dyes were discussed as well. Additionally, findings from the cytotoxicity and photostability experiments on living cells indicated the favorable biocompatibility and high photostability of the CP dyes.

  2. 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.

  3. 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 1 nM in a 70 mm diameter homogeneous phantom, and detection is feasible to near 10 pM. 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.

  4. Fluorescence-Lifetime Imaging Microscopy for Visualization of Quantum Dots’ Endocytic Pathway

    Directory of Open Access Journals (Sweden)

    Leona Damalakiene

    2016-03-01

    Full Text Available Accumulation of carboxylated polyethylene glycol (PEG CdSe/ZnSquantum dots (QDs has been monitored in living fibroblasts using confocal microscopy for fluorescence intensity and fluorescence-lifetime imaging (FLIM. The wide range of mean photoluminescence (PL lifetime values was observed for the intracellular QDs in different intracellular microenvironment, which revealed structural heterogeneity of endosomes and enabled the distinguishing among endosomes of different maturity.

  5. Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Landt Larsen, Ane; Færgeman, Nils J.

    2010-01-01

    The nematode Caenorhabditis elegans is a genetically tractable model organism to investigate sterol transport. In vivo imaging of the fluorescent sterol, dehydroergosterol (DHE), is challenged by C. elegans' high autofluorescence in the same spectral region as emission of DHE. We present a method...... homologues of Niemann-Pick C disease proteins. Our approach is generally useful for identifying fluorescent probes in the presence of high cellular autofluorescence....

  6. Portal vein territory identification using indocyanine green fluorescence imaging: Technical details and short-term outcomes.

    Science.gov (United States)

    Kobayashi, Yuta; Kawaguchi, Yoshikuni; Kobayashi, Kosuke; Mori, Kazuhiro; Arita, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Kokudo, Norihiro

    2017-12-01

    Portal vein (PV) territory identification during liver resection may be performed using indocyanine green (ICG) fluorescence imaging technique. However, the technical details of the fluorescence staining technique have not been fully elucidated. This study was performed to demonstrate the technical details of PV territory identification using fluorescence imaging and evaluates the short-term outcomes. From 2011 to 2015, 105 underwent liver resection at the University of Tokyo Hospital with one of the following fluorescence staining techniques by transhepatic PV injection or intravenous injection of ICG: single staining (n = 36), multiple staining (n = 31), counterstaining (n = 22), negative staining (n = 13), or paradoxical negative staining (n = 3). The PV territory was identified as a region with fluorescence or a defect of fluorescence using one of the five staining techniques. ICG was administered by transhepatic PV injection in all but the negative staining technique, which employed intravenous injection. No adverse events associated with the ICG administration occurred. The mortality, postoperative total morbidity, and the major complication (Clavien-Dindo grade ≥III) rates were 0.0%, 14.3%, and 7.6%. We have demonstrated the technical details of five types of fluorescence staining techniques. These techniques are safe to perform and facilitate clear visualization of the PV territory in real time, enhancing the efficacy of anatomical removal of such territories. © 2017 Wiley Periodicals, Inc.

  7. CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

    Directory of Open Access Journals (Sweden)

    Nan Guo

    2014-10-01

    Full Text Available Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art.

  8. Dual modality live cell imaging with multiple-wavelength digital holography and epi-fluorescence

    Science.gov (United States)

    Mann, Christopher J.; Bingham, Philip R.; Lin, Henry K.; Paquit, Vincent C.; Gleason, Shaun S.

    2011-03-01

    We apply multiple-wavelength digital holography in combination with epi-fluorescence microscopy in order to generate quantitative phase and fluorescence information from cell samples. While digital holography provides high precision morphological information, the addition of fluorescence supplies the specificity needed to identify cellular constituents. By the application of multiple-wavelength digital holography it is possible to obtain the complete wavefront data deterministically and in real-time. We demonstrate this dual-mode imaging capability through the investigation of living cells. [Figure not available: see fulltext.

  9. mScarlet: a bright monomeric red fluorescent protein for cellular imaging.

    Science.gov (United States)

    Bindels, Daphne S; Haarbosch, Lindsay; van Weeren, Laura; Postma, Marten; Wiese, Katrin E; Mastop, Marieke; Aumonier, Sylvain; Gotthard, Guillaume; Royant, Antoine; Hink, Mark A; Gadella, Theodorus W J

    2017-01-01

    We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.

  10. Fluorescence Images of DNA-Bound YOYO between Coupled Silver Particles

    OpenAIRE

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

    2007-01-01

    Oligonucleotide-bound silver particles were coupled through hybridization with target complementary oligonucleotides. YOYO molecules were intercalated into DNA duplexes bound between the coupled metal particles. Fluorescence images of YOYO molecules were monitored by scanning confocal microscopy. Relative to the free single YOYO, the emission brightness of the image was enhanced 80-fold after intercalating the fluorophores into the DNA duplexes between the coupled silver particles. Some image...

  11. Ultrasmall Organic Nanoparticles with Aggregation-Induced Emission and Enhanced Quantum Yield for Fluorescence Cell Imaging

    OpenAIRE

    Xu, Suying; Bai, Xilin; Ma, Jingwen; Xu, Minmin; Hu, Gaofei; James, Tony D.; Wang, Leyu

    2016-01-01

    The use of fluorescence probes for biomedical imaging has attracted significant attention over recent years owing to their high resolution at cellular level. The probes are available in many formats including small particle size based imaging agents which are considered to be promising candidates, due to their excellent stabilities. Yet, concerns over the potential cytotoxicity effects of inorganic luminescent particles have led to questions about their suitability for imaging applications. E...

  12. Characterizing fluorescent imaging properties of antibodies conjugated to IRDye800CW for use in imaging of head and neck cancer

    Science.gov (United States)

    Foster, Robert C.; Krell, Asher M.; Chung, Thomas K.; Warram, Jason M.; Zinn, Kurt R.; Rosenthal, Eben L.

    2014-03-01

    Introduction: Proteins conjugated to the near infrared (NIR) moieties for detection of head and neck cancers are being translated to the clinic. However, little is known about the fluorescent properties of IRDye800CW after conjugation to antibodies. We investigated factors that may alter the real-time observed fluorescence of antibody conjugated dye and the rate of fluorescent signal loss. Methods: Signal loss was examined using three FDA approved monoclonal antibodies conjugated to IRDye800CW (LICOR) over a period of 15 days. Temperature effects on fluorescence were examined for conjugated dye in both solution and a mouse tumor model. Samples were cooled to -20°C then warmed to predetermined temperatures up to 60°C with imaging performed using the PEARL Impulse (LI-COR) and LUNA (Novadaq) systems. Results: Short term fluorescent signal loss (< 1 hour) was linear, while long term loss (15 days) was exponential with significant increases in rate observed with light exposure and increased temperatures. Cooling of tumor tissue at -20°C was shown to significantly increase tumor fluorescence on both imaging modalities when compared to room temperature (p=0.008, p=0.019). Concurrently the ratio of tumor to background fluorescent signal (TBR) increased with decreasing temperature with statistically significant increases seen at -20°C and 4°C (p=0.0015, p=0.03). Conclusions: TBR is increased with decreasing sample temperature, suggesting that the clinical exam of fluorescently labeled tissues may be improved at cooler temperatures. Our results indicate that both the rate of signal loss and the change in fluorescence with temperature observed for IRDye800CW are independent of the conjugating antibody.

  13. Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

    Energy Technology Data Exchange (ETDEWEB)

    Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz; Lukasik, Beata; Garner, Logan E.; Chworos, Arkadiusz

    2017-05-01

    Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining was determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.

  14. Two-photon excited fluorescence lifetime imaging microscopy for FRET study on protein interactions

    Science.gov (United States)

    Qu, Junle; Lin, Ziyang; Liu, Lixin; Guo, Xuan; Chen, Danni; Niu, Hanben

    2005-01-01

    Two-photon excited fluorescence lifetime imaging (2P-FLIM) provides a more direct and precise approach to fluorescence resonance energy transfer (FRET), which allows studying the dynamic behavior of protein-protein interactions in living cells. In this paper, we describe the combination of a Leica TCS SP2 laser scanning microscope and a time-correlated single photon counting (TCSPC) lifetime imaging module developed by Becker & Hickl for two-photon excited fluorescence lifetime imaging. This 2P-FLIM system was used for FRET study on the interaction of heat shock protein hsp27 with p38 MAP kinase in the single living cell. Results show that the reduction in donor (CFP) lifetime in the presence of acceptor (YFP) reveals interactions between the two proteins.

  15. Mechanotransduction in Endothelial Cells Studied with Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chien Shu [Departments of Bioengineering and Medicine and Institute of Engineering in Medicine, University of California, San Diego, La Jolla, California 92093-0427 (United States)

    2011-01-01

    Mechanotransduction involves the conversion of mechanical stimuli to intracellular signaling to modulate gene and protein expressions and hence cellular functions in endothelial cells, thus playing importance roles in the regulation of homeostasis in health and disease. The aim of this paper is to investigate the dynamics of mechanotransduction in endothelial cells by the use of fluorescent resonance energy transfer (FRET) to study the temporal and spatial activation of Src kinase and focal adhesion kinase, both of which play critical roles in many cellular processes. The results have contributed to the elucidation of the roles of these two important signaling molecules and their interactions in mediating mechanotransduction.

  16. Near-infrared fluorescent peptide probes for imaging of tumor in vivo and their biotoxicity evaluation.

    Science.gov (United States)

    Liu, Liwei; Lin, Guimiao; Yin, Feng; Law, Wing-Cheung; Yong, Ken-Tye

    2016-04-01

    Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) fluorescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells. © 2016 Wiley Periodicals, Inc.

  17. Wide-field flexible endoscope for simultaneous color and NIR fluorescence image acquisition during surveillance colonoscopy

    Science.gov (United States)

    García-Allende, P. Beatriz; Nagengast, Wouter B.; Glatz, Jürgen; Ntziachristos, Vasilis

    2013-03-01

    Colorectal cancer (CRC) is the third most common form of cancer and, despite recent declines in both incidence and mortality, it still remains the second leading cause of cancer-related deaths in the western world. Colonoscopy is the standard for detection and removal of premalignant lesions to prevent CRC. The major challenges that physicians face during surveillance colonoscopy are the high adenoma miss-rates and the lack of functional information to facilitate decision-making concerning which lesions to remove. Targeted imaging with NIR fluorescence would address these limitations. Tissue penetration is increased in the NIR range while the combination with targeted NIR fluorescent agents provides molecularly specific detection of cancer cells, i.e. a red-flag detection strategy that allows tumor imaging with optimal sensitivity and specificity. The development of a flexible endoscopic fluorescence imaging method that can be integrated with standard medical endoscopes and facilitates the clinical use of this potential is described in this work. A semi-disposable coherent fiber optic imaging bundle that is traditionally employed in the exploration of biliary and pancreatic ducts is proposed, since it is long and thin enough to be guided through the working channel of a traditional video colonoscope allowing visualization of proximal lesions in the colon. A custom developed zoom system magnifies the image of the proximal end of the imaging bundle to fill the dimensions of two cameras operating in parallel providing the simultaneous color and fluorescence video acquisition.

  18. Dual Probe with Fluorescent and Magnetic Properties for Imaging Solid Tumor Xenografts

    Directory of Open Access Journals (Sweden)

    Liang Shan

    2007-03-01

    Full Text Available A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR fluorescent transferrin conjugate (TfNIR on the surface of contrast agent-encapsulated cationic liposome (Lip-CA. This probe was used for magnetic resonance imaging (MRI and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.

  19. Spectral Behavior of White Pigment Mixtures Using Reflectance, Ultraviolet-Fluorescence Spectroscopy, and Multispectral Imaging.

    Science.gov (United States)

    Pronti, Lucilla; Felici, Anna Candida; Ménager, Matthieu; Vieillescazes, Cathy; Piacentini, Mario

    2017-01-01

    Reflectance spectroscopy, ultraviolet (UV)-fluorescence spectroscopy, and multispectral imaging have been widely employed for pigment identification on paintings. From ancient times to the present, lead white, zinc white, and titanium white have been the most important white pigments used for paintings and they are used as pigment markers for dating a work of art. The spectral behavior of these pigments is reported in several scientific papers and websites, but those of their mixtures are quite unknown. We present a combined nondestructive approach for identifying mixtures of lead white, zinc white, and titanium white as powder and dispersed in two different binder media (egg yolk and linseed oil) by using reflectance spectroscopy, spectrofluorimetry, multispectral reflectance and UV-fluorescence imaging. We propose a novel approach for mapping the presence of white pigments in paintings by false color images obtained from multispectral reflectance and UV-fluorescence images. We found that the presence of lead white mixed with either zinc white or titanium white is highly detectable. Zinc white mixed with lead white or titanium white can be identified due to its UV-fluorescence emission, whereas titanium white in association with lead white or zinc white is distinguishable by its reflectance spectral features. In most cases, the UV-fluorescence analyses also permit the recognition of the binder media in which the pigments are dispersed.

  20. 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.

  1. Method for Imaging Live-Cell RNA Using an RNA Aptamer and a Fluorescent Probe.

    Science.gov (United States)

    Sato, Shin-Ichi; Yatsuzuka, Kenji; Katsuda, Yousuke; Uesugi, Motonari

    2018-01-01

    Live-cell imaging of mRNA dynamics is increasingly important to understanding spatially restricted gene expression. We recently developed a convenient and versatile method that uses a gene-specific RNA aptamer and a fluorescent probe to enable spatiotemporal imaging of endogenous mRNAs in living cells. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. The new RNA-imaging technology might be useful for live-cell imaging of any RNA molecules.

  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. In vivo assessment of wound re-epithelialization by UV fluorescence excitation imaging

    Science.gov (United States)

    Wang, Ying; Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Williams, Maura; Farinelli, William; Anderson, R. R.; Franco, Walfre

    2017-02-01

    Background and Objectives: We have previously demonstrated the efficacy of a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds in vitro. This system can image highly-proliferating cellular processes (295/340 nm excitation/emission wavelengths) to study epithelialization in a cultured wound model. The objective of the current work is to evaluate the suitability of u-FEI for monitoring wound re-epithelialization in vivo. Study Design: Full-thickness wounds were created in the tail of rats and imaged weekly using u-FEI at 295/340nm excitation/emission wavelengths. Histology was used to investigate the correlation between the spatial distribution and intensity of fluorescence and the extent of wound epithelialization. In addition, the expression of the nuclear protein Ki67 was used to confirm the association between the proliferation of keratinocyte cells and the intensity of fluorescence. Results: Keratinocytes forming neo-epidermis exhibited higher fluorescence intensity than the keratinocytes not involved in re-epithelialization. In full-thickness wounds the fluorescence first appeared at the wound edge where keratinocytes initiated the epithelialization process. Fluorescence intensity increased towards the center as the keratinocytes partially covered the wound. As the wound healed, fluorescence decreased at the edges and was present only at the center as the keratinocytes completely covered the wound at day 21. Histology demonstrated that changes in fluorescence intensity from the 295/340nm band corresponded to newly formed epidermis. Conclusions: u-FEI at 295/340nm allows visualization of proliferating keratinocyte cells during re-epithelialization of wounds in vivo, potentially providing a quantitative, objective and simple method for evaluating wound closure in the clinic.

  4. Carbon nanomaterials: multi-functional agents for biomedical fluorescence and Raman imaging.

    Science.gov (United States)

    Bartelmess, J; Quinn, S J; Giordani, S

    2015-07-21

    Carbon based nanomaterials have emerged over the last few years as important agents for biomedical fluorescence and Raman imaging applications. These spectroscopic techniques utilize either fluorescently labelled carbon nanomaterials or the intrinsic photophysical properties of the carbon nanomaterial. In this review article we present the utilization and performance of several classes of carbon nanomaterials, namely carbon nanotubes, carbon nanohorns, carbon nanoonions, nanodiamonds and different graphene derivatives, which are currently employed for in vitro as well as in vivo imaging in biology and medicine. A variety of different approaches, imaging agents and techniques are examined and the specific properties of the various carbon based imaging agents are discussed. Some theranostic carbon nanomaterials, which combine diagnostic features (i.e. imaging) with cell specific targeting and therapeutic approaches (i.e. drug delivery or photothermal therapy), are also included in this overview.

  5. Imaging of Bacterial and Fungal Cells Using Fluorescent Carbon Dots Prepared from Carica papaya Juice.

    Science.gov (United States)

    Kasibabu, Betha Saineelima B; D'souza, Stephanie L; Jha, Sanjay; Kailasa, Suresh Kumar

    2015-07-01

    In this paper, we have described a simple hydrothermal method for preparation of fluorescent carbon dots (C-dots) using Carica papaya juice as a precursor. The synthesized C-dots show emission peak at 461 nm with a quantum yield of 7.0 %. The biocompatible nature of C-dots was confirmed by a cytotoxicity assay on E. coli. The C-dots were used as fluorescent probes for imaging of bacterial (Bacillus subtilis) and fungal (Aspergillus aculeatus) cells and emitted green and red colors under different excitation wavelengths, which indicates that the C-dots can be used as a promising material for cell imaging.

  6. Wide-field TCSPC-based fluorescence lifetime imaging (FLIM) microscopy

    Science.gov (United States)

    Suhling, Klaus; Hirvonen, Liisa M.; Becker, Wolfgang; Smietana, Stefan; Netz, Holger; Milnes, James; Conneely, Thomas; Le Marois, Alix; Jagutzki, Ottmar

    2016-05-01

    Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise, robust and mature technique to measure photon arrival times in applications such as fluorescence spectroscopy and microscopy, light detection and ranging (lidar) and optical tomography. Wide-field TCSPC detection techniques, where the position and the arrival time of the photons are recorded simultaneously, have seen several advances in the last few years, from the microsecond to the picosecond time scale. Here, we summarise some of our recent work in this field with emphasis on microsecond resolution phosphorescence lifetime imaging (PLIM) and nanosecond fluorescence lifetime imaging (FLIM) microscopy.

  7. Chlorophyll fluorescence imaging for the noninvasive assessment of anthocyanins in whole grape (Vitis vinifera L.) bunches.

    Science.gov (United States)

    Agati, Giovanni; Traversi, Maria Laura; Cerovic, Zoran G

    2008-01-01

    The distribution of anthocyanins in grape (Vitis vinifera L.) bunches from the Sangiovese cultivar was measured nondestructively by chlorophyll fluorescence imaging using two excitation light bands at 550 and 650 nm in sequence. The pixel intensity in the derived logarithm of the fluorescence excitation ratio image was directly related, by an exponential function (r2 = 0.93), to the anthocyanin concentration of berry extracts. The method will be useful for the assessment of the heterogeneity of anthocyanin accumulation in berries that is known to depend on physiologic and climatic factors. It can also represent a new, rapid and noninvasive technique for the assessment of grape ripening and the appropriate time of harvest.

  8. Fluorescent nanoprobes for sensing and imaging of metal ions: recent advances and future perspectives.

    Science.gov (United States)

    Zhang, JingJing; Cheng, FangFang; Li, JingJing; Zhu, Jun-Jie; Lu, Yi

    2016-06-01

    Recent advances in nanoscale science and technology have generated nanomaterials with unique optical properties. Over the past decade, numerous fluorescent nanoprobes have been developed for highly sensitive and selective sensing and imaging of metal ions, both in vitro and in vivo. In this review, we provide an overview of the recent development of the design and optical properties of the different classes of fluorescent nanoprobes based on noble metal nanomaterials, upconversion nanoparticles, semiconductor quantum dots, and carbon-based nanomaterials. We further detail their application in the detection and quantification of metal ions for environmental monitoring, food safety, medical diagnostics, as well as their use in biomedical imaging in living cells and animals.

  9. Cost-effective and compact wide-field fluorescent imaging on a cell-phone.

    Science.gov (United States)

    Zhu, Hongying; Yaglidere, Oguzhan; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

    2011-01-21

    We demonstrate wide-field fluorescent and darkfield imaging on a cell-phone with compact, light-weight and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. For this purpose, we used battery powered light-emitting diodes (LEDs) to pump the sample of interest from the side using butt-coupling, where the pump light was guided within the sample cuvette to uniformly excite the specimen. The fluorescent emission from the sample was then imaged using an additional lens that was positioned right in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to our detection path, an inexpensive plastic colour filter was sufficient to create the dark-field background required for fluorescent imaging, without the need for a thin-film interference filter. We validate the performance of this platform by imaging various fluorescent micro-objects in 2 colours (i.e., red and green) over a large field-of-view (FOV) of ∼81 mm(2) with a raw spatial resolution of ∼20 μm. With additional digital processing of the captured cell-phone images, through the use of compressive sampling theory, we demonstrate ∼2 fold improvement in our resolving power, achieving ∼10 μm resolution without a trade-off in our FOV. Further, we also demonstrate darkfield imaging of non-fluorescent specimen using the same interface, where this time the scattered light from the objects is detected without the use of any filters. The capability of imaging a wide FOV would be exceedingly important to probe large sample volumes (e.g., >0.1 mL) of e.g., blood, urine, sputum or water, and for this end we also demonstrate fluorescent imaging of labeled white-blood cells from whole blood samples, as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts. Weighing only ∼28 g (∼1 ounce), this compact and cost-effective fluorescent imaging platform

  10. An x-ray fluorescence imaging system for gold nanoparticle detection.

    Science.gov (United States)

    Ricketts, K; Guazzoni, C; Castoldi, A; Gibson, A P; Royle, G J

    2013-11-07

    Gold nanoparticles (GNPs) may be used as a contrast agent to identify tumour location and can be modified to target and image specific tumour biological parameters. There are currently no imaging systems in the literature that have sufficient sensitivity to GNP concentration and distribution measurement at sufficient tissue depth for use in in vivo and in vitro studies. We have demonstrated that high detecting sensitivity of GNPs can be achieved using x-ray fluorescence; furthermore this technique enables greater depth imaging in comparison to optical modalities. Two x-ray fluorescence systems were developed and used to image a range of GNP imaging phantoms. The first system consisted of a 10 mm(2) silicon drift detector coupled to a slightly focusing polycapillary optic which allowed 2D energy resolved imaging in step and scan mode. The system has sensitivity to GNP concentrations as low as 1 ppm. GNP concentrations different by a factor of 5 could be resolved, offering potential to distinguish tumour from non-tumour. The second system was designed to avoid slow step and scan image acquisition; the feasibility of excitation of the whole specimen with a wide beam and detection of the fluorescent x-rays with a pixellated controlled drift energy resolving detector without scanning was investigated. A parallel polycapillary optic coupled to the detector was successfully used to ascertain the position where fluorescence was emitted. The tissue penetration of the technique was demonstrated to be sufficient for near-surface small-animal studies, and for imaging 3D in vitro cellular constructs. Previous work demonstrates strong potential for both imaging systems to form quantitative images of GNP concentration.

  11. Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

    Science.gov (United States)

    Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Gorpas, Dimitris; Fatakdawala, Hussain; Marcu, Laura

    2014-01-01

    Abstract. We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures. PMID:24898604

  12. Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

    Science.gov (United States)

    Wiesmann, Veit; Bergler, Matthias; Palmisano, Ralf; Prinzen, Martin; Franz, Daniela; Wittenberg, Thomas

    2017-03-18

    Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.

  13. Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

    Science.gov (United States)

    Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Gorpas, Dimitris; Fatakdawala, Hussain; Marcu, Laura

    2014-06-01

    We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures.

  14. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    Science.gov (United States)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  15. Detection of microbial biofilms on food processing surfaces: hyperspectral fluorescence imaging study

    Science.gov (United States)

    Jun, Won; Kim, Moon S.; Chao, Kaunglin; Lefcourt, Alan M.; Roberts, Michael S.; McNaughton, James L.

    2009-05-01

    We used a portable hyperspectral fluorescence imaging system to evaluate biofilm formations on four types of food processing surface materials including stainless steel, polypropylene used for cutting boards, and household counter top materials such as formica and granite. The objective of this investigation was to determine a minimal number of spectral bands suitable to differentiate microbial biofilm formation from the four background materials typically used during food processing. Ultimately, the resultant spectral information will be used in development of handheld portable imaging devices that can be used as visual aid tools for sanitation and safety inspection (microbial contamination) of the food processing surfaces. Pathogenic E. coli O157:H7 and Salmonella cells were grown in low strength M9 minimal medium on various surfaces at 22 +/- 2 °C for 2 days for biofilm formation. Biofilm autofluorescence under UV excitation (320 to 400 nm) obtained by hyperspectral fluorescence imaging system showed broad emissions in the blue-green regions of the spectrum with emission maxima at approximately 480 nm for both E. coli O157:H7 and Salmonella biofilms. Fluorescence images at 480 nm revealed that for background materials with near-uniform fluorescence responses such as stainless steel and formica cutting board, regardless of the background intensity, biofilm formation can be distinguished. This suggested that a broad spectral band in the blue-green regions can be used for handheld imaging devices for sanitation inspection of stainless, cutting board, and formica surfaces. The non-uniform fluorescence responses of granite make distinctions between biofilm and background difficult. To further investigate potential detection of the biofilm formations on granite surfaces with multispectral approaches, principal component analysis (PCA) was performed using the hyperspectral fluorescence image data. The resultant PCA score images revealed distinct contrast between

  16. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

    Science.gov (United States)

    Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

    2016-01-01

    Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

  17. Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves.

    Science.gov (United States)

    Broess, Koen; Borst, Jan Willem; van Amerongen, Herbert

    2009-05-01

    This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F (0)- and the F (m)-state. The corresponding average lifetimes are approximately 250 ps and approximately 1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of approximately 500 nm in the focal (xy) plane and 2 microm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed.

  18. Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging.

    Science.gov (United States)

    de Almeida, Rodrigo F M; Loura, Luís M S; Prieto, Manuel

    2009-02-01

    Membrane microdomains and their involvement in cellular processes are part of the current paradigm of biomembranes. However, a better characterization of domains, namely lipid rafts, is needed. In this review, it is shown how the use of time-resolved fluorescence, with the adequate parameters and probes, helps elucidating the type, number, fraction, composition and size of lipid phases and domains in multicomponent model systems. The determination of phase diagrams for lipid mixtures containing sphingolipids and/or cholesterol is exemplified. The use of fluorescence quenching and Förster resonance energy transfer (FRET) are also illustrated. Strategies for studying protein-induced domains are presented. The advantages of using single point microscopic decays and fluorescence lifetime imaging microscopy (FLIM) in systems with three-phase coexistence are explained. Finally, the introduction of FLIM allows studies in live cell membranes, and the nature of the microdomains observed is readily elucidated due to the information retrieved from fluorescence lifetimes.

  19. Benchtop and animal validation of a portable fluorescence microscopic imaging system for potential use in cholecystectomy.

    Science.gov (United States)

    Ye, Jian; Liu, Guanghui; Liu, Peng; Zhang, Shiwu; Shao, Pengfei; Smith, Zachary J; Liu, Chenhai; Xu, Ronald X

    2018-02-01

    We propose a portable fluorescence microscopic imaging system (PFMS) for intraoperative display of biliary structure and prevention of iatrogenic injuries during cholecystectomy. The system consists of a light source module, a camera module, and a Raspberry Pi computer with an LCD. Indocyanine green (ICG) is used as a fluorescent contrast agent for experimental validation of the system. Fluorescence intensities of the ICG aqueous solution at different concentration levels are acquired by our PFMS and compared with those of a commercial Xenogen IVIS system. We study the fluorescence detection depth by superposing different thicknesses of chicken breast on an ICG-loaded agar phantom. We verify the technical feasibility for identifying potential iatrogenic injury in cholecystectomy using a rat model in vivo. The proposed PFMS system is portable, inexpensive, and suitable for deployment in resource-limited settings. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  20. Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

    Science.gov (United States)

    Kočišová, Eva; Praus, Petr; Bok, Jiří; Bonneau, Stéphanie; Sureau, Franck

    2015-09-01

    Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.

  1. Excitation-resolved multispectral method for imaging pharmacokinetic parameters in dynamic fluorescent molecular tomography

    Science.gov (United States)

    Chen, Maomao; Zhou, Yuan; Su, Han; Zhang, Dong; Luo, Jianwen

    2017-04-01

    Imaging of the pharmacokinetic parameters in dynamic fluorescence molecular tomography (DFMT) can provide three-dimensional metabolic information for biological studies and drug development. However, owing to the ill-posed nature of the FMT inverse problem, the relatively low quality of the parametric images makes it difficult to investigate the different metabolic processes of the fluorescent targets with small distances. An excitation-resolved multispectral DFMT method is proposed; it is based on the fact that the fluorescent targets with different concentrations show different variations in the excitation spectral domain and can be considered independent signal sources. With an independent component analysis method, the spatial locations of different fluorescent targets can be decomposed, and the fluorescent yields of the targets at different time points can be recovered. Therefore, the metabolic process of each component can be independently investigated. Simulations and phantom experiments are carried out to evaluate the performance of the proposed method. The results demonstrated that the proposed excitation-resolved multispectral method can effectively improve the reconstruction accuracy of the parametric images in DFMT.

  2. 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.

  3. 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

  4. Image scanning fluorescence emission difference microscopy based on a detector array.

    Science.gov (United States)

    Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

    2017-06-01

    We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  5. Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

    Science.gov (United States)

    Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

    2015-03-01

    With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

  6. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

    OpenAIRE

    Shen, Yi; Rosendale, Morgane; Campbell, Robert E.; Perrais, David

    2014-01-01

    Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultan...

  7. Fluorescent Nanoparticle Imaging Allows Noninvasive Evaluation of Immune Cell Modulation in Esophageal Dysplasia

    Directory of Open Access Journals (Sweden)

    Peiman Habibollahi

    2014-05-01

    Full Text Available Esophageal tumors provide unique challenges and opportunities for developing and testing surveillance imaging technology for different tumor microenvironment components, including assessment of immune cell modulation, with the ultimate goal of promoting early detection and response evaluation. In this context, accessibility through the lumen using a minimally invasive approach provides a means for repetitive evaluation longitudinally by combining fluorescent endoscopic imaging technology with novel fluorescent nanoparticles that are phagocytized by immune cells in the microenvironment. The agent we developed for imaging is synthesized from Feraheme (ferumoxytol, a Food and Drug Administration-approved monocrystaline dextran-coated iron oxide nanoparticle, which we conjugated to a near-infrared fluorochrome, CyAL5.5. We demonstrate a high level of uptake of the fluorescent nanoparticles by myeloid-derived suppressor cells (MDSCs in the esophagus and spleen of L2Cre;p120ctnflox/flox mice. These mice develop esophageal dysplasia leading to squamous cell carcinoma; we have previously demonstrated that dysplastic and neoplastic esophageal lesions in these mice have an immune cell infiltration that is dominated by MDSCs. In the L2Cre;p120ctnflox/flox mice, evaluation of the spleen reveals that nearly 80% of CD45+ leukocytes that phagocytized the nanoparticle were CD11b+Gr1+ MDSCs. After dexamethasone treatment, we observed concordant decreased fluorescent signal from esophageal lesions during fluorescent endoscopy and decreased CyAL5.5-fluorescent-positive immune cell infiltration in esophageal dysplastic lesions by fluorescence-activated cell sorting analysis. Our observations suggest that this translatable technology may be used for the early detection of dysplastic changes and the serial assessment of immunomodulatory therapy and to visualize changes in MDSCs in the esophageal tumor microenvironment.

  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. Coherent fiber bundle based integrated photoacoustic, ultrasound and fluorescence imaging (PAUSFI) for endoscopy and diagnostic bio-imaging applications

    Science.gov (United States)

    James, Joseph; Murukeshan, V. M.; Sathiyamoorthy, K.; Woh, Lye Sun

    2014-08-01

    Recent research in diagnostic imaging and sensing focuses on deriving complementary information from the diagnosed site. From that perspective it is imperative to devise new imaging platforms where multiple distinct modalities are used either simultaneously or sequentially. Increased efforts have been devoted towards establishing such multi-modal imaging systems, which house and operate more than two imaging modalities within a single instrumentation set-up. In this context, we propose a novel multi-modal imaging platform using non-ionizing radiation that has been successfully conceptualized, established and experimentally demonstrated. This proposed GRIN lensed fiber-optic microscope and linear array transducer based PAUSFI (photoacoustic, ultrasound and fluorescence imaging) system makes use of non-ionizing radiation sources to map optical and acoustic heterogeneities (complementary information) along the depth of the tissue at multi-scale resolution (microscopic to mesoscopic). The fiber-optic assembly enables the system to perform minimally invasive remote light delivery and high resolution fluorescence and photoacoustic imaging of inaccessible areas of intact tissues or intra body cavities. It is expected that the proposed multi-modal imaging system could open up niches in bio-imaging research in the near future.

  10. Photoacoustic imaging of the near-infrared fluorescent protein iRFP in vivo

    Science.gov (United States)

    Krumholz, Arie; Filonov, Grigory S.; Xia, Jun; Yao, Junjie; Verkhusha, Vladislav V.; Wang, Lihong V.

    2012-02-01

    Genetically encoded probes powerfully and non-invasively target specific tissues, cells, and subcellular locations. iRFP, a novel near-infrared fluorescent protein with low quantum yield whose absorption and fluorescence maxima are located at wavelengths longer than the Q-band of hemoglobin absorption, is ideal for PAT. Here, we report on an in vitro comparison of iRFP with other far-red fluorescent proteins, and its use in imaging a mouse tumor xenograft model. In an in vivo experiment, we stably transfected iRFP into MTLn3 adenocarcinoma cells and injected them into the mammary fat pad of female SCID/NCr mice, then imaged the resulting tumors two and three weeks post injection. The contrast increase from the protein expression was high enough to clearly separate the tumor region from the rest of the animal.

  11. Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Sun, Yuansheng; Day, Richard N; Periasamy, Ammasi

    2011-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET ). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ~2 d. PMID:21886099

  12. 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...... to permit planar laser-induced fluorescence imaging, in which large quantities of poorly atomized fuel impinges on the internal surfaces of the combustor. Results reveal high variability in atomization quality. In the absence of flame, small droplets are confined to areas of recirculating flow, whereas......-induced fluorescence is a useful tool for the analysis of all stages of altitude relight. Copyright © 2013 by R. W. Read, J. W. Rogerson, and S. Hochgreb....

  13. 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.

  14. Carbon dots of different composition and surface functionalization: cytotoxicity issues relevant to fluorescence cell imaging.

    Science.gov (United States)

    Wang, Yanli; Anilkumar, Parambath; Cao, Li; Liu, Jia-Hui; Luo, Pengju G; Tackett, Kenneth N; Sahu, Sushant; Wang, Ping; Wang, Xin; Sun, Ya-Ping

    2011-11-01

    Nanoscale carbon particles have emerged as versatile precursors for a new class of highly fluorescent nanomaterials that resemble semiconductor quantum dots. The surface-passivated fluorescent carbon nanoparticles, dubbed 'carbon dots', were already demonstrated for their potential optical bioimaging applications in vitro and in vivo. In this study, we conducted a systematic cytotoxicity evaluation on the carbon dots prepared by various combinations of precursor carbon nanoparticles and molecules for the particle surface functionalization. The results suggested that the cytotoxicity of carbon dots was dependent on the selection of surface passivation molecules. Those dots showing more significant cytotoxicity at higher concentrations were also evaluated for their effects on the fluorescence imaging of live cells. The implications of the results on the eventual use of carbon dots as cell imaging agents are discussed.

  15. A dark-field scanning spectroscopy platform for localized scatter and fluorescence imaging of tissue

    Science.gov (United States)

    Krishnaswamy, Venkataramanan; Laughney, Ashley M.; Paulsen, Keith D.; Pogue, Brian W.

    2011-03-01

    Tissue ultra-structure and molecular composition provide native contrast mechanisms for discriminating across pathologically distinct tissue-types. Multi-modality optical probe designs combined with spatially confined sampling techniques have been shown to be sensitive to this type of contrast but their extension to imaging has only been realized recently. A modular scanning spectroscopy platform has been developed to allow imaging localized morphology and molecular contrast measures in breast cancer surgical specimens. A custom designed dark-field telecentric scanning spectroscopy system forms the core of this imaging platform. The system allows imaging localized elastic scatter and fluorescence measures over fields of up to 15 mm x 15 mm at 100 microns resolution in tissue. Results from intralipid and blood phantom measurements demonstrate the ability of the system to quantify localized scatter parameters despite significant changes in local absorption. A co-registered fluorescence spectroscopy mode is also demonstrated in a protophorphyrin-IX phantom.

  16. Image guided surgery using near-infrared fluorescence: road to clinical translation of novel probes for real time tumor visualization

    Science.gov (United States)

    Hoogstins, Charlotte E. S.; Handgraaf, Henricus J. M.; Boogerd, Leonora S. F.; Burggraaf, Jacobus; Vahrmeijer, Alexander L.

    2017-02-01

    Fluorescence imaging is a novel intraoperative technique for guiding oncologic surgeons toward radical tumor resections. Application of various fluorescent agents in exploratory clinical trials has already yielded promising results. The field of fluorescence imaging must now move beyond the proof-of-concept phase toward clinical application and implementation. This shift encompasses several hurdles, including standardization, advanced-phase study endpoints, regulatory affairs and routine implementation, which need to be addressed by the community in close collaboration with all stakeholders. These challenges, and the possible actions to overcome them and promote and accelerate clinical implementation of fluorescence imaging, are summarized in this paper.

  17. Automated Image Analysis of HER2 Fluorescence In Situ Hybridization to Refine Definitions of Genetic Heterogeneity in Breast Cancer Tissue

    Directory of Open Access Journals (Sweden)

    Gedmante Radziuviene

    2017-01-01

    Full Text Available Human epidermal growth factor receptor 2 gene- (HER2- targeted therapy for breast cancer relies primarily on HER2 overexpression established by immunohistochemistry (IHC with borderline cases being further tested for amplification by fluorescence in situ hybridization (FISH. Manual interpretation of HER2 FISH is based on a limited number of cells and rather complex definitions of equivocal, polysomic, and genetically heterogeneous (GH cases. Image analysis (IA can extract high-capacity data and potentially improve HER2 testing in borderline cases. We investigated statistically derived indicators of HER2 heterogeneity in HER2 FISH data obtained by automated IA of 50 IHC borderline (2+ cases of invasive ductal breast carcinoma. Overall, IA significantly underestimated the conventional HER2, CEP17 counts, and HER2/CEP17 ratio; however, it collected more amplified cells in some cases below the lower limit of GH definition by manual procedure. Indicators for amplification, polysomy, and bimodality were extracted by factor analysis and allowed clustering of the tumors into amplified, nonamplified, and equivocal/polysomy categories. The bimodality indicator provided independent cell diversity characteristics for all clusters. Tumors classified as bimodal only partially coincided with the conventional GH heterogeneity category. We conclude that automated high-capacity nonselective tumor cell assay can generate evidence-based HER2 intratumor heterogeneity indicators to refine GH definitions.

  18. Automated Image Analysis of HER2 Fluorescence In Situ Hybridization to Refine Definitions of Genetic Heterogeneity in Breast Cancer Tissue.

    Science.gov (United States)

    Radziuviene, Gedmante; Rasmusson, Allan; Augulis, Renaldas; Lesciute-Krilaviciene, Daiva; Laurinaviciene, Aida; Clim, Eduard; Laurinavicius, Arvydas

    2017-01-01

    Human epidermal growth factor receptor 2 gene- (HER2-) targeted therapy for breast cancer relies primarily on HER2 overexpression established by immunohistochemistry (IHC) with borderline cases being further tested for amplification by fluorescence in situ hybridization (FISH). Manual interpretation of HER2 FISH is based on a limited number of cells and rather complex definitions of equivocal, polysomic, and genetically heterogeneous (GH) cases. Image analysis (IA) can extract high-capacity data and potentially improve HER2 testing in borderline cases. We investigated statistically derived indicators of HER2 heterogeneity in HER2 FISH data obtained by automated IA of 50 IHC borderline (2+) cases of invasive ductal breast carcinoma. Overall, IA significantly underestimated the conventional HER2, CEP17 counts, and HER2/CEP17 ratio; however, it collected more amplified cells in some cases below the lower limit of GH definition by manual procedure. Indicators for amplification, polysomy, and bimodality were extracted by factor analysis and allowed clustering of the tumors into amplified, nonamplified, and equivocal/polysomy categories. The bimodality indicator provided independent cell diversity characteristics for all clusters. Tumors classified as bimodal only partially coincided with the conventional GH heterogeneity category. We conclude that automated high-capacity nonselective tumor cell assay can generate evidence-based HER2 intratumor heterogeneity indicators to refine GH definitions.

  19. Fluorescent carbon nanoparticles derived from natural materials of mango fruit for bio-imaging probes

    Science.gov (United States)

    Jeong, Chan Jin; Roy, Arup Kumer; Kim, Sung Han; Lee, Jung-Eun; Jeong, Ji Hoon; Insik; Park, Sung Young

    2014-11-01

    Water soluble fluorescent carbon nanoparticles (FCP) obtained from a single natural source, mango fruit, were developed as unique materials for non-toxic bio-imaging with different colors and particle sizes. The prepared FCPs showed blue (FCP-B), green (FCP-G) and yellow (FCP-Y) fluorescence, derived by the controlled carbonization method. The FCPs demonstrated hydrodynamic diameters of 5-15 nm, holding great promise for clinical applications. The biocompatible FCPs demonstrated great potential in biological fields through the results of in vitro imaging and in vivo biodistribution. Using intravenously administered FCPs with different colored particles, we precisely defined the clearance and biodistribution, showing rapid and efficient urinary excretion for safe elimination from the body. These findings therefore suggest the promising possibility of using natural sources for producing fluorescent materials.Water soluble fluorescent carbon nanoparticles (FCP) obtained from a single natural source, mango fruit, were developed as unique materials for non-toxic bio-imaging with different colors and particle sizes. The prepared FCPs showed blue (FCP-B), green (FCP-G) and yellow (FCP-Y) fluorescence, derived by the controlled carbonization method. The FCPs demonstrated hydrodynamic diameters of 5-15 nm, holding great promise for clinical applications. The biocompatible FCPs demonstrated great potential in biological fields through the results of in vitro imaging and in vivo biodistribution. Using intravenously administered FCPs with different colored particles, we precisely defined the clearance and biodistribution, showing rapid and efficient urinary excretion for safe elimination from the body. These findings therefore suggest the promising possibility of using natural sources for producing fluorescent materials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04805a

  20. High-speed multispectral fluorescence lifetime imaging implementation for in vivo applications

    OpenAIRE

    Shrestha, Sebina; Applegate, Brian E.; Park, Jesung; Xiao, Xudong; Pande, Paritosh; Javier A. Jo

    2010-01-01

    Fluorescence lifetime imaging microscopy (FLIM) offers a noninvasive approach for characterizing the biochemical composition of biological tissue. In recent years, there has been an increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of robust, fast, and cost-effective FLIM instrumentation suitable for in vivo tissue imaging. Unfortunately, the predominant multispectral FLIM approaches su...

  1. Through-skull vasculature assessment using fluorescence brain imaging on murine models at around 800 nm

    Science.gov (United States)

    Le, Hanh N. D.; Gau, Yung-Tian A.; Rahmim, Arman; Wong, Dean F.; Bergles, Dwight E.; Kang, Jin U.

    2017-02-01

    We describe a scanning near-infrared fluorescence imager for through-skull non-invasive brain imaging on live murine models. The captured photoluminescence feature through scattering media was enhanced using a high sensitivity scientific CMOS sensor with the obtained spatial resolution of 15.63 μm, depth of field of 5 mm and an average local signal-to-noise ratio of 37.5 dB.

  2. Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography (Conference Presentation)

    Science.gov (United States)

    Andrews, Natalie; Davis, Samuel; Hay, Carys; Kumar, Sunil; Ramel, Marie-Christine; Bugeon, Laurence; McGinty, James; Dallman, Margaret J.; French, Paul M. W.

    2017-02-01

    Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime optical projection tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Förster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.

  3. Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging.

    Science.gov (United States)

    Pyle, Joseph R; Chen, Jixin

    2017-01-01

    Super-resolution imaging of single DNA molecules via point accumulation for imaging in nanoscale topography (PAINT) has great potential to visualize fine DNA structures with nanometer resolution. In a typical PAINT video acquisition, dye molecules (YOYO-1) in solution sparsely bind to the target surfaces (DNA) whose locations can be mathematically determined by fitting their fluorescent point spread function. Many YOYO-1 molecules intercalate into DNA and remain there during imaging, and most of them have to be temporarily or permanently fluorescently bleached, often stochastically, to allow for the visualization of a few fluorescent events per DNA per frame of the video. Thus, controlling the fluorescence on-off rate is important in PAINT. In this paper, we study the photobleaching of YOYO-1 and its correlation with the quality of the PAINT images. At a low excitation laser power density, the photobleaching of YOYO-1 is too slow and a minimum required power density was identified, which can be theoretically predicted with the proposed method in this report.

  4. Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach

    DEFF Research Database (Denmark)

    Brewer, Jonathan R.; Thoke, Henrik Seir; Stock, Robeto

    2017-01-01

    We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the tem...

  5. Polymerized LB Films Imaged with a Combined Atomic Force Microscope-Fluorescence Microscope

    NARCIS (Netherlands)

    Putman, C.A.J.; 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

  6. Fluorescence lifetime imaging using a single photon avalanche diode array sensor (Conference Presentation)

    Science.gov (United States)

    Wargocki, Piotr M.; Spence, David J.; Goldys, Ewa M.; Charbon, Edoardo; Bruschini, Claudio E.; Antalović, Ivan Michel; Burri, Samuel

    2017-02-01

    Single photon detectors allows us work with the weakest fluorescence signals. Single photon arrays, combined with ps-controlled gating allow us to create image maps of fluorescence lifetimes, which can be used for in-vivo discrimination of tissue activity. Here we present fluorescence lifetime imaging using the `SwissSPAD' sensor, a 512-by-128-pixel array of gated single photon detectors, fabricated in a standard high-voltage 0.35 μm CMOS process. We present a protocol for spatially resolved lifetime measurements where the lifetime can be retrieved for each pixel. We demonstrate the system by imaging patterns of Fluorescein and Rhodamine B on test slides, as well as measuring mixed samples to retrieve both components of the decay lifetime. The single photon sensitivity of the sensor creates a valuable instrument to perform live cell or live animal (in vivo) measurements of the weak autofluorescent signals, for example distinguishing unlabelled free and bound NADH. Our ultimate goal is to create a real time fluorescence lifetime imaging system, possibly integrated into augmented reality goggles, which could allow immediate discrimination of in vivo tissues.

  7. Video-rate two-photon excited fluorescence lifetime imaging system with interleaved digitization.

    Science.gov (United States)

    Dow, Ximeng Y; Sullivan, Shane Z; Muir, Ryan D; Simpson, Garth J

    2015-07-15

    A fast (up to video rate) two-photon excited fluorescence lifetime imaging system based on interleaved digitization is demonstrated. The system is compatible with existing beam-scanning microscopes with minor electronics and software modification. Proof-of-concept demonstrations were performed using laser dyes and biological tissue.

  8. Real-time micro-scale temperature imaging at low cost based on fluorescent intensity ratio

    Science.gov (United States)

    Xiong, Jianghao; Zhao, Mingshu; Han, Xiaotian; Cao, Zhongmin; Wei, Xiantao; Chen, Yonghu; Duan, Changkui; Yin, Min

    2017-02-01

    Real-time temperature imaging with high spatial resolution has been a challenging task but also one with wide potential applications. To achieve this task, temperature sensor is critical. Fluorescent materials stand out to be promising candidates due to their quick response and strong temperature dependence. However, former reported temperature imaging techniques with fluorescent materials are mainly based on point by point scanning, which cannot fulfill the requirement of real-time monitoring. Based on fluorescent intensity ratio (FIR) of two emission bands of SrB4O7:Sm2+, whose spatial distributions were simultaneously recorded by two cameras with special filters separately, real-time temperature imaging with high spatial resolution has been realized with low cost. The temperature resolution can reach about 2 °C in the temperature range from 120 to 280 °C the spatial resolution is about 2.4 μm and the imaging time is as fast as one second. Adopting this system, we observed the dynamic change of a micro-scale thermal distribution on a printed circuit board (PCB). Different applications and better performance could also be achieved on this system with appropriate fluorescent materials and high sensitive CCD detectors according to the experimental environment.

  9. Development and validation of a custom made indocyanine green fluorescence lymphatic vessel imager

    Science.gov (United States)

    Pallotta, Olivia J.; van Zanten, Malou; McEwen, Mark; Burrow, Lynne; Beesley, Jack; Piller, Neil

    2015-06-01

    Lymphoedema is a chronic progressive condition often producing significant morbidity. An in-depth understanding of an individual's lymphatic architecture is valuable both in the understanding of underlying pathology and for targeting and tailoring treatment. Severe lower limb injuries resulting in extensive loss of soft tissue require transposition of a flap consisting of muscle and/or soft tissue to close the defect. These patients are at risk of lymphoedema and little is known about lymphatic regeneration within the flap. Indocyanine green (ICG), a water-soluble dye, has proven useful for the imaging of lymphatic vessels. When injected into superficial tissues it binds to plasma proteins in lymph. By exposing the dye to specific wavelengths of light, ICG fluoresces with near-infrared light. Skin is relatively transparent to ICG fluorescence, enabling the visualization and characterization of superficial lymphatic vessels. An ICG fluorescence lymphatic vessel imager was manufactured to excite ICG and visualize real-time fluorescence as it travels through the lymphatic vessels. Animal studies showed successful ICG excitation and detection using this imager. Clinically, the imager has assisted researchers to visualize otherwise hidden superficial lymphatic pathways in patients postflap surgery. Preliminary results suggest superficial lymphatic vessels do not redevelop in muscle flaps.

  10. Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Joseph R. Pyle

    2017-11-01

    Full Text Available Super-resolution imaging of single DNA molecules via point accumulation for imaging in nanoscale topography (PAINT has great potential to visualize fine DNA structures with nanometer resolution. In a typical PAINT video acquisition, dye molecules (YOYO-1 in solution sparsely bind to the target surfaces (DNA whose locations can be mathematically determined by fitting their fluorescent point spread function. Many YOYO-1 molecules intercalate into DNA and remain there during imaging, and most of them have to be temporarily or permanently fluorescently bleached, often stochastically, to allow for the visualization of a few fluorescent events per DNA per frame of the video. Thus, controlling the fluorescence on–off rate is important in PAINT. In this paper, we study the photobleaching of YOYO-1 and its correlation with the quality of the PAINT images. At a low excitation laser power density, the photobleaching of YOYO-1 is too slow and a minimum required power density was identified, which can be theoretically predicted with the proposed method in this report.

  11. Fluorescent magnetic Fe3 O4 /rare Earth colloidal nanoparticles for dual-modality imaging.

    Science.gov (United States)

    Zhu, Haie; Shang, Yalei; Wang, Wenhao; Zhou, Yingjie; Li, Penghui; Yan, Kai; Wu, Shuilin; Yeung, Kelvin W K; Xu, Zushun; Xu, Haibo; Chu, Paul K

    2013-09-09

    Fluorescent magnetic colloidal nanoparticles (FMCNPs) are produced by a two-step, seed emulsifier-free emulsion polymerization in the presence of oleic acid and sodium undecylenate-modified Fe3 O4 nanoparticles (NPs). The Fe3 O4 /poly(St-co-GMA) nanoparticles are first synthesized as the seed and Eu(AA)3 Phen is copolymerized with the remaining St and GMA to form the fluorescent polymer shell in the second step. The uniform core-shell structured FMCNPs with a mean diameter of 120 nm exhibit superparamagnetism with saturation magnetization of 1.92 emu/g. Red luminescence from the FMCNPs is confirmed by the salient fluorescence emission peaks of europium ions at 594 and 619 nm as well as 2-photon confocal scanning laser microscopy. The in vitro cytotoxicity test conducted using the MTT assay shows good cytocompatibility and the T2 relaxivity of the FMCNPs is 353.86 mM(-1) S(-1) suggesting its potential in magnetic resonance imaging (MRI). In vivo MRI studies based on a rat model show significantly enhanced T2 -weighted images of the liver after administration and prussian blue staining of the liver tissue slice reveals accumulation of FMCNPs in the organ. The cytocompatibility, superparamagnetism, and excellent fluorescent properties of FMCNPs make them suitable for biological imaging probes in MRI and optical imaging. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A fluorescent imaging technique for quantifying spray deposits on plant leaves

    Science.gov (United States)

    Because of the unique characteristics of electrostatically-charged sprays, use of traditional methods to quantify deposition from these sprays has been challenging. A new fluorescent imaging technique was developed to quantify spray deposits from electrostatically-charged sprays on natural plant lea...

  13. 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...

  14. Label-free in vivo imaging of human leukocytes using two-photon excited endogenous fluorescence

    Science.gov (United States)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-04-01

    We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

  15. Spectral and temporal multiplexing for multispectral fluorescence and reflectance imaging using two color sensors.

    Science.gov (United States)

    Dimitriadis, Nikolas; Grychtol, Bartłomiej; Theuring, Martin; Behr, Tobias; Sippel, Christian; Deliolanis, Nikolaos C

    2017-05-29

    Fluorescence imaging can reveal functional, anatomical or pathological features of high interest in medical interventions. We present a novel method to record and display in video rate multispectral color and fluorescence images over the visible and near infrared range. The fast acquisition in multiple channels is achieved through a combination of spectral and temporal multiplexing in a system with two standard color sensors. Accurate color reproduction and high fluorescence unmixing performance are experimentally demonstrated with a prototype system in a challenging imaging scenario. Through spectral simulation and optimization we show that the system is sensitive to all dyes emitting in the visible and near infrared region without changing filters and that the SNR of multiple unmixed components can be kept high if parameters are chosen well. We propose a sensitive per-pixel metric of unmixing quality in a single image based on noise propagation and present a method to visualize the high-dimensional data in a 2D graph, where up to three fluorescent components can be distinguished and segmented.

  16. Airborne laser induced fluorescence imaging. Innovative technology summary report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-06-01

    Laser-Induced Fluorescence (LIF) was demonstration as part of the Fernald Environmental Management Project (FEMP) Plant 1 Large Scale Demonstration and Deployment Project (LSDDP) sponsored by the US Department of Energy (DOE) Office of Science and Technology, Deactivation and Decommissioning Focus Area located at the Federal Energy Technology Center (FETC) in Morgantown, West Virginia. The demonstration took place on November 19, 1996. In order to allow the contaminated buildings undergoing deactivation and decommissioning (D and D) to be opened to the atmosphere, radiological surveys of floors, walls and ceilings must take place. After successful completion of the radiological clearance survey, demolition of the building can continue. Currently, this process is performed by collecting and analyzing swipe samples for radiological analysis. Two methods are used to analyze the swipe samples: hand-held frisker and laboratory analysis. For the purpose of this demonstration, the least expensive method, swipe samples analyzed by hand-held frisker, is the baseline technology. The objective of the technology demonstration was to determine if the baseline technology could be replaced using LIF.

  17. Ultrahigh sensitivity endoscopic camera using a new CMOS image sensor: providing with clear images under low illumination in addition to fluorescent images.

    Science.gov (United States)

    Aoki, Hisae; Yamashita, Hiromasa; Mori, Toshiyuki; Fukuyo, Tsuneo; Chiba, Toshio

    2014-11-01

    We developed a new ultrahigh-sensitive CMOS camera using a specific sensor that has a wide range of spectral sensitivity characteristics. The objective of this study is to present our updated endoscopic technology that has successfully integrated two innovative functions; ultrasensitive imaging as well as advanced fluorescent viewing. Two different experiments were conducted. One was carried out to evaluate the function of the ultrahigh-sensitive camera. The other was to test the availability of the newly developed sensor and its performance as a fluorescence endoscope. In both studies, the distance from the endoscopic tip to the target was varied and those endoscopic images in each setting were taken for further comparison. In the first experiment, the 3-CCD camera failed to display the clear images under low illumination, and the target was hardly seen. In contrast, the CMOS camera was able to display the targets regardless of the camera-target distance under low illumination. Under high illumination, imaging quality given by both cameras was quite alike. In the second experiment as a fluorescence endoscope, the CMOS camera was capable of clearly showing the fluorescent-activated organs. The ultrahigh sensitivity CMOS HD endoscopic camera is expected to provide us with clear images under low illumination in addition to the fluorescent images under high illumination in the field of laparoscopic surgery.

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

    Science.gov (United States)

    Mayer-Cumblidge, M Uljana [Richland, WA; Cao, Haishi [Richland, WA

    2010-08-17

    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.

  19. Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

    NARCIS (Netherlands)

    Haring, M.T.; Liv Hamarat, N.; Zonnevylle, A.C.; Narvaez Gonzalez, A.C.; Voortman, L.M.; Kruit, P.; Hoogenboom, J.P.

    2017-01-01

    In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of

  20. Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

    DEFF Research Database (Denmark)

    Fidorra, Matthias; Garcia, Alejandra; Ipsen, John Hjort

    2009-01-01

    We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks...

  1. Silica micro/nanospheres for theranostics: from bimodal MRI and fluorescent imaging probes to cancer therapy

    Directory of Open Access Journals (Sweden)

    Shanka Walia

    2015-02-01

    Full Text Available Nano-theranostics offer remarkable potential for future biomedical technology with simultaneous applications for diagnosis and therapy of disease sites. Through smart and careful chemical modifications of the nanoparticle surface, these can be converted to multifunctional tiny objects which in turn can be used as vehicle for delivering multimodal imaging agents and therapeutic material to specific target sites in vivo. In this sense, bimodal imaging probes that simultaneously enable magnetic resonance imaging and fluorescence imaging have gained tremendous attention because disease sites can be characterized quick and precisely through synergistic multimodal imaging. But such hybrid nanocomposite materials have limitations such as low chemical stability (magnetic component and harsh cytotoxic effects (fluorescent component and, hence, require a biocompatible protecting agent. Silica micro/nanospheres have shown promise as protecting agent due to the high stability and low toxicity. This review will cover a full description of MRI-active and fluorescent multifunctional silica micro/nanospheres including the design of the probe, different characterization methods and their application in imaging and treatment in cancer.

  2. [Intensity loss of two-photon excitation fluorescence microscopy images of mouse oocyte chromosomes].

    Science.gov (United States)

    Zhao, Feng-Ying; Wu, Hong-Xin; Chen, Die-Yan; Ma, Wan-Yun

    2014-07-01

    As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed. Mouse oocytes with a large diameter, which play an important role in clinical and biological fields, have obvious absorption and scattering effects. In the present paper, we performed compensation for two-photon fluorescence images of mouse oocyte chromosomes. Using volume as a parameter, the attenuation degree of these chromosomes was also studied. The result of our data suggested that there exists a severe axial intensity loss in two-photon microscopic images of mouse oocytes due to the absorption and scattering effects. It is necessary to make compensation for these images of mouse oocyte chromosomes obtained from two-photon microscopic system. It will be specially needed in studying the quantitative three-dimensional information of mouse oocytes.

  3. Characterization of E coli biofim formations on baby spinach leaf surfaces using hyperspectral fluorescence imaging

    Science.gov (United States)

    Cho, Hyunjeong; Baek, Insuck; Oh, Mirae; Kim, Sungyoun; Lee, Hoonsoo; Kim, Moon S.

    2017-05-01

    Bacterial biofilm formed by pathogens on fresh produce surfaces is a food safety concern because the complex extracellular matrix in the biofilm structure reduces the reduction and removal efficacies of washing and sanitizing processes such as chemical or irradiation treatments. Therefore, a rapid and nondestructive method to identify pathogenic biofilm on produce surfaces is needed to ensure safe consumption of fresh, raw produce. This research aimed to evaluate the feasibility of hyperspectral fluorescence imaging for detecting Escherichia.coli (ATCC 25922) biofilms on baby spinach leaf surfaces. Samples of baby spinach leaves were immersed and inoculated with five different levels (from 2.6x104 to 2.6x108 CFU/mL) of E.coli and stored at 4°C for 24 h and 48 h to induce biofilm formation. Following the two treatment days, individual leaves were gently washed to remove excess liquid inoculums from the leaf surfaces and imaged with a hyperspectral fluorescence imaging system equipped with UV-A (365 nm) and violet (405 nm) excitation sources to evaluate a spectral-image-based method for biofilm detection. The imaging results with the UV-A excitation showed that leaves even at early stages of biofilm formations could be differentiated from the control leaf surfaces. This preliminary investigation demonstrated the potential of fluorescence imaging techniques for detection of biofilms on leafy green surfaces.

  4. Portable hyperspectral fluorescence imaging system for detection of biofilms on stainless steel surfaces

    Science.gov (United States)

    Jun, Won; Lee, Kangjin; Millner, Patricia; Sharma, Manan; Chao, Kuanglin; Kim, Moon S.

    2008-04-01

    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 used in the manufacture of food processing equipment. Stainless steel coupons were immersed in bacterium cultures, such as E. coli, Pseudomonas pertucinogena, Erwinia chrysanthemi, and Listeria innocula. Following a 1-week exposure, biofilm formations were assessed using fluorescence imaging. In addition, the effects on biofilm formation from both tryptic soy broth (TSB) and M9 medium with casamino acids (M9C) were examined. TSB grown cells enhance biofilm production compared with M9C-grown cells. Hyperspectral fluorescence images of the biofilm samples, in response to ultraviolet-A (320 to 400 nm) excitation, were acquired from approximately 416 to 700 nm. Visual evaluation of individual images at emission peak wavelengths in the blue revealed the most contrast between biofilms and stainless steel coupons. Two-band ratios compared with the single-band images increased the contrast between the biofilm forming area and stainless steel coupon surfaces. The 444/588 nm ratio images exhibited the greatest contrast between the biofilm formations and stainless coupon surfaces.

  5. Experimental validation of L-shell x-ray fluorescence computed tomography imaging: phantom study.

    Science.gov (United States)

    Bazalova-Carter, Magdalena; Ahmad, Moiz; Xing, Lei; Fahrig, Rebecca

    2015-10-01

    Thanks to the current advances in nanoscience, molecular biochemistry, and x-ray detector technology, x-ray fluorescence computed tomography (XFCT) has been considered for molecular imaging of probes containing high atomic number elements, such as gold nanoparticles. The commonly used XFCT imaging performed with K-shell x rays appears to have insufficient imaging sensitivity to detect the low gold concentrations observed in small animal studies. Low energy fluorescence L-shell x rays have exhibited higher signal-to-background ratio and appeared as a promising XFCT mode with greatly enhanced sensitivity. The aim of this work was to experimentally demonstrate the feasibility of L-shell XFCT imaging and to assess its achievable sensitivity. We built an experimental L-shell XFCT imaging system consisting of a miniature x-ray tube and two spectrometers, a silicon drift detector (SDD), and a CdTe detector placed at [Formula: see text] with respect to the excitation beam. We imaged a 28-mm-diameter water phantom with 4-mm-diameter Eppendorf tubes containing gold solutions with concentrations of 0.06 to 0.1% Au. While all Au vials were detectable in the SDD L-shell XFCT image, none of the vials were visible in the CdTe L-shell XFCT image. The detectability limit of the presented L-shell XFCT SDD imaging setup was 0.007% Au, a concentration observed in small animal studies.

  6. Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence.

    Science.gov (United States)

    Laptenok, Sergey P; Borst, Jan Willem; Mullen, Katharine M; van Stokkum, Ivo H M; Visser, Antonie J W G; van Amerongen, Herbert

    2010-07-21

    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 involved in the energy-transfer process are monitored. This contrasts with current methods that measure either steady-state fluorescence of donor and acceptor molecules or time-resolved fluorescence of donor molecules, and thereby probe a mixture of donor molecules that are involved in FRET and those that are fluorescent but not involved in FRET. The absence of FRET can, for instance, be due to unwanted acceptor bleaching or incomplete maturing of visible proteins that should act as acceptor molecules. In addition, parameters describing the rise of acceptor fluorescence and the decay of donor fluorescence can be determined via simultaneous global analysis of multiple FLIM images, thereby increasing the reliability of the analysis. In the present study, plant protoplasts transfected with fusions of visible fluorescent proteins are used to illustrate the new data analysis method. It is demonstrated that the distances estimated with the present method are substantially smaller than those estimated from the average donor lifetimes, due to a fraction of non-transferring donor molecules. Software to reproduce the presented results is provided in an open-source and freely available package called "TIMP" for "The R project for Statistical Computing".

  7. Sentinel node mapping guided by indocyanine green fluorescence imaging in gastric cancer.

    Science.gov (United States)

    Tajima, Yusuke; Yamazaki, Kimiyasu; Masuda, Yuki; Kato, Masanori; Yasuda, Daisuke; Aoki, Takeshi; Kato, Takashi; Murakami, Masahiko; Miwa, Mitsuharu; Kusano, Mitsuo

    2009-01-01

    In this study, we determined the possible usefulness of sentinel node (SN) mapping guided by indocyanine green (ICG) fluorescence imaging in the management of gastric cancer. ICG fluorescence imaging system has recently been developed for obtaining biochemical information from living tissues. Our series consisted of 56 patients with gastric cancer who underwent standard gastrectomy with lymphadenectomy. Two milliliters of ICG solution (0.5%) was injected into the submucosa around the tumor endoscopically before the operation or into the subserosa intraoperatively. ICG fluorescence imaging was conducted using a charge-coupled device camera with a light-emitting diode having a wavelength of 760 nm as the light source and a cut filter to filter out light with wavelengths below 820 nm as the detector. SNs were detected in 54 (96.4%) of the 56 patients, and the mean number of SNs was 7.2 +/- 7.0. Even SNs that were not green in color could be easily and clearly visualized by ICG fluorescence imaging. cT1-stage cancers were associated with a significantly higher accuracy rate (97.2% vs. 72.2%, P = 0.0127) than cT2-or cT3-stage cancers. Preoperative ICG injection was associated with a significantly higher incidence of cT1-stage cancers (87.1% vs. 40.0%, P = 0.0004), a larger mean number of SNs (9.9 +/- 7.5 vs. 4.1 +/- 5.0, P cancer. Our data suggest that SN mapping guided by ICG fluorescence imaging might be useful for predicting the metastatic status in lymph nodes in cases of gastric cancer, especially those with cT1-stage cancer.

  8. Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.

    Science.gov (United States)

    Ni, Qiang; Mehta, Sohum; Zhang, Jin

    2018-01-01

    Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity. © 2017 Federation of European Biochemical Societies.

  9. 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

    2011-06-06

    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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Monosodium glutamate derived tricolor fluorescent carbon nanoparticles for cell-imaging application.

    Science.gov (United States)

    Zheng, Nannan; Ding, Sha; Zhou, Xingping

    2016-06-01

    Fluorescent carbon nanoparticle (FCN) is a new type of carbon-based materials. Because of its wide raw material sources, excellent optical properties and good biocompatibility, FCN is getting more and more attentions. However, its synthesis from resources at low cost under mild conditions is still a challenge. Here we report a novel and simple method derived from monosodium glutamate carbonization to make tricolor fluorescent carbon nanoparticles with an average size below 10nm, a high yield up to 35.2% based on the carbon content in the resource, a long life-time of 3.71ns, and a high fluorescence quantum yield up to 51.5% by using quinine sulfate as the standard substance. We discovered that the fluorescent stability of the FCNs was very excellent under UV irradiation for hours in aqueous solutions of pH ranged from 2.0 to 9.0. The cell viability tested under a pretty high concentration of FCNs indicated their safety for biological applications. Based on their high fluorescence quantum efficiency and the advantages mentioned above, these FCNs were then used for cell imaging and exhibited a perfect performance under 3 kinds of excitation bands (UV, blue, and green lights). Thus, they can be practically applied to immune labeling and imaging in vivo in the near future. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Classification of corn kernels contaminated with aflatoxins using fluorescence and reflectance hyperspectral images analysis

    Science.gov (United States)

    Zhu, Fengle; Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Brown, Robert; Bhatnagar, Deepak; Cleveland, Thomas

    2015-05-01

    Aflatoxins are secondary metabolites produced by certain fungal species of the Aspergillus genus. Aflatoxin contamination remains a problem in agricultural products due to its toxic and carcinogenic properties. Conventional chemical methods for aflatoxin detection are time-consuming and destructive. This study employed fluorescence and reflectance visible near-infrared (VNIR) hyperspectral images to classify aflatoxin contaminated corn kernels rapidly and non-destructively. Corn ears were artificially inoculated in the field with toxigenic A. flavus spores at the early dough stage of kernel development. After harvest, a total of 300 kernels were collected from the inoculated ears. Fluorescence hyperspectral imagery with UV excitation and reflectance hyperspectral imagery with halogen illumination were acquired on both endosperm and germ sides of kernels. All kernels were then subjected to chemical analysis individually to determine aflatoxin concentrations. A region of interest (ROI) was created for each kernel to extract averaged spectra. Compared with healthy kernels, fluorescence spectral peaks for contaminated kernels shifted to longer wavelengths with lower intensity, and reflectance values for contaminated kernels were lower with a different spectral shape in 700-800 nm region. Principal component analysis was applied for data compression before classifying kernels into contaminated and healthy based on a 20 ppb threshold utilizing the K-nearest neighbors algorithm. The best overall accuracy achieved was 92.67% for germ side in the fluorescence data analysis. The germ side generally performed better than endosperm side. Fluorescence and reflectance image data achieved similar accuracy.

  12. Demonstration of plant fluorescence by imaging technique and Intelligent FluoroSensor

    Science.gov (United States)

    Lenk, Sándor; Gádoros, Patrik; Kocsányi, László; Barócsi, Attila

    2015-10-01

    Photosynthesis is a process that converts carbon-dioxide into organic compounds, especially into sugars, using the energy of sunlight. The absorbed light energy is used mainly for photosynthesis initiated at the reaction centers of chlorophyll-protein complexes, but part of it is lost as heat and chlorophyll fluorescence. Therefore, the measurement of the latter can be used to estimate the photosynthetic activity. The basic method, when illuminating intact leaves with strong light after a dark adaptation of at least 20 minutes resulting in a transient change of fluorescence emission of the fluorophore chlorophyll-a called `Kautsky effect', is demonstrated by an imaging setup. The experimental kit includes a high radiant blue LED and a CCD camera (or a human eye) equipped with a red transmittance filter to detect the changing fluorescence radiation. However, for the measurement of several fluorescence parameters, describing the plant physiological processes in detail, the variation of several excitation light sources and an adequate detection method are needed. Several fluorescence induction protocols (e.g. traditional Kautsky, pulse amplitude modulated and excitation kinetic), are realized in the Intelligent FluoroSensor instrument. Using it, students are able to measure different plant fluorescence induction curves, quantitatively determine characteristic parameters and qualitatively interpret the measured signals.

  13. Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging.

    Science.gov (United States)

    Wang, Dan; Qian, Jun; He, Sailing; Park, Jin Sun; Lee, Kwang-Sup; Han, Sihai; Mu, Ying

    2011-09-01

    We report polymeric nanomicelles doped with organic fluorophores (StCN, (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]-acrylonitrile), which have the property of aggregation-enhanced fluorescence. The fluorescent nanomicelles have two unique features: (1) They give much brighter fluorescence emission than mono-fluorophores. (2) The nanomicelles with amphiphilic copolymers [e.g., phospholipids-PEG (polyethylene glycol)] make the encapsulated fluorophores more stable in various bio-environments and easy for further conjugation with bio-molecules. After chemical and optical characterization, these fluorescent nanomicelles are utilized as efficient optical probes for in vivo sentinel lymph node (SLN) mapping of mice. The StCN-encapsulated nanomicelles, as well as their bioconjugates with arginine-glycine-aspartic acid (RGD) peptides, are used to target subcutaneously xenografted tumors in mice, and in vivo fluorescence images demonstrate the potential to use PEGylated phospholipid nanomicelles with aggregation-enhanced fluorescence as bright nanoprobes for in vivo diagnosis of tumors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Boosting accuracy of automated classification of fluorescence microscope images for location proteomics

    Directory of Open Access Journals (Sweden)

    Huang Kai

    2004-06-01

    Full Text Available Abstract Background Detailed knowledge of the subcellular location of each expressed protein is critical to a full understanding of its function. Fluorescence microscopy, in combination with methods for fluorescent tagging, is the most suitable current method for proteome-wide determination of subcellular location. Previous work has shown that neural network classifiers can distinguish all major protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Building on these results, we evaluate here new classifiers and features to improve the recognition of protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Results We report here a thorough comparison of the performance on this problem of eight different state-of-the-art classification methods, including neural networks, support vector machines with linear, polynomial, radial basis, and exponential radial basis kernel functions, and ensemble methods such as AdaBoost, Bagging, and Mixtures-of-Experts. Ten-fold cross validation was used to evaluate each classifier with various parameters on different Subcellular Location Feature sets representing both 2D and 3D fluorescence microscope images, including new feature sets incorporating features derived from Gabor and Daubechies wavelet transforms. After optimal parameters were chosen for each of the eight classifiers, optimal majority-voting ensemble classifiers were formed for each feature set. Comparison of results for each image for all eight classifiers permits estimation of the lower bound classification error rate for each subcellular pattern, which we interpret to reflect the fraction of cells whose patterns are distorted by mitosis, cell death or acquisition errors. Overall, we obtained statistically significant improvements in classification accuracy over the best previously published results, with the overall error rate being reduced by one-third to one-half and with the average

  15. Boosting accuracy of automated classification of fluorescence microscope images for location proteomics.

    Science.gov (United States)

    Huang, Kai; Murphy, Robert F

    2004-06-18

    Detailed knowledge of the subcellular location of each expressed protein is critical to a full understanding of its function. Fluorescence microscopy, in combination with methods for fluorescent tagging, is the most suitable current method for proteome-wide determination of subcellular location. Previous work has shown that neural network classifiers can distinguish all major protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Building on these results, we evaluate here new classifiers and features to improve the recognition of protein subcellular location patterns in both 2D and 3D fluorescence microscope images. We report here a thorough comparison of the performance on this problem of eight different state-of-the-art classification methods, including neural networks, support vector machines with linear, polynomial, radial basis, and exponential radial basis kernel functions, and ensemble methods such as AdaBoost, Bagging, and Mixtures-of-Experts. Ten-fold cross validation was used to evaluate each classifier with various parameters on different Subcellular Location Feature sets representing both 2D and 3D fluorescence microscope images, including new feature sets incorporating features derived from Gabor and Daubechies wavelet transforms. After optimal parameters were chosen for each of the eight classifiers, optimal majority-voting ensemble classifiers were formed for each feature set. Comparison of results for each image for all eight classifiers permits estimation of the lower bound classification error rate for each subcellular pattern, which we interpret to reflect the fraction of cells whose patterns are distorted by mitosis, cell death or acquisition errors. Overall, we obtained statistically significant improvements in classification accuracy over the best previously published results, with the overall error rate being reduced by one-third to one-half and with the average accuracy for single 2D images being higher than

  16. Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

    Directory of Open Access Journals (Sweden)

    Mély Yves

    2008-09-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

  17. One-Pot Synthesis of Fe3O4@PS@P(AEMH-FITC) Magnetic Fluorescent Nanocomposites for Bimodal Imaging.

    Science.gov (United States)

    Wang, Xuandong; Liu, Huiyu; Jun, Ren; Fu, Changhui; Li, Linlin; Li, Tianlong; Tang, Fangqiong; Meng, Xianwei

    2016-03-01

    Magnetic fluorescent nanocomposites have attracted much attention because of their merging magnetic and fluorescent properties for biomedical application. However, the procedure of synthesis of magnetic fluorescent nanocomposites is always complicated. In addition, the properties of fluorescent component could be easily influenced by magnetic component, retaining both of the magnetic and fluorescent properties into one single nanoparticle considered to be a significant challenge. Herein, we report one-pot method to synthesize multifunctional magnetic fluorescent Fe3O4@PS@P(AEMH-FITC) nanocomposites for bimodal imaging. The asprepared Fe3O4@PS@P(AEMH-FITC) nanocomposites with well-define spherical core/shell structure were stable properties. Moreover, the Fe3O4@PS@P(AEMH-FITC) nanocomposites displayed efficient fluorescent and magnetic properties, respectively. Meanwhile, the magnetic resonance imaging (MRI) and HePG2 cancer cell fluorescent images experiment results suggested that Fe3O4@PS@P(AEMH-FITC) nanocomposites could be used as MRI contrast agents and Fluorescence Imaging (FLI) agents for bioimaging application. Our investigation paves a facile avenue for synthesized magnetic fluorescent nanostructures with well biocompatibility for potential bioimaging application in MRI and FLI.

  18. 3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.

    Energy Technology Data Exchange (ETDEWEB)

    Nieman, Linda T.; Sinclair, Michael B.; Davidson, George S.; Van Benthem, Mark Hilary; Haaland, David Michael; Timlin, Jerilyn Ann; Sasaki, Darryl Yoshio; Bachand, George David; Jones, Howland D. T.

    2007-02-01

    A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

  19. Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

    Science.gov (United States)

    Chen, Xucai; Wang, Jianjun; Versluis, Michel; de Jong, Nico; Villanueva, Flordeliza S.

    2013-06-01

    High speed imaging has application in a wide area of industry and scientific research. In medical research, high speed imaging has the potential to reveal insight into mechanisms of action of various therapeutic interventions. Examples include ultrasound assisted thrombolysis, drug delivery, and gene therapy. Visual observation of the ultrasound, microbubble, and biological cell interaction may help the understanding of the dynamic behavior of microbubbles and may eventually lead to better design of such delivery systems. We present the development of a high speed bright field and fluorescence imaging system that incorporates external mechanical waves such as ultrasound. Through collaborative design and contract manufacturing, a high speed imaging system has been successfully developed at the University of Pittsburgh Medical Center. We named the system "UPMC Cam," to refer to the integrated imaging system that includes the multi-frame camera and its unique software control, the customized modular microscope, the customized laser delivery system, its auxiliary ultrasound generator, and the combined ultrasound and optical imaging chamber for in vitro and in vivo observations. This system is capable of imaging microscopic bright field and fluorescence movies at 25 × 106 frames per second for 128 frames, with a frame size of 920 × 616 pixels. Example images of microbubble under ultrasound are shown to demonstrate the potential application of the system.

  20. Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

    Science.gov (United States)

    Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

    2017-02-01

    Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

  1. Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope.

    Science.gov (United States)

    Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D

    2013-02-01

    We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0-400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

  2. 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. © 2013 Elsevier B.V. All rights reserved.

  3. Near-infrared fluorescence image quality test methods for standardized performance evaluation

    Science.gov (United States)

    Kanniyappan, Udayakumar; Wang, Bohan; Yang, Charles; Ghassemi, Pejhman; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

    2017-03-01

    Near-infrared fluorescence (NIRF) imaging has gained much attention as a clinical method for enhancing visualization of cancers, perfusion and biological structures in surgical applications where a fluorescent dye is monitored by an imaging system. In order to address the emerging need for standardization of this innovative technology, it is necessary to develop and validate test methods suitable for objective, quantitative assessment of device performance. Towards this goal, we develop target-based test methods and investigate best practices for key NIRF imaging system performance characteristics including spatial resolution, depth of field and sensitivity. Characterization of fluorescence properties was performed by generating excitation-emission matrix properties of indocyanine green and quantum dots in biological solutions and matrix materials. A turbid, fluorophore-doped target was used, along with a resolution target for assessing image sharpness. Multi-well plates filled with either liquid or solid targets were generated to explore best practices for evaluating detection sensitivity. Overall, our results demonstrate the utility of objective, quantitative, target-based testing approaches as well as the need to consider a wide range of factors in establishing standardized approaches for NIRF imaging system performance.

  4. Fluorescence images of DNA-bound YOYO between coupled silver particles.

    Science.gov (United States)

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

    2007-11-06

    Oligonucleotide-bound silver particles were coupled through hybridization with target complementary oligonucleotides. YOYO molecules were intercalated into DNA duplexes bound between the coupled metal particles. Fluorescence images of YOYO molecules were monitored by scanning confocal microscopy. Relative to the free single YOYO, the emission brightness of the image was enhanced 80-fold after intercalating the fluorophores into the DNA duplexes between the coupled silver particles. Some images of the labeled metal particle dimers were observed to be dumbbell-shaped, suggesting that the stretching of intercalated YOYO molecules was restricted because of the orientation effect of fluorophores. The shortened lifetime of YOYO molecules between the coupled metal particles indicated that the fluorescence was enhanced via a near-field interaction mechanism between the fluorophore and the metal nanoparticle.

  5. Zwitterion functionalized gold nanoclusters for multimodal near infrared fluorescence and photoacoustic imaging

    Science.gov (United States)

    Shen, Danjin; Henry, Maxime; Trouillet, Vanessa; Comby-Zerbino, Clothilde; Bertorelle, Franck; Sancey, Lucie; Antoine, Rodolphe; Coll, Jean-Luc; Josserand, Véronique; Le Guével, Xavier

    2017-05-01

    Gold nanoclusters (Au NCs) are an emerging type of theranostic agents combining therapeutic and imaging features with reduced toxicity. Au NCs stabilized by a zwitterion ligand with a fine control of the metal core size and the ligand coverage were synthesized by wet chemistry. Intense fluorescence signal is reported for the highest ligand coverage, whereas photoacoustic signal is stronger for the largest metal core. The best Au NC candidate with an average molecular weight of 17 kDa could be detected with high sensitivity on a 2D-near-infrared imaging instrument (limit of detection (LOD) = 2.3 μ M ) and by photoacoustic imaging. In vitro and in vivo experiments demonstrate an efficient cell uptake in U87 cell lines, a fast renal clearance (t1 /2 α = 6.5 ± 1.3 min), and a good correlation between near infrared fluorescence and photoacoustic measurements to follow the early uptake of Au NCs in liver.

  6. Overview of the optical properties of fluorescent nanoparticles for optical imaging

    Directory of Open Access Journals (Sweden)

    Federico Boschi

    2017-08-01

    Full Text Available Nanoparticles (NPs include a wide group of small structures composed by very different materials and characterized by peculiar properties that make them suitable for many applications, especially imaging and drug delivery. In this overview we focus on the optical properties of fluorescent NPs available for in vivo, in vitro and ex vivo preclinical studies and detectable with the optical imaging technique alone or in combination with microscopic confocal imaging. We summarize here the basic principles of the optical detection of fluorescent NPs, elucidate which are the current issues to be resolved and possible solutions to achieve the highest sensitivity and specificity for an unbiased analysis. So far NPs application in clinic is in evaluation due to safety questions still unaddressed but in the future they could dramatically improve both preclinical research and patient clinical care. 

  7. Overview of the optical properties of fluorescent nanoparticles for optical imaging.

    Science.gov (United States)

    Boschi, Federico; De Sanctis, Francesco

    2017-08-29

    Nanoparticles (NPs) include a wide group of small structures composed by very different materials and characterized by peculiar properties that make them suitable for many applications, especially imaging and drug delivery. In this overview we focus on the optical properties of fluorescent NPs available for in vivo, in vitro and ex vivo preclinical studies and detectable with the optical imaging technique alone or in combination with microscopic confocal imaging. We summarize here the basic principles of the optical detection of fluorescent NPs, elucidate which are the current issues to be resolved and possible solutions to achieve the highest sensitivity and specificity for an unbiased analysis. So far NPs application in clinic is in evaluation due to safety questions still unaddressed but in the future they could dramatically improve both preclinical research and patient clinical care.

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Interactions of Insolation and Shading on Ability to Use Fluorescence Imaging to Detect Fecal Contaminated Spinach

    Directory of Open Access Journals (Sweden)

    Alan M. Lefcourt

    2017-10-01

    Full Text Available Fecal contamination of produce in fields is a recognized food safety risk, and it is a requirement that fields be surveyed for evidence of fecal contamination. It may be possible to increase the efficacy of such surveys using imaging techniques that rely on detection of fluorescence responses of fecal material to UV excitation. However, fluorescence responses are easily masked by ambient illumination. This study investigated the potential of using a shroud to reduce the impact of ambient illumination on responses measured using relatively inexpensive optical components. During periods of near peak insolation, even with full shrouding, results indicate that reliable detection would be problematic. Towards dusk, effective imaging could be accomplished even with a gap of 250 cm at the bottom of the shroud. Results suggest that imaging using relatively inexpensive components could provide the basis for detection of fecal contamination in produce fields if surveys were conducted during dawn or dusk, or at night.

  10. Fluorescence lifetime imaging of endogenous molecules in live mouse cancer models (Conference Presentation)

    Science.gov (United States)

    Svindrych, Zdenek; Wang, Tianxiong; Hu, Song; Periasamy, Ammasi

    2017-02-01

    NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.

  11. Leaf Gas Exchange and Chlorophyll a Fluorescence Imaging of Rice Leaves Infected with Monographella albescens.

    Science.gov (United States)

    Tatagiba, Sandro Dan; DaMatta, Fábio Murilo; Rodrigues, Fabrício Ávila

    2015-02-01

    This study was intended to analyze the photosynthetic performance of rice leaf blades infected with Monographella albescens by combining chlorophyll (Chl) a fluorescence images with gas exchange and photosynthetic pigment pools. The net CO2 assimilation rate, stomatal conductance, transpiration rate, total Chl and carotenoid pools, and Chl a/b ratio all decreased but the internal CO2 concentration increased in the inoculated plants compared with their noninoculated counterparts. The first detectable changes in the images of Chl a fluorescence from the leaves of inoculated plants were already evident at 24 h after inoculation (hai) and increased dramatically as the leaf scald lesions expanded. However, these changes were negligible for the photosystem II photochemical efficiency (Fv/Fm) at 24 hai, in contrast to other Chl fluorescence traits such as the photochemical quenching coefficient, yield of photochemistry, and yield for dissipation by downregulation; which, therefore, were much more sensitive than the Fv/Fm ratio in assessing the early stages of fungal infection. It was also demonstrated that M. albescens was able to impair the photosynthetic process in both symptomatic and asymptomatic leaf areas. Overall, it was proven that Chl a fluorescence imaging is an excellent tool to describe the loss of functionality of the photosynthetic apparatus occurring in rice leaves upon infection by M. albescens.

  12. In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters

    Science.gov (United States)

    Wang, Jianling; Zhang, Gen; Li, Qiwei; Jiang, Hui; Liu, Chongyang; Amatore, Christian; Wang, Xuemei

    2013-01-01

    Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors.

  13. Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples.

    Science.gov (United States)

    Swoger, Jim; Pampaloni, Francesco; Stelzer, Ernst H K

    2014-01-01

    In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems; that is, they are used in a cellular context that is defined by hard and flat surfaces. However, a physiological context is not found in single cells cultivated on coverslips. It requires the complex three-dimensional (3D) relationship of cells cultivated in extracellular matrix (ECM) gels, tissue sections, or in naturally developing organisms. In fact, the number of applications of 3D cell cultures in basic research as well as in drug discovery and toxicity testing has been increasing over the past few years. Unfortunately, the imaging of highly scattering multicellular specimens is still challenging. The main issues are the limited optical penetration depth, the phototoxicity, and the fluorophore bleaching. Light-sheet-based fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The basic idea is to illuminate the specimen from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. We discuss the basic principles of LSFM and methods for the preparation, embedding, and imaging of 3D specimens used in the life sciences in an implementation of LSFM known as the single (or selective) plane illumination microscope (SPIM).

  14. Ambient light effect on the uniformity of image intensifier fluorescence screen brightness

    Science.gov (United States)

    Qiu, YaFeng; Chang, BenKang; Sun, LianJun; Zhang, JunJu; Gao, YouTang; Fu, RongGuo

    2008-02-01

    When testing the uniformity of Image intensifier fluorescence screen brightness, the million scale CCD brightness meter is used. Due to the distance between the meter and fluorescence screen, the effect of ambient light on the testing result is essential to the design of testing system. Test with super second generation tube, input a constant voltage to insure the fluorescence screen brightness to be constant. Collect the brightness of the same fluorescence screen in different ambient luminance environment of 1×102Lx, 1×101Lx, 1Lx, 1×10-1Lx, 1×10-2Lx, 1×10-3Lx. Study the results with software MATLAB. It is concluded as: In ambient luminance environment of 1×10-1Lx the CCD has the best result. The testing result in ambient luminance environment of above 1×103Lx show untrue image. The testing result in ambient luminance environment of below 1×10-3Lx shows its own noise image and is unbelievable either.

  15. Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

    Science.gov (United States)

    Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

    2017-07-01

    The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

  16. Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

    Science.gov (United States)

    Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

    2011-07-06

    Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

  17. Multifunctional AS1411-functionalized fluorescent gold nanoparticles for targeted cancer cell imaging and efficient photodynamic therapy.

    Science.gov (United States)

    Ai, Jun; Xu, Yuanhong; Lou, Baohua; Li, Dan; Wang, Erkang

    2014-01-01

    Herein, one multifunctional AS1411-functionalized fluorescent gold nanoparticles (named NAANPs) is synthesized and successfully applied for both targeted cancer cell imaging and efficient photodynamic therapy (PDT). The NAANPs are obtained by functionalizing the gold nanoparticles with AS1411 aptamer and then bound with one porphyrin derivative N-methylmesoporphyrin IX (NMM). Using HeLa cells over expressing nucleolin as representative cancer cells, the formed NAANPs can target to the cell surface via the specific AS1411-nucleolin interaction, which can discriminate the cancer cells from normal ones (e.g. HEK293) unambiguously. That the fluorescence intensity of NMM increased significantly upon binding to AS1411 G-quadruplex makes the NAANPs appropriate fluorescence reagent for cell imaging. Meanwhile, NMM can also be used as a photosensitizer, thus irradiation of the NAANPs by the white light from a common electric torch can lead to efficient production of cytotoxic reactive oxygen species for establishing a new type of PDT to cancer cells. Gold nanoparticles play the roles of both carrier and enhancer of the functional groups onto the cells. In addition, they not only possess inherently certain cytotoxicity to the cancer cells, but also boost the cellular uptake of the fluorescent groups. As a result, the efficiency of both the targeted cell imaging and PDT could be ensured. © 2013 Elsevier B.V. All rights reserved.

  18. Video Object Tracking in Neural Axons with Fluorescence Microscopy Images

    Directory of Open Access Journals (Sweden)

    Liang Yuan

    2014-01-01

    tracking. In this paper, we describe two automated tracking methods for analyzing neurofilament movement based on two different techniques: constrained particle filtering and tracking-by-detection. First, we introduce the constrained particle filtering approach. In this approach, the orientation and position of a particle are constrained by the axon’s shape such that fewer particles are necessary for tracking neurofilament movement than object tracking techniques based on generic particle filtering. Secondly, a tracking-by-detection approach to neurofilament tracking is presented. For this approach, the axon is decomposed into blocks, and the blocks encompassing the moving neurofilaments are detected by graph labeling using Markov random field. Finally, we compare two tracking methods by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and the experimental results show that both methods demonstrate good performance in comparison with the existing approaches, and the tracking accuracy of the tracing-by-detection approach is slightly better between the two.

  19. Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema

    Science.gov (United States)

    Tan, I.-Chih; Maus, Erik A.; Rasmussen, John C.; Marshall, Milton V.; Fife, Caroline E.; Smith, Latisha A.; Sevick-Muraca, Eva M.

    2011-03-01

    Treatment of lymphatic disease is complicated and controversial, due in part to the limited understanding of the lymphatic system. Lymphedema (LE) is a frequent complication after surgical resection and radiation treatment in cancer survivors, and is especially debilitating in regions where treatment options are limited. Although some extremity LE can be effectively treated with manual lymphatic drainage (MLD) therapy or compression devices to direct proximal lymph transport, head and neck LE is more challenging, due to complicated geometry and complex lymphatic structure in head and neck region. Herein, we describe the compassionate use of an investigatory technique of near-infrared (NIR) fluorescence imaging to understand the lymphatic anatomy and function, and to help direct MLD in a patient with head and neck LE. Immediately after 9 intradermal injections of 25 μg indocyanine green each around the face and neck region, NIR fluorescence images were collected using a custom-built imaging system with diffused excitation light illumination. These images were then used to direct MLD therapy. In addition, 3-dimensional (3D) surface profilometry was used to monitor response to therapy. NIR fluorescence images of functioning lymphatic vessels and abnormal structures were obtained. Precise geometries of facial structures were obtained using 3D profilometry, and detection of small changes in edema between therapy sessions was achieved. NIR fluorescence imaging provides a mapping of lymphatic architecture to direct MLD therapy and thus improve treatment efficacy in the head and neck LE, while 3D profilometry allowed longitudinal assessment of edema to evaluate the efficacy of therapy.

  20. Imaging of whole tumor cut sections using a novel scanning beam confocal fluorescence MACROscope

    Science.gov (United States)

    Constantinou, Paul; Vukovic, Vojislav; Haugland, Hans K.; Nicklee, Trudey; Hedley, David W.; Wilson, Brian C.

    2001-07-01

    Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at micrometers resolutions. We have used a novel confocal scanning laser MACROscopeTM (CSLM) capable of acquiring images over fields of view up to 2 cm X 2 cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxia-inducible transcription factor 1 alpha (HIF-1(alpha) ), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

  1. 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.

  2. Highly fluorescent semiconducting polymer dots for single-molecule imaging and biosensing

    Science.gov (United States)

    Sun, Wei; Yu, Jiangbo; Ye, Fangmao; Rong, Yu; Chiu, Daniel T.

    2013-09-01

    This paper describes the preparation of semiconducting polymer dots (Pdots) and their application for single-molecule imaging and biosensing. The Pdots possessed high fluorescence brightness, with a 16 nm Pdot being ~9 times brighter than 13 nm Quantum Dots (Qdots). The surface of Pdots was successfully conjugated with streptavidin, which made Pdots suitable for specific subcellular labeling and targeting. The interior composition of Pdots was also successfully modified, through which the Pdots obtained additional functionalities. We demonstrated the utility of gold nanoparticle embedded Pdots in dual-modality imaging. We also demonstrated that Rhodamine B embedded Pdots were able to function as ratiometric temperature sensor in live-cell imaging mode.

  3. Assessing lymphatic response to treatments in head and neck cancer using near-infrared fluorescence imaging

    Science.gov (United States)

    Tan, I.-Chih; Karni, Ron J.; Rasmussen, John C.; Sevick-Muraca, Eva M.

    2014-05-01

    Care for head and neck (HN) cancer could be improved with better mapping of lymphatic drainage pathways in HN region as well as understanding the effect of the cancer treatments on lymphatics. In this study, near-infrared fluorescence imaging is being used to visualize the lymphatics in human subjects diagnosed with HN cancer before and after treatments. Imaging results show the lymphatic architecture and contractile function in HN. Reformation of lymphatics during the course of cancer care was also seen in the longitudinal imaging. This allows us to better understand the lymphatics in HN cancer patients.

  4. Full-field supercritical angle fluorescence microscopy for live cell imaging

    Science.gov (United States)

    Barroca, Thomas; Balaa, Karla; Delahaye, Julie; Lévêque-Fort, Sandrine; Fort, Emmanuel

    2011-08-01

    We introduce a full-field fluorescence imaging technique with axial confinement of about 100nm at the sample/substrate interface. Contrary to standard surface imaging techniques, this confinement is obtained through emission filtering. This technique is based on supercritical emission selectivity. It can be implemented on any epifluorescence microscope with a commercial high numerical aperture objective and offers a real-time surface imaging capability. This technique is of particular interest for live cell membrane and adhesion studies. Using human embryonic kidney cells, we show that one can observe simultaneously the surface and in-depth cell phenomena.

  5. Nanoscale fluorescence lifetime imaging of an optical antenna with a single diamond NV center.

    Science.gov (United States)

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

    2013-08-14

    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.

  6. Differential laser-induced perturbation spectroscopy and fluorescence imaging for biological and materials sensing

    Science.gov (United States)

    Burton, Dallas Jonathan

    The field of laser-based diagnostics has been a topic of research in various fields, more specifically for applications in environmental studies, military defense technologies, and medicine, among many others. In this dissertation, a novel laser-based optical diagnostic method, differential laser-induced perturbation spectroscopy (DLIPS), has been implemented in a spectroscopy mode and expanded into an imaging mode in combination with fluorescence techniques. The DLIPS method takes advantage of deep ultraviolet (UV) laser perturbation at sub-ablative energy fluences to photochemically cleave bonds and alter fluorescence signal response before and after perturbation. The resulting difference spectrum or differential image adds more information about the target specimen, and can be used in combination with traditional fluorescence techniques for detection of certain materials, characterization of many materials and biological specimen, and diagnosis of various human skin conditions. The differential aspect allows for mitigation of patient or sample variation, and has the potential to develop into a powerful, noninvasive optical sensing tool. The studies in this dissertation encompass efforts to continue the fundamental research on DLIPS including expansion of the method to an imaging mode. Five primary studies have been carried out and presented. These include the use of DLIPS in a spectroscopy mode for analysis of nitrogen-based explosives on various substrates, classification of Caribbean fruit flies versus Caribbean fruit flies that have been irradiated with gamma rays, and diagnosis of human skin cancer lesions. The nitrogen-based explosives and Caribbean fruit flies have been analyzed with the DLIPS scheme using the imaging modality, providing complementary information to the spectroscopic scheme. In each study, a comparison between absolute fluorescence signals and DLIPS responses showed that DLIPS statistically outperformed traditional fluorescence techniques

  7. Sentinel node mapping guided by indocyanine green fluorescence imaging during laparoscopic surgery in gastric cancer.

    Science.gov (United States)

    Tajima, Yusuke; Murakami, Masahiko; Yamazaki, Kimiyasu; Masuda, Yuki; Kato, Masanori; Sato, Atsushi; Goto, Satoru; Otsuka, Koji; Kato, Takashi; Kusano, Mitsuo

    2010-07-01

    Indocyanine green (ICG) fluorescence imaging has recently been reported as a new method for sentinel node (SN) mapping in several types of cancers. In this study, we determined the possible usefulness of SN mapping guided by ICG fluorescence imaging during laparoscopy-assisted gastrectomy (LAG) for gastric cancer. Our series consisted of 77 patients with cT1- or cT2-stage gastric cancer who had undergone LAG (LAG group; 38 patients) or conventional open gastrectomy (OG group; 39 patients). Intraoperative SN mapping guided by ICG fluorescence imaging was conducted with a charge-coupled device camera with a light-emitting diode as the light source and a cut filter as the detector. The detection rate and mean number of fluorescent nodes (FNs) were 94.7% and 7.9, respectively, in the LAG group, and 94.9% and 7.2, respectively, in the OG group. The accuracy and false-negative rates were 97.2% (35 of 36 cases) and 25% (1 of 4), respectively, in the LAG group, and 91.9% (34 of 37) and 23.1% (3 of 13), respectively, in the OG group. Among 33 LAG group patients and 27 OG group patients without FN metastasis, lymph node metastasis was found only in non-SNs located in the same lymphatic basin as the detected FNs. SN mapping guidance by ICG fluorescence imaging could be useful for predicting the lymph node metastasis in gastric cancer, even during LAG. Our data suggest that dissection of the lymphatic basin containing FNs with laparoscopic surgery may be a promising approach as a new type of minimally invasive surgery for patients with cT1- or cT2-stage gastric cancer having no metastasis in FNs.

  8. pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery.

    Science.gov (United States)

    Li, Shengliang; Hu, Kelei; Cao, Weipeng; Sun, Yun; Sheng, Wang; Li, Feng; Wu, Yan; Liang, Xing-Jie

    2014-11-21

    To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a phenylboronic acid-modified poly(lactic acid)-poly(ethyleneimine)(PLA-PEI) copolymer loaded with doxorubicin (Dox) for intracellular imaging and pH-responsive drug delivery. The nanoparticles exhibited superior fluorescence properties, such as fluorescence stability, no blinking and excitation-dependent fluorescence behavior. The Dox-loaded fluorescent nanoparticles showed pH-responsive drug release and were more effective in suppressing the proliferation of MCF-7 cells. In addition, the biocompatible fluorescent nanoparticles could be used as a tool for intracellular imaging and drug delivery, and the process of endosomal escape was traced by real-time imaging. These pH-responsive and biocompatible fluorescent polymer nanoparticles, based on phenylboronic acid, are promising tools for intracellular imaging and drug delivery.

  9. Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Ryo Higuchi-Sanabria

    Full Text Available Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

  10. Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

    Science.gov (United States)

    Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

    2016-01-01

    Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

  11. High-resolution imaging in a deep turbid medium based on an ultrasound-switchable fluorescence technique

    Science.gov (United States)

    Yuan, Baohong; Uchiyama, Seiichi; Liu, Yuan; Nguyen, Kytai T.; Alexandrakis, George

    2012-07-01

    The spatial resolution of fluorescence imaging techniques in deep optically turbid media such as tissues is limited by photon diffusion. To break the diffusion limit and achieve high-resolution and deep-tissue fluorescence imaging, a fundamentally different method was demonstrated based on a concept of ultrasound-switchable fluorescence. The results showed that a small fluorescent tube with a diameter of ˜180 μm at a depth of ˜20 mm in an optical scattering medium (μs'≈3.2 and μa≈0.026 cm-1) can be clearly imaged with a size of ˜260 μm. The depth-to-resolution ratio is shown to be about one order of magnitude better than other deep-tissue fluorescence imaging techniques.

  12. Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

    Science.gov (United States)

    DSouza, Alisha V.; Lin, Huiyun; Henderson, Eric R.; Samkoe, Kimberley S.; Pogue, Brian W.

    2016-08-01

    There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here.

  13. Twenty years of fluorescence imaging of intracellular Chloride.

    Directory of Open Access Journals (Sweden)

    Daniele eArosio

    2014-08-01

    Full Text Available Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride fluxes. These molecular mechanisms are not evenly distributed and regulated over the neuron surface and this fact can lead to a compartmentalized control of the intracellular concentration of chloride. The inhibitory drive provided by the activity of the GABAA receptors depends on the direction and strength of the associated currents, which are ultimately dictated by the gradient of chloride, the main charge carrier flowing through the GABAA channel. Thus, the intracellular distribution of chloride determines the local strength of ionotropic inhibition and influences the interaction between converging excitation and inhibition. The importance of chloride regulation is also underlined by its involvement in several brain pathologies, including epilepsy and disorders of the autistic spectra.The full comprehension of the physiological meaning of GABAergic activity on neurons requires the measure of the spatiotemporal dynamics of chloride fluxes across the membrane. Nowadays, there are several available tools for the task, and both synthetic and genetically encoded indicators have been successfully used for chloride imaging. Here, we will review the available sensors analyzing their properties and outlining desirable future developments.

  14. Diffuse reflectance and fluorescence multispectral imaging system for assessment of skin

    Science.gov (United States)

    Saknite, Inga; Jakovels, Dainis; Spigulis, Janis

    2014-05-01

    The diffuse reflectance multispectral imaging technique has been used for distant mapping of in vivo skin chromophores (hemoglobin and melanin). The fluorescence multispectral imaging is not so common for skin applications due to complicity of data acquisition and processing, but could provide additional information about skin fluorophores. Both techniques are compatible, and could be combined into a multimodal solution. The multispectral imaging system Nuance based on liquid crystal tunable filters was adapted for diffuse reflectance and fluorescence spectral imaging of in vivo skin. Uniform illumination was achieved by LED ring light. Combination of four LEDs (warm white, 770 nm, 830 nm and 890 nm) was used to support diffuse reflectance mode in spectral range 450-950 nm. 405 nm LEDs were used for excitation of skin autofluorescence. Multispectral imaging system was adapted for spectral working range of 450-950 nm with scanning step of 10 nm and spectral resolution of 15 nm. An average field of view was 50x35 mm in size with spatial resolution 0,05 mm (the pixel size). Due to spectrally different illumination intensity and system sensitivity, various exposure times (from 7…500 ms) were used for each image acquisition. The proposed approach was tested for different skin lesions: benign nevus, hemangioma, basalioma and halo nevus. Spectral image cubes of different skin lesions were acquired and analyzed to test its diagnostic potential.

  15. Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

    Science.gov (United States)

    Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

    2017-07-01

    Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

  16. Tetraphenylethene-based aggregation-induced emission fluorescent organic nanoparticles: facile preparation and cell imaging application.

    Science.gov (United States)

    Zhang, Xiqi; Liu, Meiying; Yang, Bin; Zhang, Xiaoyong; Wei, Yen

    2013-12-01

    Tetraphenylethene-based (TPE) aggregation-induced emission fluorescent organic nanoparticles (FONs) were facilely prepared via Schiff base condensation with ɛ-polylysine (Ply) and subsequent reduction to form stable CN covalent bond. Thus obtained TPE-Ply FONs were characterized by a series of techniques including fluorescent spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of TPE-Ply FONs were further investigated to explore their potential biomedical application. We demonstrated that such FONs showed high water dispersibility, intense fluorescence, uniform morphology (100-200nm) and excellent biocompatibility, making them promising for cell imaging application. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. The preparation of ethylenediamine-modified fluorescent carbon dots and their use in imaging of cells.

    Science.gov (United States)

    Dong, Wei; Zhou, Siqi; Dong, Yan; Wang, Jingwen; Ge, Xin; Sui, Lili

    2015-09-01

    In this work, fluorescent carbon dots (CDs) were synthesized using a hydrothermal method with glucose as the carbon source and were surface-modified with ethylenediamine. The properties of as-prepared CDs were analyzed by transmission electron microscopy (TEM), Fourier transform infrared (FTIR), ultraviolet-visible light (UV/vis) absorption and fluorescent spectra. Furthermore, CDs conjugated with mouse anti-(human carcinoembryonic antigen) (CEA) monoclonal antibody were successful employed in the biolabeling and fluorescent imaging of human gastric carcinoma cells. In addition, the cytotoxicity of CDs was also tested using human gastric carcinoma cells. There was no apparent cytotoxicity on human gastric carcinoma cells. These results suggest the potential application of the as-prepared CDs in bioimaging and related fields. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection

    Science.gov (United States)

    Eibl, Matthias; Karpf, Sebastian; Hakert, Hubertus; Weng, Daniel; Huber, Robert

    2017-02-01

    Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. We present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.

  19. Ultrasmall Organic Nanoparticles with Aggregation-Induced Emission and Enhanced Quantum Yield for Fluorescence Cell Imaging.

    Science.gov (United States)

    Xu, Suying; Bai, Xilin; Ma, Jingwen; Xu, Minmin; Hu, Gaofei; James, Tony D; Wang, Leyu

    2016-08-02

    The use of fluorescence probes for biomedical imaging has attracted significant attention over recent years owing to their high resolution at cellular level. The probes are available in many formats including small particle size based imaging agents which are considered to be promising candidates, due to their excellent stabilities. Yet, concerns over the potential cytotoxicity effects of inorganic luminescent particles have led to questions about their suitability for imaging applications. Exploration of alternatives inspired us to use organic fluorophores with aggregation-induced emission (AIE), prepared by functionalizing the amine group on tetraphenylethene with 3,5-bis(trifluoromethyl)phenyl isocyanate. The as-synthesized novel AIE fluorophore (TPE-F) display enhanced quantum yield and longer lifetime as compared with its counterparts (4,4',4″,4‴-(ethene-1,1,2,2-tetrayl)tetraaniline, TPE-AM). Furthermore, the TPE-F was encapsulated into small-size organic nanoparticles (NPs; dynamic light scattering size, ∼10 nm) with polysuccinimide (PSI). The biocompatibility, excellent stability, bright fluorescence, and selective cell targeting of these NPs enable the as-prepared TPE-F NPs to be suitable for specific fluorescence cell imaging.

  20. Bio-degradable highly fluorescent conjugated polymer nanoparticles for bio-medical imaging applications.

    Science.gov (United States)

    Repenko, Tatjana; Rix, Anne; Ludwanowski, Simon; Go, Dennis; Kiessling, Fabian; Lederle, Wiltrud; Kuehne, Alexander J C

    2017-09-07

    Conjugated polymer nanoparticles exhibit strong fluorescence and have been applied for biological fluorescence imaging in cell culture and in small animals. However, conjugated polymer particles are hydrophobic and often chemically inert materials with diameters ranging from below 50 nm to several microns. As such, conjugated polymer nanoparticles cannot be excreted through the renal system. This drawback has prevented their application for clinical bio-medical imaging. Here, we present fully conjugated polymer nanoparticles based on imidazole units. These nanoparticles can be bio-degraded by activated macrophages. Reactive oxygen species induce scission of the conjugated polymer backbone at the imidazole unit, leading to complete decomposition of the particles into soluble low molecular weight fragments. Furthermore, the nanoparticles can be surface functionalized for directed targeting. The approach opens a wide range of opportunities for conjugated polymer particles in the fields of medical imaging, drug-delivery, and theranostics.Conjugated polymer nanoparticles have been applied for biological fluorescence imaging in cell culture and in small animals, but cannot readily be excreted through the renal system. Here the authors show fully conjugated polymer nanoparticles based on imidazole units that can be bio-degraded by activated macrophages.

  1. Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

    Science.gov (United States)

    Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

    2014-02-01

    Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

  2. Automated Cart with VIS/NIR Hyperspectral Reflectance and Fluorescence Imaging Capabilities

    Directory of Open Access Journals (Sweden)

    Alan M. Lefcourt

    2016-12-01

    Full Text Available A system to take high-resolution Visible/Near Infra-Red (VIS/NIR hyperspectral reflectance and fluorescence images in outdoor fields using ambient lighting or a pulsed laser (355 nm, respectively, for illumination purposes was designed, built, and tested. Components of the system include a semi-autonomous cart, a gated-intensified camera, a spectral adapter, a frequency-triple Nd:YAG (Neodymium-doped Yttrium Aluminium Garnet laser, and optics to convert the Gaussian laser beam into a line-illumination source. The front wheels of the cart are independently powered by stepper motors that support stepping or continuous motion. When stepping, a spreadsheet is used to program parameters of image sets to be acquired at each step. For example, the spreadsheet can be used to set delays before the start of image acquisitions, acquisition times, and laser attenuation. One possible use of this functionality would be to establish acquisition parameters to facilitate the measurement of fluorescence decay-curve characteristics. The laser and camera are mounted on an aluminum plate that allows the optics to be calibrated in a laboratory setting and then moved to the cart. The system was validated by acquiring images of fluorescence responses of spinach leaves and dairy manure.

  3. In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique

    Science.gov (United States)

    Xu, Heng; Rice, Brad W.

    2009-11-01

    Spectral unmixing is a useful technique in fluorescence imaging for reducing the effects of native tissue autofluorescence and separating multiple fluorescence probes. While spectral unmixing methods are well established in fluorescence microscopy, they typically rely on precharacterized in-vitro spectra for each fluorophore. However, there are unique challenges for in-vivo applications, since the tissue absorption and scattering can have a significant impact on the measured spectrum of the fluorophore, and therefore make the in-vivo spectra substantially different to that of in vitro. In this work, we introduce a spectral unmixing algorithm tailored for in-vivo optical imaging that does not rely on precharacterized spectral libraries. It is derived from a multivariate curve resolution (MCR) method, which has been widely used in studies of chemometrics and gene expression. Given multispectral images and a few straightforward constraints such as non-negativity, the algorithm automatically finds the signal distribution and the pure spectrum of each component. Signal distribution maps help separate autofluorescence from other probes in the raw images and hence provide better quantification and localization for each probe. The algorithm is demonstrated with an extensive set of in-vivo experiments using near-infrared dyes and quantum dots in both epi-illumination and transillumination geometries.

  4. Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

    Science.gov (United States)

    Pandey, Vikas; Gupta, Shalini; Elangovan, Ravikrishnan

    2018-01-01

    Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid–solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

  5. [Nondestructive imaging of elements distribution in biomedical samples by X-ray fluorescence computed tomography].

    Science.gov (United States)

    Yang, Qun; Deng, Biao; Lü, Wei-Wei; Du, Guo-Hao; Yan, Fu-Hua; Xiao, Ti-Qiao; Xu, Hong-Jie

    2011-10-01

    X-ray fluorescence computed tomography is a stimulated emission tomography that allows nondestructive reconstruction of the elements distribution in the sample, which is important for biomedical investigations. Owing to the high flux density and easy energy tunability of highly collimated synchrotron X-rays, it is possible to apply X-ray fluorescence CT to biomedical samples. Reported in the present paper, an X-ray fluorescence CT system was established at Shanghai Synchrotron Radiation Facility for the investigations of trace elements distribution inside biomedical samples. By optimizing the experiment setup, the spatial resolution was improved and the data acquisition process was obviously speeded up. The maximum-likelihood expectation-maximization algorithm was introduced for the image reconstruction, which remarkably improved the imaging accuracy of element distributions. The developed system was verified by the test sample and medical sample respectively. The results showed that the distribution of interested elements could be imaged correctly, and the spatial resolution of 150 m was achieved. In conclusion, the developed system could be applied to the research on large-size biomedical samples, concerning imaging accuracy, spatial resolution and data collection time.

  6. Combined acoustic-photoacoustic and fluorescence imaging catheter for the detection of the atherosclerotic plaque

    Science.gov (United States)

    Abran, Maxime; Matteau-Pelletier, Carl; Zerouali-Boukhal, Karim; Tardif, Jean-Claude; Lesage, Frédéric

    2011-03-01

    In industrialized countries, cardiovascular diseases remain the main cause of mortality. The detection of atherosclerosis and its associated plaque using imaging techniques allows studying the efficacy of new drugs in vivo. Intravascular ultrasound (IVUS) imaging has been demonstrated to be a powerful tool to uncover structural information of atherosclerotic plaques. Recently, intravascular photoacoustic (IVPA) has been combined with IVUS imaging to add functional and/or molecular information. The IVPA/IVUS combination has been demonstrated in phantoms and ex vivo tissues to provide relevant information about the composition of the plaque, as well as its vulnerability. In this work, we extend previous work by developing a combined IVPA/IVUS system using a rotating ultrasound transducer in a catheter to which an optical fiber is attached. In addition, a third modality was included through fluorescence detection in the same fiber at a distinct wavelength from PA, opening the door to complementary information using fluorescence activatable probes. Cylindrical silicon phantoms with inclusions containing fluorophores or ink were used to validate the system. Bleaching of the fluorophore by the pulsed laser used for photoacoustic was quantified. IVUS images were obtained continuously and used to co-register photoacoustic and fluorescence signals.

  7. Nanostructures Derived from Starch and Chitosan for Fluorescence Bio-Imaging

    Directory of Open Access Journals (Sweden)

    Yinxue Zu

    2016-07-01

    Full Text Available Fluorescent nanostructures (NSs derived from polysaccharides have drawn great attention as novel fluorescent probes for potential bio-imaging applications. Herein, we reported a facile alkali-assisted hydrothermal method to fabricate polysaccharide NSs using starch and chitosan as raw materials. Transmission electron microscopy (TEM demonstrated that the average particle sizes are 14 nm and 75 nm for starch and chitosan NSs, respectively. Fourier transform infrared (FT-IR spectroscopy analysis showed that there are a large number of hydroxyl or amino groups on the surface of these polysaccharide-based NSs. Strong fluorescence with an excitation-dependent emission behaviour was observed under ultraviolet excitation. Interestingly, the photostability of the NSs was found to be superior to fluorescein and rhodamine B. The quantum yield of starch NSs could reach 11.12% under the excitation of 360 nm. The oxidative metal ions including Cu(II, Hg(IIand Fe(III exhibited a quench effect on the fluorescence intensity of the prepared NSs. Both of the two kinds of the multicoloured NSs showed a maximum fluorescence intensity at pH 7, while the fluorescence intensity decreased dramatically when they were put in an either acidic or basic environment (at pH 3 or 11. The cytotoxicity study of starch NSs showed that low cell cytotoxicity and 80% viability was found after 24 h incubation, when their concentration was less than 10 mg/mL. The study also showed the possibility of using the multicoloured starch NSs for mouse melanoma cells and guppy fish imaging.

  8. Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

    Science.gov (United States)

    Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

    2016-09-10

    A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Oxygen-generating hybrid nanoparticles to enhance fluorescent/photoacoustic/ultrasound imaging guided tumor photodynamic therapy.

    Science.gov (United States)

    Gao, Shi; Wang, Guohao; Qin, Zainen; Wang, Xiangyu; Zhao, Guoqing; Ma, Qingjie; Zhu, Lei

    2017-01-01

    Photodynamic therapy (PDT) is a promising tumor treatment modality that can convert oxygen into cytotoxic singlet oxygen (SO) via photosensitizer to ablate tumor growth. However, the uncontrolled cancer cell proliferation during tumor development and the oxygen consumption during PDT always result in an insufficient oxygen level in tumors, which can adversely affect the PDT efficiency in turn. We designed an oxygen-generating PDT nanocomplex by encapsulating a manganese dioxide nanoparticle (MnO 2 NP) in an indocyanine green (ICG) modified hyaluronic acid nanoparticle (HANP) to overcome this limitation. Because of the excellent fluorescent and photoacoustic properties, the tumor accumulation of the ICG-HANP/MnO 2 (IHM) nanocomplex was monitored by fluorescent imaging and photoacoustic imaging after intravenous administration into the SCC7 tumor-bearing mouse model. Both high fluorescent and photoacoustic signals were detected and found peak at 6 h post-injection (tumor-muscle ratio: 4.03 ± 0.36 for fluorescent imaging and 2.93 ± 0.13 for photoacoustic imaging). In addition, due to the high reactivity of MnO 2 NP to H 2 O 2 , an unfavorable tumor cell metabolic, the oxygen content in the tumor is elevated 2.25 ± 0.07 times compared to that without IHM treatment as ultrasound imaging confirmed. After laser irradiation, significant tumor growth inhibition was observed in the IHM-treated group compared to the ICG-HANP-treated group, attributed to the beneficial oxygen-generating property of IHM for PDT. It is expected that the design of IHM will provide an alternative way of improving clinical PDT efficacy and will be widely applied in cancer theranostics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Fluorescence in situ hybridization (FISH) signal analysis using automated generated projection images.

    Science.gov (United States)

    Wang, Xingwei; Chen, Xiaodong; Li, Yuhua; Liu, Hong; Li, Shibo; Zhang, Roy R; Zheng, Bin

    2012-01-01

    Fluorescence in situ hybridization (FISH) tests provide promising molecular imaging biomarkers to more accurately and reliably detect and diagnose cancers and genetic disorders. Since current manual FISH signal analysis is low-efficient and inconsistent, which limits its clinical utility, developing automated FISH image scanning systems and computer-aided detection (CAD) schemes has been attracting research interests. To acquire high-resolution FISH images in a multi-spectral scanning mode, a huge amount of image data with the stack of the multiple three-dimensional (3-D) image slices is generated from a single specimen. Automated preprocessing these scanned images to eliminate the non-useful and redundant data is important to make the automated FISH tests acceptable in clinical applications. In this study, a dual-detector fluorescence image scanning system was applied to scan four specimen slides with FISH-probed chromosome X. A CAD scheme was developed to detect analyzable interphase cells and map the multiple imaging slices recorded FISH-probed signals into the 2-D projection images. CAD scheme was then applied to each projection image to detect analyzable interphase cells using an adaptive multiple-threshold algorithm, identify FISH-probed signals using a top-hat transform, and compute the ratios between the normal and abnormal cells. To assess CAD performance, the FISH-probed signals were also independently visually detected by an observer. The Kappa coefficients for agreement between CAD and observer ranged from 0.69 to 1.0 in detecting/counting FISH signal spots in four testing samples. The study demonstrated the feasibility of automated FISH signal analysis that applying a CAD scheme to the automated generated 2-D projection images.

  11. Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity

    Science.gov (United States)

    Levitt, James A.; Chung, Pei-Hua; Suhling, Klaus

    2015-09-01

    Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ɛ, is around 5 in lipid droplets and 25FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.

  12. Photosensitizer-conjugated gold nanorods for enzyme-activatable fluorescence imaging and photodynamic therapy.

    Science.gov (United States)

    Jang, Boseung; Choi, Yongdoo

    2012-01-01

    We report on the development of photosensitizer-conjugated gold nanorods (MMP2P-GNR) in which photosensitizers were conjugated onto the surface of gold nanorods (GNR) via a protease-cleavable peptide linker. We hypothesized that fluorescence and phototoxicity of the conjugated photosensitizers would be suppressed in their native state, becoming activated only after cleavage by the target protease matrix metalloprotease-2 (MMP2). Quantitative analysis of the fluorescence and singlet oxygen generation (SOG) demonstrated that the MMP2P-GNR conjugate emitted fluorescence intensity corresponding to 0.4% ± 0.01% and an SOG efficiency of 0.89% ± 1.04% compared to free pyropheophorbide-a. From the in vitro cell studies using HT1080 cells that overexpress MMP2 and BT20 cells that lack MMP2, we observed that fluorescence and SOG was mediated by the presence or absence of MMP2 in these cell lines. This novel activatable photosensitizing system may be useful for protease-mediated fluorescence imaging and subsequent photodynamic therapy for various cancers.

  13. Fluorescence microscopy image noise reduction using a stochastically-connected random field model.

    Science.gov (United States)

    Haider, S A; Cameron, A; Siva, P; Lui, D; Shafiee, M J; Boroomand, A; Haider, N; Wong, A

    2016-02-17

    Fluorescence microscopy is an essential part of a biologist's toolkit, allowing assaying of many parameters like subcellular localization of proteins, changes in cytoskeletal dynamics, protein-protein interactions, and the concentration of specific cellular ions. A fundamental challenge with using fluorescence microscopy is the presence of noise. This study introduces a novel approach to reducing noise in fluorescence microscopy images. The noise reduction problem is posed as a Maximum A Posteriori estimation problem, and solved using a novel random field model called stochastically-connected random field (SRF), which combines random graph and field theory. Experimental results using synthetic and real fluorescence microscopy data show the proposed approach achieving strong noise reduction performance when compared to several other noise reduction algorithms, using quantitative metrics. The proposed SRF approach was able to achieve strong performance in terms of signal-to-noise ratio in the synthetic results, high signal to noise ratio and contrast to noise ratio in the real fluorescence microscopy data results, and was able to maintain cell structure and subtle details while reducing background and intra-cellular noise.

  14. Water-soluble BODIPY-based fluorescent probe for mitochondrial imaging (Conference Presentation)

    Science.gov (United States)

    Sui, Binglin; Tang, Simon; Woodward, Adam W.; Kim, Bosung; Belfield, Kevin D.

    2016-03-01

    A new mitochondrial targeting fluorescent probe is designed, synthesized, characterized, and investigated. The probe is composed of three moieties, a BODIPY platform working as the fluorophore, two triphenylphosphonium (TPP) groups serving as mitochondrial targeting moiety, and two long highly hydrophilic polyethylene glycol (PEG) chains to increase its water solubility and reduce its cytotoxicity. As a mitochondria-selective fluorescent probe, the probe exhibits a series of desirable advantages compared with other reported fluorescent mitochondrial probes. It is readily soluble in aqueous media and emits very strong fluorescence. Photophysical determination experiments show that the photophysical properties of the probe are independent of solvent polarity and it has high quantum yield in various solvents examined. The probe also has good photostability and pH insensitivity over a broad pH range. Results obtained from cell viability tests indicate that the cytotoxicity of the probe is very low. Confocal fluorescence microscopy colocalization experiments reveal that this probe possesses excellent mitochondrial targeting ability and it is suitable for imaging mitochondria in living cells.

  15. Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells

    Science.gov (United States)

    Rück, Angelika; Hauser, Carmen; Mosch, Simone; Kalinina, Sviatlana

    2014-09-01

    Fluorescence-guided diagnosis of tumor tissue is in many cases insufficient, because false positive results interfere with the outcome. Improvement through observation of cell metabolism might offer the solution, but needs a detailed understanding of the origin of autofluorescence. With respect to this, spectrally resolved multiphoton fluorescence lifetime imaging was investigated to analyze cell metabolism in metabolic phenotypes of malignant and nonmalignant oral mucosa cells. The time-resolved fluorescence characteristics of NADH were measured in cells of different origins. The fluorescence lifetime of bound and free NADH was calculated from biexponential fitting of the fluorescence intensity decay within different spectral regions. The mean lifetime was increased from nonmalignant oral mucosa cells to different squamous carcinoma cells, where the most aggressive cells showed the longest lifetime. In correlation with reports in the literature, the total amount of NADH seemed to be less for the carcinoma cells and the ratio of free/bound NADH was decreased from nonmalignant to squamous carcinoma cells. Moreover for squamous carcinoma cells a high concentration of bound NADH was found in cytoplasmic organelles (mainly mitochondria). This all together indicates that oxidative phosphorylation and a high redox potential play an important role in the energy metabolism of these cells.

  16. Mass Spectrometric Imaging of Red Fluorescent Protein in Breast Tumor Xenografts

    Science.gov (United States)

    Chughtai, Kamila; Jiang, Lu; Post, Harm; Winnard, Paul T.; Greenwood, Tiffany R.; Raman, Venu; Bhujwalla, Zaver M.; Heeren, Ron M. A.; Glunde, Kristine

    2013-05-01

    Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yankelevich, Diego R. [Department of Electrical and Co