Sample records for biological fluorescence imaging

  1. Fluorescence confocal endomicroscopy in biological imaging (United States)

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


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

  2. Ion beam induced fluorescence imaging in biological systems

    International Nuclear Information System (INIS)

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


    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

  3. Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

    International Nuclear Information System (INIS)

    Saloma, Caesar; Palmes-Saloma, Cynthia; Kondoh, Hisato


    Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 μm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 μm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested. (author)

  4. Fluorescence Image Segmentation by using Digitally Reconstructed Fluorescence Images


    Blumer, Clemens; Vivien, Cyprien; Oertner, Thomas G; Vetter, Thomas


    In biological experiments fluorescence imaging is used to image living and stimulated neurons. But the analysis of fluorescence images is a difficult task. It is not possible to conclude the shape of an object from fluorescence images alone. Therefore, it is not feasible to get good manual segmented nor ground truth data from fluorescence images. Supervised learning approaches are not possible without training data. To overcome this issues we propose to synthesize fluorescence images and call...

  5. Multimodal fluorescence imaging spectroscopy

    NARCIS (Netherlands)

    Stopel, Martijn H W; Blum, Christian; Subramaniam, Vinod; Engelborghs, Yves; Visser, Anthonie J.W.G.


    Multimodal fluorescence imaging is a versatile method that has a wide application range from biological studies to materials science. Typical observables in multimodal fluorescence imaging are intensity, lifetime, excitation, and emission spectra which are recorded at chosen locations at the sample.

  6. Differential laser-induced perturbation spectroscopy and fluorescence imaging for biological and materials sensing (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. Development of Two Color Fluorescent Imager and Integrated Fluidic System for Nanosatellite Biology Applications (United States)

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


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

  8. Fluorescent carbon dots and nanodiamonds for biological imaging: preparation, application, pharmacokinetics and toxicity. (United States)

    Liu, Jia-Hui; Yang, Sheng-Tao; Chen, Xin-Xin; Wang, Haifang


    The rapid advancement of nanotechnology has brought us some new types of fluorescent probes, which are indispensable for bioimaging in life sciences. Because of their innate biocompatibility, good resistance against photobleaching, long fluorescence lifetime and wide fluorescence spectral region, fluorescent carbon quantum dots (C-Dots) and nanosized diamonds (nanodiamonds, NDs) are gradually evolving into promising reagents for bioimaging. In this review, we summarize the recent achievements in fluorescent C-Dots and NDs with emphases on their preparation, properties, imaging application, pharmacokinetics and toxicity. Perspectives on further investigations and opportunities to develop C-Dots and NDs into the safer and more sensitive imaging probes for both living cells and animal models are discussed.

  9. Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine. (United States)

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


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

  10. Intravascular near-infrared fluorescence molecular imaging of atherosclerosis: toward coronary arterial visualization of biologically high-risk plaques (United States)

    Calfon, Marcella A.; Vinegoni, Claudio; Ntziachristos, Vasilis; Jaffer, Farouc A.


    New imaging methods are urgently needed to identify high-risk atherosclerotic lesions prior to the onset of myocardial infarction, stroke, and ischemic limbs. Molecular imaging offers a new approach to visualize key biological features that characterize high-risk plaques associated with cardiovascular events. While substantial progress has been realized in clinical molecular imaging of plaques in larger arterial vessels (carotid, aorta, iliac), there remains a compelling, unmet need to develop molecular imaging strategies targeted to high-risk plaques in human coronary arteries. We present recent developments in intravascular near-IR fluorescence catheter-based strategies for in vivo detection of plaque inflammation in coronary-sized arteries. In particular, the biological, light transmission, imaging agent, and engineering principles that underlie a new intravascular near-IR fluorescence sensing method are discussed. Intravascular near-IR fluorescence catheters appear highly translatable to the cardiac catheterization laboratory, and thus may offer a new in vivo method to detect high-risk coronary plaques and to assess novel atherosclerosis biologics.

  11. Synthesis, biological evaluation, and live cell imaging of novel fluorescent duocarmycin analogs. (United States)

    Tietze, Lutz F; Behrendt, Frank; Pestel, Galina F; Schuberth, Ingrid; Mitkovski, Mišo


    For a better understanding of the mode of action of duocarmycin and its analogs, the novel fluorescent duocarmycin derivatives 13-15 and 17b-19b were synthesized, and their bioactivity as well as their cellular uptake investigated using confocal laser scanning microscopy (CLSM) in live-cell imaging experiments. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  12. Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering.

    Directory of Open Access Journals (Sweden)

    Isaac Nuñez

    Full Text Available The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here

  13. Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering. (United States)

    Nuñez, Isaac; Matute, Tamara; Herrera, Roberto; Keymer, Juan; Marzullo, Timothy; Rudge, Timothy; Federici, Fernán


    The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under

  14. Carbon nanotubes allow capture of krypton, barium and lead for multichannel biological X-ray fluorescence imaging. (United States)

    Serpell, Christopher J; Rutte, Reida N; Geraki, Kalotina; Pach, Elzbieta; Martincic, Markus; Kierkowicz, Magdalena; De Munari, Sonia; Wals, Kim; Raj, Ritu; Ballesteros, Belén; Tobias, Gerard; Anthony, Daniel C; Davis, Benjamin G


    The desire to study biology in situ has been aided by many imaging techniques. Among these, X-ray fluorescence (XRF) mapping permits observation of elemental distributions in a multichannel manner. However, XRF imaging is underused, in part, because of the difficulty in interpreting maps without an underlying cellular 'blueprint'; this could be supplied using contrast agents. Carbon nanotubes (CNTs) can be filled with a wide range of inorganic materials, and thus can be used as 'contrast agents' if biologically absent elements are encapsulated. Here we show that sealed single-walled CNTs filled with lead, barium and even krypton can be produced, and externally decorated with peptides to provide affinity for sub-cellular targets. The agents are able to highlight specific organelles in multiplexed XRF mapping, and are, in principle, a general and versatile tool for this, and other modes of biological imaging.

  15. Combined application of dynamic light scattering imaging and fluorescence intravital microscopy in vascular biology

    International Nuclear Information System (INIS)

    Kalchenko, V; Harmelin, A; Ziv, K; Addadi, Y; Madar-Balakirski, N; Neeman, M; Meglinski, I


    The dynamic light scattering imaging (DLSI) system combined with the conventional fluorescence intravital microscope (FIM) has been applied for the examination of blood and lymph vessels in the mouse ear in vivo. While the CCD camera can be shared by both techniques the combined application of DLSI and FIM allows rapid switching between the modalities. In current study temporal speckles fluctuations are used for rendering blood vessels structure and monitoring blood perfusion with the higher spatial resolution, whereas FIM provides the images of lymphatic vessels. The results clearly demonstrate that combined application of DLSI and FIM approaches provides synchronic in vivo images of blood and lymph vessels with higher contrast and specificity. The use of this new dual-modal diagnostic system is particularly important and has a great potential to significantly expand the capabilities of vascular diagnostics providing synchronic in vivo images of blood and lymph vessels

  16. Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities. (United States)

    Vergucht, Eva; Brans, Toon; Beunis, Filip; Garrevoet, Jan; Bauters, Stephen; De Rijcke, Maarten; Deruytter, David; Janssen, Colin; Riekel, Christian; Burghammer, Manfred; Vincze, Laszlo


    Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

  17. Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight (United States)

    Weinstein, S.; Pane, D.; Warren-Rhodes, K.; Cockell, C.; Ernst, L. A.; Minkley, E.; Fisher, G.; Emani, S.; Wettergreen, D. S.; Wagner, M.


    We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

  18. Synthesis and biological investigation of PIM mimics carrying biotin or a fluorescent label for cellular imaging. (United States)

    Front, Sophie; Bourigault, Marie-Laure; Rose, Stéphanie; Noria, Ségueni; Quesniaux, Valérie F J; Martin, Olivier R


    Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM-biotin conjugate binds to macrophage membranes with rapid kinetics.

  19. Fluorescence and Spectral Imaging

    Directory of Open Access Journals (Sweden)

    Ralph S. DaCosta


    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.

  20. One-step fabrication of PEGylated fluorescent nanodiamonds through the thiol-ene click reaction and their potential for biological imaging (United States)

    Huang, Hongye; Liu, Meiying; Tuo, Xun; Chen, Junyu; Mao, Liucheng; Wen, Yuanqing; Tian, Jianwen; Zhou, Naigen; Zhang, Xiaoyong; Wei, Yen


    Over the past years, fluorescent carbon nanoparticles have got growing interest for biological imaging. Fluorescent nanodiamonds (FNDs) are novel fluorescent carbon nanoparticles with multitudinous useful properties, including remarkable fluorescence properties, extremely low toxicity and high refractive index. However, facile preparation of FNDs with designable properties and functions from non-fluorescent detonation nanodiamonds (DNDs) has demonstrated to be challengeable. In this work, we reported for the first time that preparation of Polyethylene glycol (PEG) functionalized FNDs through a one-step thiol-ene click reaction using thiol containing PEG (PEG-SH) as the coating agent. Based on the characterization results, we demonstrated that PEG-SH could be efficiently introduced on DNDs to obtain FNDs through the thiol-ene click chemistry. The resultant FND-PEG composites showed high water dispersibility, strong fluorescence and low cytotoxicity. Moreover, FND-PEG composites could be internalized by cells and displayed good cell dyeing performance. All of these features implied that FND-PEG composites are of great potential for biological imaging. Taken together, a facile one-step strategy based on the one-step thiol-ene click reaction has been developed for efficient preparation of FND-PEG composites from non-fluorescent DNDs. The strategy should be also useful for fabrication of many other functional FNDs via using different thiol containing compounds for the universality of thiol-ene click reaction.

  1. Multi Spectral Fluorescence Imager (MSFI) (United States)

    Caron, Allison


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

  2. A novel self-catalyzed photoATRP strategy for preparation of fluorescent hydroxyapatite nanoparticles and their biological imaging (United States)

    Jiang, Ruming; Liu, Meiying; Huang, Hongye; Huang, Long; Huang, Qiang; Wen, Yuanqing; Cao, Qian-yong; Tian, Jianwen; Zhang, Xiaoyong; Wei, Yen


    Hydroxyapatite (HAp), as an important biomaterial for the regeneration and reconstruction of bone tissue, has attracted more and more attention of researchers and scientists due to its unique structure and compositions. However, the preparation of fluorescent HAp with controllable morphology has achieved only limited success. In this work, we reported a novel strategy to construct the water dispersible fluorescent HAp nanorods via the combination of ligand exchange and metal-free atom transfer radical polymerization (ATRP). The Br-containing fluorescent HAp nanorods with controllable size and morphology were first prepared through hydrothermal treatment. A multifunctional organic molecule (named as PTH-Br) with aggregation-induced emission feature was immobilized on the surface of hydrophobic HAp nanorods through ligand exchange reaction. The PTH-Br could be used as the initiator and catalyst for surface-initiated metal-free ATRP using poly(ethylene glycol) methacrylate as monomer to obtain hydrophilic fluorescent HAp polymer nanoparticles. This strategy successfully endowed HAp nanorods excellent fluorescence properties and favorable water dispersibility but well preserved their regular morphology. Biological assays demonstrated that the HAp-PTH-poly(PEGMA) nanoparticles exhibited good biocompatibility and efficient cell uptake performance. Taken together, we have developed a rather facile strategy based on the surface ligand exchange reaction and metal-free photoATRP to fabricate fluorescent HAp with controllable size and morphology, high water dispersibility and biological properties. These HAp-PTH-poly(PEGMA) nanoparticles should be novel and promising candidates for biomedical applications.

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

    CSIR Research Space (South Africa)

    Mabaso, M


    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...

  4. Fluorescent microthermographic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Barton, D.L.


    In the early days of microelectronics, design rules and feature sizes were large enough that sub-micron spatial resolution was not needed. Infrared or IR thermal techniques were available that calculated the object`s temperature from infrared emission. There is a fundamental spatial resolution limitation dependent on the wavelengths of light being used in the image formation process. As the integrated circuit feature sizes began to shrink toward the one micron level, the limitations imposed on IR thermal systems became more pronounced. Something else was needed to overcome this limitation. Liquid crystals have been used with great success, but they lack the temperature measurement capabilities of other techniques. The fluorescent microthermographic imaging technique (FMI) was developed to meet this need. This technique offers better than 0.01{degrees}C temperature resolution and is diffraction limited to 0.3 {mu}m spatial resolution. While the temperature resolution is comparable to that available on IR systems, the spatial resolution is much better. The FMI technique provides better spatial resolution by using a temperature dependent fluorescent film that emits light at 612 nm instead of the 1.5 {mu}m to 12 {mu}m range used by IR techniques. This tutorial starts with a review of blackbody radiation physics, the process by which all heated objects emit radiation to their surroundings, in order to understand the sources of information that are available to characterize an object`s surface temperature. The processes used in infrared thermal imaging are then detailed to point out the limitations of the technique but also to contrast it with the FMI process. The FMI technique is then described in detail, starting with the fluorescent film physics and ending with a series of examples of past applications of FMI.

  5. Oligothiophenes as Fluorescent Markers for Biological Applications

    Directory of Open Access Journals (Sweden)

    Antonio Manetto


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

  6. Boronic acids for fluorescence imaging of carbohydrates. (United States)

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


    "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.

  7. Fluorescence Imaging Reveals Surface Contamination (United States)

    Schirato, Richard; Polichar, Raulf


    In technique to detect surface contamination, object inspected illuminated by ultraviolet light to make contaminants fluoresce; low-light-level video camera views fluorescence. Image-processing techniques quantify distribution of contaminants. If fluorescence of material expected to contaminate surface is not intense, tagged with low concentration of dye.

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

    Directory of Open Access Journals (Sweden)

    Gregor P. C. Drummen


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

  9. Recent developments in multimodality fluorescence imaging probes

    Directory of Open Access Journals (Sweden)

    Jianhong Zhao


    Full Text Available Multimodality optical imaging probes have emerged as powerful tools that improve detection sensitivity and accuracy, important in disease diagnosis and treatment. In this review, we focus on recent developments of optical fluorescence imaging (OFI probe integration with other imaging modalities such as X-ray computed tomography (CT, magnetic resonance imaging (MRI, positron emission tomography (PET, single-photon emission computed tomography (SPECT, and photoacoustic imaging (PAI. The imaging technologies are briefly described in order to introduce the strengths and limitations of each techniques and the need for further multimodality optical imaging probe development. The emphasis of this account is placed on how design strategies are currently implemented to afford physicochemically and biologically compatible multimodality optical fluorescence imaging probes. We also present studies that overcame intrinsic disadvantages of each imaging technique by multimodality approach with improved detection sensitivity and accuracy. KEY WORDS: Optical imaging, Fluorescence, Multimodality, Near-infrared fluorescence, Nanoprobe, Computed tomography, Magnetic resonance imaging, Positron emission tomography, Single-photon emission computed tomography, Photoacoustic imaging

  10. Hyperspectral small animal fluorescence imaging: spectral selection imaging (United States)

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


    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.

  11. Assessing Photosynthesis by Fluorescence Imaging (United States)

    Saura, Pedro; Quiles, Maria Jose


    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…

  12. Trace Element Mapping of a Biological Specimen by a Full-Field X-ray Fluorescence Imaging Microscope with a Wolter Mirror

    International Nuclear Information System (INIS)

    Hoshino, Masato; Yamada, Norimitsu; Ishino, Toyoaki; Namiki, Takashi; Watanabe, Norio; Aoki, Sadao


    A full-field X-ray fluorescence imaging microscope with a Wolter mirror was applied to the element mapping of alfalfa seeds. The X-ray fluorescence microscope was built at the Photon Factory BL3C2 (KEK). X-ray fluorescence images of several growing stages of the alfalfa seeds were obtained. X-ray fluorescence energy spectra were measured with either a solid state detector or a CCD photon counting method. The element distributions of iron and zinc which were included in the seeds were obtained using a photon counting method

  13. Single Molecule Fluorescence: from Physical Fascination to Biological Relevance


    Segers-Nolten, Gezina M.J.


    Confocal fluorescence microscopy is particularly well-known from the beautiful images that have been obtained with this technique from cells. Several cellular components could be nicely visualized simultaneously by staining them with different fluorophores. Not only for ensemble applications but also in single molecule research confocal fluorescence microscopy became a popular technique. In this thesis the possibilities are shown to study a complicated biological process, which is Nucleotide ...

  14. Facile synthesis of polymeric fluorescent organic nanoparticles based on the self-polymerization of dopamine for biological imaging. (United States)

    Shi, Yingge; Jiang, Ruming; Liu, Meiying; Fu, Lihua; Zeng, Guangjian; Wan, Qing; Mao, Liucheng; Deng, Fengjie; Zhang, Xiaoyong; Wei, Yen


    Polymeric fluorescent organic nanoparticles (polymer-FONs) have raised considerable research attention for biomedical applications owing to their advantages as compared with fluorescent inorganic nanoparticles and small organic molecules. In this study, we presented an efficient, facile and environment-friendly strategy to produce polymer-FONs, which relied on the self-polymerization of dopamine and polyethyleneimine (PEI) in rather mild conditions. To obtain the final polymer-FONs, aldehyde group-containing copolymers (named as poly(UA-co-PEGMA)) were synthesized by reversible addition-fragmentation chain-transfer polymerization using polyethylene glycol methyl ether methacrylate (PEGMA) and 1-undecen-10-al (UA) as monomers. The dopamine was conjugated onto poly(UA-co-PEGMA) through a multicomponent reaction between UA and dopamine to obtain poly(UA-co-PEGMA)-DA, which was further utilized for preparation of polymer-FONs through self-polymerization of dopamine and PEI. 1 H nuclear magnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy and fluorescence spectroscopy were employed to characterize the structure, morphology, compositions and optical properties of these polymer-FONs. Cell viability and cell uptake behavior results suggested that these polymer-FONs possess good biocompatibility and can be potentially utilized for biomedical applications. More importantly, the method can be also applied to fabricate many other multifunctional polymer-FONs with great potential for biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Performance evaluation of spot detection algorithms in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M


    Full Text Available triggered the development of a highly sophisticated imaging tool known as fluorescence microscopy. This is used to visualise and study intracellular processes. The use of fluorescence microscopy and a specific staining method make biological molecules... was first used in astronomical applications [2] to detect isotropic objects, and was then introduced to biological applications [3]. Olivio-Marin[3] approached the problem of feature extraction based on undecimated wavelet representation of the image...

  16. Fluorescence based molecular in vivo imaging

    International Nuclear Information System (INIS)

    Ebert, Bernd


    Molecular imaging represents a modern research area that allows the in vivo study of molecular biological process kinetics using appropriate probes and visualization methods. This methodology may be defined- apart from the contrast media injection - as non-abrasive. In order to reach an in vivo molecular process imaging as accurate as possible the effects of the used probes on the biological should not be too large. The contrast media as important part of the molecular imaging can significantly contribute to the understanding of molecular processes and to the development of tailored diagnostics and therapy. Since more than 15 years PTB is developing optic imaging systems that may be used for fluorescence based visualization of tissue phantoms, small animal models and the localization of tumors and their predecessors, and for the early recognition of inflammatory processes in clinical trials. Cellular changes occur during many diseases, thus the molecular imaging might be of importance for the early diagnosis of chronic inflammatory diseases. Fluorescent dyes can be used as unspecific or also as specific contrast media, which allow enhanced detection sensitivity

  17. Midrange affinity fluorescent Zn(II) sensors of the Zinpyr family: syntheses, characterization, and biological imaging applications. (United States)

    Nolan, Elizabeth M; Jaworski, Jacek; Racine, Maryann E; Sheng, Morgan; Lippard, Stephen J


    The syntheses and photophysical characterization of ZP9, 2-{2-chloro-6-hydroxy-3-oxo-5-[(2-{[pyridin-2-ylmethyl-(1H-pyrrol-2-ylmethyl)amino]methyl}phenylamino)methyl]-3H-xanthen-9-yl}benzoic acid, and ZP10, 2-{2-chloro-6-hydroxy-5-[(2-{[(1-methyl-1H-pyrrol-2-ylmethyl)pyridin-2-ylmethylamino]methyl}phenylamino)methyl]-3-oxo-3H-xanthen-9-yl}benzoic acid, two asymmetrically derivatized fluorescein-based dyes, are described. These sensors each contain an aniline-based ligand moiety functionalized with a pyridyl-amine-pyrrole group and have dissociation constants for Zn(II) in the sub-micromolar (ZP9) and low-micromolar (ZP10) range, which we define as "midrange". They give approximately 12- (ZP9) and approximately 7-fold (ZP10) fluorescence turn-on immediately following Zn(II) addition at neutral pH and exhibit improved selectivity for Zn(II) compared to the di-(2-picolyl)amine-based Zinpyr (ZP) sensors. Confocal microscopy studies indicate that such asymmetrical fluorescein-based probes are cell permeable and Zn(II) responsive in vivo.

  18. Scattered and Fluorescent Photon Track Reconstruction in a Biological Tissue

    Directory of Open Access Journals (Sweden)

    Maria N. Kholodtsova


    Full Text Available Appropriate analysis of biological tissue deep regions is important for tumor targeting. This paper is concentrated on photons’ paths analysis in such biotissue as brain, because optical probing depth of fluorescent and excitation radiation differs. A method for photon track reconstruction was developed. Images were captured focusing on the transparent wall close and parallel to the source fibres, placed in brain tissue phantoms. The images were processed to reconstruct the photons most probable paths between two fibres. Results were compared with Monte Carlo simulations and diffusion approximation of the radiative transfer equation. It was shown that the excitation radiation optical probing depth is twice more than for the fluorescent photons. The way of fluorescent radiation spreading was discussed. Because of fluorescent and excitation radiation spreads in different ways, and the effective anisotropy factor, geff, was proposed for fluorescent radiation. For the brain tissue phantoms it were found to be 0.62±0.05 and 0.66±0.05 for the irradiation wavelengths 532 nm and 632.8 nm, respectively. These calculations give more accurate information about the tumor location in biotissue. Reconstruction of photon paths allows fluorescent and excitation probing depths determination. The geff can be used as simplified parameter for calculations of fluorescence probing depth.

  19. Multispectral fluorescence imaging techniques for nondestructive food safety inspection (United States)

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


    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.

  20. Fluorescence imaging spectrometer optical design (United States)

    Taiti, A.; Coppo, P.; Battistelli, E.


    The optical design of the FLuORescence Imaging Spectrometer (FLORIS) studied for the Fluorescence Explorer (FLEX) mission is discussed. FLEX is a candidate for the ESA's 8th Earth Explorer opportunity mission. FLORIS is a pushbroom hyperspectral imager foreseen to be embarked on board of a medium size satellite, flying in tandem with Sentinel-3 in a Sun synchronous orbit at a height of about 815 km. FLORIS will observe the vegetation fluorescence and reflectance within a spectral range between 500 and 780 nm. Multi-frames acquisitions on matrix detectors during the satellite movement will allow the production of 2D Earth scene images in two different spectral channels, called HR and LR with spectral resolution of 0.3 and 2 nm respectively. A common fore optics is foreseen to enhance by design the spatial co-registration between the two spectral channels, which have the same ground spatial sampling (300 m) and swath (150 km). An overlapped spectral range between the two channels is also introduced to simplify the spectral coregistration. A compact opto-mechanical solution with all spherical and plane optical elements is proposed, and the most significant design rationales are described. The instrument optical architecture foresees a dual Babinet scrambler, a dioptric telescope and two grating spectrometers (HR and LR), each consisting of a modified Offner configuration. The developed design is robust, stable vs temperature, easy to align, showing very high optical quality along the whole field of view. The system gives also excellent correction for transverse chromatic aberration and distortions (keystone and smile).

  1. Fluorescence lifetime imaging of skin cancer (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


    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.

  2. Dissecting Redox Biology Using Fluorescent Protein Sensors. (United States)

    Schwarzländer, Markus; Dick, Tobias P; Meyer, Andreas J; Morgan, Bruce


    Fluorescent protein sensors have revitalized the field of redox biology by revolutionizing the study of redox processes in living cells and organisms. Within one decade, a set of fundamental new insights has been gained, driven by the rapid technical development of in vivo redox sensing. Redox-sensitive yellow and green fluorescent protein variants (rxYFP and roGFPs) have been the central players. Although widely used as an established standard tool, important questions remain surrounding their meaningful use in vivo. We review the growing range of thiol redox sensor variants and their application in different cells, tissues, and organisms. We highlight five key findings where in vivo sensing has been instrumental in changing our understanding of redox biology, critically assess the interpretation of in vivo redox data, and discuss technical and biological limitations of current redox sensors and sensing approaches. We explore how novel sensor variants may further add to the current momentum toward a novel mechanistic and integrated understanding of redox biology in vivo. Antioxid. Redox Signal. 24, 680-712.

  3. Multispectral open-air intraoperative fluorescence imaging. (United States)

    Behrooz, Ali; Waterman, Peter; Vasquez, Kristine O; Meganck, Jeff; Peterson, Jeffrey D; Faqir, Ilias; Kempner, Joshua


    Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

  4. Multispectral system for medical fluorescence imaging

    International Nuclear Information System (INIS)

    Andersson, P.S.; Montan, S.; Svanberg, S.


    The principles of a powerful multicolor imaging system for tissue fluorescence diagnostics are discussed. Four individually spectrally filtered images are formed on a matrix detector by means of a split-mirror arrangement. The four images are processed in a computer, pixel by pixel, by means of mathematical operations, leading to an optimized contrast image, which enhances a selected feature. The system is being developed primarily for medical fluorescence imaging, but has wide applications in fluorescence, reflectance, and transmission monitoring related to a wide range of industrial and environmental problems. The system operation is described for the case of linear imaging on a diode array detector. Laser-induced fluorescence is used for cancer tumor and arteriosclerotic plaque demarcation using the contrast enhancement capabilities of this imaging system. Further examples of applications include fluorescing minerals and flames

  5. APD detectors for biological fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Mazeres, S.; Borrel, V.; Magenc, C.; Courrech, J.L.; Bazer-Bachi, R.


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

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

    Directory of Open Access Journals (Sweden)

    M. Monti


    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.

  7. 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:


    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.

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

    Directory of Open Access Journals (Sweden)

    Enzo Lombi

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

  9. High speed fluorescence imaging with compressed ultrafast photography (United States)

    Thompson, J. V.; Mason, J. D.; Beier, H. T.; Bixler, J. N.


    Fluorescent lifetime imaging is an optical technique that facilitates imaging molecular interactions and cellular functions. Because the excited lifetime of a fluorophore is sensitive to its local microenvironment,1, 2 measurement of fluorescent lifetimes can be used to accurately detect regional changes in temperature, pH, and ion concentration. However, typical state of the art fluorescent lifetime methods are severely limited when it comes to acquisition time (on the order of seconds to minutes) and video rate imaging. Here we show that compressed ultrafast photography (CUP) can be used in conjunction with fluorescent lifetime imaging to overcome these acquisition rate limitations. Frame rates up to one hundred billion frames per second have been demonstrated with compressed ultrafast photography using a streak camera.3 These rates are achieved by encoding time in the spatial direction with a pseudo-random binary pattern. The time domain information is then reconstructed using a compressed sensing algorithm, resulting in a cube of data (x,y,t) for each readout image. Thus, application of compressed ultrafast photography will allow us to acquire an entire fluorescent lifetime image with a single laser pulse. Using a streak camera with a high-speed CMOS camera, acquisition rates of 100 frames per second can be achieved, which will significantly enhance our ability to quantitatively measure complex biological events with high spatial and temporal resolution. In particular, we will demonstrate the ability of this technique to do single-shot fluorescent lifetime imaging of cells and microspheres.

  10. Three Dimensional Fluorescence Microscopy Image Synthesis and Segmentation


    Fu, Chichen; Lee, Soonam; Ho, David Joon; Han, Shuo; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.


    Advances in fluorescence microscopy enable acquisition of 3D image volumes with better image quality and deeper penetration into tissue. Segmentation is a required step to characterize and analyze biological structures in the images and recent 3D segmentation using deep learning has achieved promising results. One issue is that deep learning techniques require a large set of groundtruth data which is impractical to annotate manually for large 3D microscopy volumes. This paper describes a 3D d...

  11. A study on a portable fluorescence imaging system (United States)

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


    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.

  12. Aorta Fluorescence Imaging by Using Confocal Microscopy


    Wang, Chun-Yang; Tsai, Jui-che; Chuang, Ching-Cheng; Hsieh, Yao-Sheng; Sun, Chia-Wei


    The activated leukocyte attacked the vascular endothelium and the associated increase in VEcadherin number was observed in experiments. The confocal microscopic system with a prism-based wavelength filter was used for multiwavelength fluorescence measurement. Multiwavelength fluorescence imaging based on the VEcadherin within the aorta segment of a rat was achieved. The confocal microscopic system capable of fluorescence detection of cardiovascular tissue is a useful tool for measuring the bi...

  13. Segmentation and classification of cell cycle phases in fluorescence imaging. (United States)

    Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan


    Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

  14. Fluorescence optical imaging in anticancer drug delivery. (United States)

    Etrych, Tomáš; Lucas, Henrike; Janoušková, Olga; Chytil, Petr; Mueller, Thomas; Mäder, Karsten


    In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted tissues or cells have been intensively studied. Understanding their pharmacokinetic properties is crucial for the successful transition of this research into clinical practice. Among others, fluorescence imaging has become one of the most commonly used imaging tools in pre-clinical research. The development of increasing numbers of suitable fluorescent dyes excitable in the visible to near-infrared wavelengths of the spectrum has significantly expanded the applicability of fluorescence imaging. This paper focuses on the potential applications and limitations of non-invasive imaging techniques in the field of drug delivery, especially in anticancer therapy. Fluorescent imaging at both the cellular and systemic levels is discussed in detail. Additionally, we explore the possibility for simultaneous treatment and imaging using theranostics and combinations of different imaging techniques, e.g., fluorescence imaging with computed tomography. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Jones, Carolyn; Suhling, Klaus


    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 μm which is consistent with a theoretical model based on the full width at half maximum of the emission spectrum proposed by Toptygin

  16. Fluorescence imaging of soybean flavonol isolines (United States)

    Kim, Moon S.; Lee, Edward H.; Mulchi, Charles L.; McMurtrey, James E., III; Chappelle, Emmett W.; Rowland, Randy A.


    Experiments were conducted to characterize the fluorescence emission of leaves from four soybean ('Harosoy') plants containing different concentrations of flavonols (kaempferol glycosides). The investigation utilized genetically mutated soybean flavonol isolines grown in a constant environment, thus limiting factors known to affect fluorescence emission characteristics other than different kaempferol glycosides concentrations. Flavonol isolines included OX922, OX941, OX942, OX944. The first two isolines contain kaempferol (K) glycosides; K3, K6, and K9, and the latter two did not have K3, K6, and K9. A fluorescence imaging system (FIS) was used to characterize steady state florescence images of the sample leaves measured at wavelengths centered at 450, 550, 680, and 740 nm with an excitation at 360 nm. Images taken with FIS greatly complement non-imaging fluorescence measurements by characterizing the spatial variation of fluorescence within leaves. We also acquired fluorescence emission spectra to characterize spectral features of the soybean flavonol isolines. The emission spectral shape of the fluorescence emission characteristics were not significantly different between the soybeans that contain kaempferol glycosides and the ones that do not contain kaempferol glycosides. Typical emission maxima of green vegetation in the blue, green, red, and far-red bands were noticed in all four soybean isolines. However, plants containing kaempferol glycosides, OX922 and OX941 had significantly lower intensities throughout the wavelength regions. These results imply that fluorescence emission intensities in the fluorescence emission bands studied are significantly affected by the presence and absence of kaempferol glycosides concentrations (UV radiation screening compounds). Pure kaempferol glycoside dissolved in solution show minimal fluorescence emission when excited with the absorption maximum radiation at 365 nm. However, a broad band emission can be seen in the green

  17. Laser-induced fluorescence imaging of bacteria (United States)

    Hilton, Peter J.


    This paper outlines a method for optically detecting bacteria on various backgrounds, such as meat, by imaging their laser induced auto-fluorescence response. This method can potentially operate in real-time, which is many times faster than current bacterial detection methods, which require culturing of bacterial samples. This paper describes the imaging technique employed whereby a laser spot is scanned across an object while capturing, filtering, and digitizing the returned light. Preliminary results of the bacterial auto-fluorescence are reported and plans for future research are discussed. The results to date are encouraging with six of the eight bacterial strains investigated exhibiting auto-fluorescence when excited at 488 nm. Discrimination of these bacterial strains against red meat is shown and techniques for reducing background fluorescence discussed.

  18. 3-D Image Analysis of Fluorescent Drug Binding

    Directory of Open Access Journals (Sweden)

    M. Raquel Miquel


    Full Text Available Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIPY FL-prazosin (QAPB was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or genetic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in the histogram. In the presence of 10 μM phenoxybenzamine (blocking agent, the QAPB (50 nM histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that of control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.

  19. Fluorescence Imaging of Fast Retrograde Axonal Transport in Living Animals

    Directory of Open Access Journals (Sweden)

    Dawid Schellingerhout


    Full Text Available Our purpose was to enable an in vivo imaging technology that can assess the anatomy and function of peripheral nerve tissue (neurography. To do this, we designed and tested a fluorescently labeled molecular probe based on the nontoxic C fragment of tetanus toxin (TTc. TTc was purified, labeled, and subjected to immunoassays and cell uptake assays. The compound was then injected into C57BL/6 mice (N = 60 for in vivo imaging and histologic studies. Image analysis and immunohistochemistry were performed. We found that TTc could be labeled with fluorescent moieties without loss of immunoreactivity or biologic potency in cell uptake assays. In vivo fluorescent imaging experiments demonstrated uptake and retrograde transport of the compound along the course of the sciatic nerve and in the spinal cord. Ex vivo imaging and immunohistochemical studies confirmed the presence of TTc in the sciatic nerve and spinal cord, whereas control animals injected with human serum albumin did not exhibit these features. We have demonstrated neurography with a fluorescently labeled molecular imaging contrast agent based on the TTc.

  20. Submicron, soft x-ray fluorescence imaging

    International Nuclear Information System (INIS)

    La Fontaine, B.; MacDowell, A.A.; Tan, Z.; White, D.L.; Taylor, G.N.; Wood, O.R. II; Bjorkholm, J.E.; Tennant, D.M.; Hulbert, S.L.


    Submicron fluorescence imaging of soft x-ray aerial images, using a high resolution fluorescent crystal is reported. Features as small as 0.1 μm were observed using a commercially available single-crystal phosphor, STI-F10G (Star Tech Instruments Inc. P. O. Box 2536, Danbury, CT 06813-2536), excited with 139 A light. Its quantum efficiency was estimated to be 5--10 times that of sodium salicylate and to be constant over a broad spectral range from 30 to 400 A. A comparison with a terbium-activated yttrium orthosilicate fluorescent crystal is also presented. Several applications, such as the characterization of the aerial images produced by deep ultraviolet or extreme ultraviolet lithographic exposure tools, are envisaged


    Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J


    A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

  2. Fluorescein Derivatives in Intravital Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Michael S. Roberts


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

  3. Fluorescence optical imaging in anticancer drug delivery

    Czech Academy of Sciences Publication Activity Database

    Etrych, Tomáš; Lucas, H.; Janoušková, Olga; Chytil, Petr; Mueller, T.; Mäder, K.


    Roč. 226, 28 March (2016), s. 168-181 ISSN 0168-3659 R&D Projects: GA ČR(CZ) GA15-02986S; GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 Keywords : fluorescence imaging * drug delivery * theranostics Subject RIV: CD - Macromolecular Chemistry Impact factor: 7.786, year: 2016

  4. Recent Progress in Synthesis and Functionalization of Multimodal Fluorescent-Magnetic Nanoparticles for Biological Applications

    Directory of Open Access Journals (Sweden)

    Raquel Serrano García


    Full Text Available There is a great interest in the development of new nanomaterials for multimodal imaging applications in biology and medicine. Multimodal fluorescent-magnetic based nanomaterials deserve particular attention as they can be used as diagnostic and drug delivery tools, which could facilitate the diagnosis and treatment of cancer and many other diseases. This review focuses on the recent developments of magnetic-fluorescent nanocomposites and their biomedical applications. The recent advances in synthetic strategies and approaches for the preparation of fluorescent-magnetic nanocomposites are presented. The main biomedical uses of multimodal fluorescent-magnetic nanomaterials, including biological imaging, cancer therapy and drug delivery, are discussed, and prospects of this field are outlined.

  5. Quantitative fluorescence microscopy and image deconvolution. (United States)

    Swedlow, Jason R


    Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used

  6. Thermally activated delayed fluorescence organic dots for two-photon fluorescence lifetime imaging (United States)

    He, Tingchao; Ren, Can; Li, Zhuohua; Xiao, Shuyu; Li, Junzi; Lin, Xiaodong; Ye, Chuanxiang; Zhang, Junmin; Guo, Lihong; Hu, Wenbo; Chen, Rui


    Autofluorescence is a major challenge in complex tissue imaging when molecules present in the biological tissue compete with the fluorophore. This issue may be resolved by designing organic molecules with long fluorescence lifetimes. The present work reports the two-photon absorption (TPA) properties of a thermally activated delayed fluorescence (TADF) molecule with carbazole as the electron donor and dicyanobenzene as the electron acceptor (i.e., 4CzIPN). The results indicate that 4CzIPN exhibits a moderate TPA cross-section (˜9 × 10-50 cm4 s photon-1), high fluorescence quantum yield, and a long fluorescence lifetime (˜1.47 μs). 4CzIPN was compactly encapsulated into an amphiphilic copolymer via nanoprecipitation to achieve water-soluble organic dots. Interestingly, 4CzIPN organic dots have been utilized in applications involving two-photon fluorescence lifetime imaging (FLIM). Our work aptly demonstrates that TADF molecules are promising candidates of nonlinear optical probes for developing next-generation multiphoton FLIM applications.

  7. 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.


    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

  8. Nonlinear excitation fluorescence microscopy: source considerations for biological applications (United States)

    Wokosin, David L.


    Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues. Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard two-photon dose is defined by 800nm, 10mW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm, 780nm, ~830nm, ~960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.

  9. Preparation and characterization of alginate based-fluorescent magnetic nanoparticles for fluorescence/magnetic resonance multimodal imaging applications (United States)

    Kwon, Yong-Su; Choi, Kee-Bong; Lim, Hyungjun; Lee, Sunghwi; Lee, Jae-Jong


    Simple and versatile methodologies have been reported that customize the surface of superparamagnetic iron oxide (SPIO) nanoparticles and impart additional fluorescence capabilities to these contrast agents. Herein, we present the rational design, synthesis, characterization, and biological applications of a new magnetic-based fluorescent probe. The dual modality imaging protocol was developed by labeling fluorophore with alginate natural polymers that have excellent biocompatibility and biodegradability, and using gelification method to form nanocomposites containing SPIO. The formation of alginate-based fluorescent magnetic (AFM) nanoparticles was observed in spherical and elliptical forms with a diameter of less than 500 nm by a transmission electron microscope (TEM). The fluorescent wavelength band in the range of 560 nm was also confirmed in the UV–visible spectrophotometer. In this study, we demonstrate that the multi-tasking design of AFM nanoparticles provides an ideal platform for building balanced dual-image probes of magnetic resonance imaging and optical imaging.

  10. Imaging efficacy of a targeted imaging agent for fluorescence endoscopy (United States)

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


    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.

  11. Open source tools for fluorescent imaging. (United States)

    Hamilton, Nicholas A


    As microscopy becomes increasingly automated and imaging expands in the spatial and time dimensions, quantitative analysis tools for fluorescent imaging are becoming critical to remove both bottlenecks in throughput as well as fully extract and exploit the information contained in the imaging. In recent years there has been a flurry of activity in the development of bio-image analysis tools and methods with the result that there are now many high-quality, well-documented, and well-supported open source bio-image analysis projects with large user bases that cover essentially every aspect from image capture to publication. These open source solutions are now providing a viable alternative to commercial solutions. More importantly, they are forming an interoperable and interconnected network of tools that allow data and analysis methods to be shared between many of the major projects. Just as researchers build on, transmit, and verify knowledge through publication, open source analysis methods and software are creating a foundation that can be built upon, transmitted, and verified. Here we describe many of the major projects, their capabilities, and features. We also give an overview of the current state of open source software for fluorescent microscopy analysis and the many reasons to use and develop open source methods. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Active mask segmentation of fluorescence microscope images. (United States)

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


    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.

  13. X-ray imaging with monochromatic synchrotron radiation. Fluorescent and phase-contrast method

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Tohoru; Itai, Yuji [Tsukuba Univ., Ibaraki (Japan). Inst. of Clinical Medicine


    To obtain the high sensitive x-ray images of biomedical object, new x-ray imaging techniques using fluorescent x-ray and phase-contrast x-ray are being developed in Japan. Fluorescent x-ray CT can detect very small amounts of specific elements in the order of ppm at one pixel, whereas phase-contrast x-ray imaging with interferometer can detect minute differences of biological object. Here, our recent experimental results are presented. (author)

  14. Active Mask Segmentation of Fluorescence Microscope Images


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


    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 mul...

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


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


    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...

  16. Biological oscillations: Fluorescence monitoring by confocal microscopy (United States)

    Chattoraj, Shyamtanu; Bhattacharyya, Kankan


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

  17. Application of Quantum Dots in Biological Imaging

    Directory of Open Access Journals (Sweden)

    Shan Jin


    Full Text Available Quantum dots (QDs are a group of semiconducting nanomaterials with unique optical and electronic properties. They have distinct advantages over traditional fluorescent organic dyes in chemical and biological studies in terms of tunable emission spectra, signal brightness, photostability, and so forth. Currently, the major type of QDs is the heavy metal-containing II-IV, IV-VI, or III-V QDs. Silicon QDs and conjugated polymer dots have also been developed in order to lower the potential toxicity of the fluorescent probes for biological applications. Aqueous solubility is the common problem for all types of QDs when they are employed in the biological researches, such as in vitro and in vivo imaging. To circumvent this problem, ligand exchange and polymer coating are proven to be effective, besides synthesizing QDs in aqueous solutions directly. However, toxicity is another big concern especially for in vivo studies. Ligand protection and core/shell structure can partly solve this problem. With the rapid development of QDs research, new elements and new morphologies have been introduced to this area to fabricate more safe and efficient QDs for biological applications.

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

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


    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 (GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

  19. An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

    International Nuclear Information System (INIS)

    Dunsby, C; Lanigan, P M P; McGinty, J; Elson, D S; Requejo-Isidro, J; Munro, I; Galletly, N; McCann, F; Treanor, B; Oenfelt, B; Davis, D M; Neil, M A A; French, P M W


    Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems

  20. Fluorescence-enhanced gadolinium-doped zinc oxide quantum dots for magnetic resonance and fluorescence imaging. (United States)

    Liu, Yanlan; Ai, Kelong; Yuan, Qinghai; Lu, Lehui


    We report here the development of Gd-doped ZnO quantum dots (QDs) as dual modal fluorescence and magnetic resonance imaging nanoprobes. They are fabricated in a simple, versatile and environmentally friendly method, not only decreasing the difficulty and complexity, but also avoiding the increase of particle's size brought about by silica coating procedure in the synthesis of nanoprobes reported previously. These nanoprobes, with exceptionally small size and enhanced fluorescence resulting from the Gd doping, can label successfully the HeLa cells in short time and present no evidence of toxicity or adverse affect on cell growth even at the concentration up to 1 mm. These results show that such nanoprobes have low toxicity, especially in comparison with the traditional PEGylated CdSe/ZnS or CdSe/CdS QDs. In MRI studies, they exert strong positive contrast effect with a large longitudinal relaxivity (r(1)) of water proton of 16 mm(-1) s(-1). Their capability of imaging HeLa cells with MRI implies that they have great potential as MRI contrast agents. Combining the high sensitivity of fluorescence imaging with high spatial resolution of MRI, We expect that the as-prepared Gd-doped Zno QDs can provide a better reliability of the collected data and find promising applications in biological, medical and other fields. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics. (United States)

    Hayashi, Shinichi; Okada, Yasushi


    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 (

  2. Selective Detection of Neurotransmitters by Fluorescence and Chemiluminescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ziqiang Wang; Edward S. Yeung


    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.

  3. Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging. (United States)

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


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

  4. Fluorescence imaging to quantify crop residue cover (United States)

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


    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.

  5. Image processing and recognition for biological images. (United States)

    Uchida, Seiichi


    This paper reviews image processing and pattern recognition techniques, which will be useful to analyze bioimages. Although this paper does not provide their technical details, it will be possible to grasp their main tasks and typical tools to handle the tasks. Image processing is a large research area to improve the visibility of an input image and acquire some valuable information from it. As the main tasks of image processing, this paper introduces gray-level transformation, binarization, image filtering, image segmentation, visual object tracking, optical flow and image registration. Image pattern recognition is the technique to classify an input image into one of the predefined classes and also has a large research area. This paper overviews its two main modules, that is, feature extraction module and classification module. Throughout the paper, it will be emphasized that bioimage is a very difficult target for even state-of-the-art image processing and pattern recognition techniques due to noises, deformations, etc. This paper is expected to be one tutorial guide to bridge biology and image processing researchers for their further collaboration to tackle such a difficult target. © 2013 The Author Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  6. Fluorescence Imaging/Agents in Tumor Resection. (United States)

    Stummer, Walter; Suero Molina, Eric


    Intraoperative fluorescence imaging allows real-time identification of diseased tissue during surgery without being influenced by brain shift and surgery interruption. 5-Aminolevulinic acid, useful for malignant gliomas and other tumors, is the most broadly explored compound approved for fluorescence-guided resection. Intravenous fluorescein sodium has recently received attention, highlighting tumor tissue based on extravasation at the blood-brain barrier (defective in many brain tumors). Fluorescein in perfused brain, unselective extravasation in brain perturbed by surgery, and propagation with edema are concerns. Fluorescein is not approved but targeted fluorochromes with affinity to brain tumor cells, in development, may offer future advantages. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Multi-spectral endogenous fluorescence imaging for bacterial differentiation (United States)

    Chernomyrdin, Nikita V.; Babayants, Margarita V.; Korotkov, Oleg V.; Kudrin, Konstantin G.; Rimskaya, Elena N.; Shikunova, Irina A.; Kurlov, Vladimir N.; Cherkasova, Olga P.; Komandin, Gennady A.; Reshetov, Igor V.; Zaytsev, Kirill I.


    In this paper, the multi-spectral endogenous fluorescence imaging was implemented for bacterial differentiation. The fluorescence imaging was performed using a digital camera equipped with a set of visual bandpass filters. Narrowband 365 nm ultraviolet radiation passed through a beam homogenizer was used to excite the sample fluorescence. In order to increase a signal-to-noise ratio and suppress a non-fluorescence background in images, the intensity of the UV excitation was modulated using a mechanical chopper. The principal components were introduced for differentiating the samples of bacteria based on the multi-spectral endogenous fluorescence images.

  8. [Development of a Fluorescence Probe for Live Cell Imaging]. (United States)

    Shibata, Aya


     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.

  9. Creating Panoramic Images for Bladder Fluorescence Endoscopy

    Directory of Open Access Journals (Sweden)

    A. Behrens


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

  10. Porphyrin-magnetite nanoconjugates for biological imaging

    LENUS (Irish Health Repository)

    Nowostawska, Malgorzata


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

  11. Smartphone microendoscopy for high resolution fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Xiangqian Hong


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

  12. Sensitive and selective tumor imaging with novel and highly activatable fluorescence probes

    International Nuclear Information System (INIS)

    Urano, Yasuteru


    Selective and sensitive tumor imaging in vivo is one of the most requested methodologies in medical sciences. Although several imaging modalities have been developed including positron emission tomography (PET) and magnetic resonance (MR) imaging for the detection of tumors, none of these modalities can activate the signals upon being accumulated or uptaken to tumor sites. Among these modalities, only optical fluorescence imaging has a marked advantage, that is, their signals can be dramatically increased upon detecting some biological features. In this short review, I will introduce some recent strategies for activatable optical fluorescence imaging of tumors, and discuss their advantages over other modalities. (author)

  13. Single Molecule Fluorescence: from Physical Fascination to Biological Relevance

    NARCIS (Netherlands)

    Segers-Nolten, Gezina M.J.


    Confocal fluorescence microscopy is particularly well-known from the beautiful images that have been obtained with this technique from cells. Several cellular components could be nicely visualized simultaneously by staining them with different fluorophores. Not only for ensemble applications but

  14. Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning. (United States)

    Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki


    We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

  15. Dynamic fluorescence imaging with molecular agents for cancer detection (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

  16. Waveguide evanescent field fluorescence microscopy & its application in cell biology (United States)

    Hassanzadeh, Abdollah

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

  17. Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: a performance overview. (United States)

    Deliolanis, Nikolaos C; Ale, Angelique; Morscher, Stefan; Burton, Neal C; Schaefer, Karin; Radrich, Karin; Razansky, Daniel; Ntziachristos, Vasilis


    A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents.

  18. Macro-/micro-environment-sensitive chemosensing and biological imaging. (United States)

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


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

  19. Multimodal quantitative phase and fluorescence imaging of cell apoptosis (United States)

    Fu, Xinye; Zuo, Chao; Yan, Hao


    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.

  20. Fluorescence image excited by a scanning UV-LED light (United States)

    Tsai, Hsin-Yi; Chen, Yi-Ju; Huang, Kuo-Cheng


    An optical scanning system using UV-LED light to induced fluorescence technology can enhance a fluorescence image significantly in a short period. It has several advantages such as lower power consumption, no scattering effect in skins, and multilayer images can be obtained to analyze skin disease. From the experiment results, the light intensity increases with increase spot size and decrease scanning speed, but the image resolution is oppositely. Moreover, the system could be widely used in clinical diagnosis and photodynamic therapy for skin disease because even the irradiated time of fluorescence substance is short but it will provide accurately positioning of fluorescence object.

  1. Characterization of a new series of fluorescent probes for imaging membrane order.

    Directory of Open Access Journals (Sweden)

    Joanna M Kwiatek

    Full Text Available Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.

  2. Video-rate optical flow corrected intraoperative functional fluorescence imaging

    NARCIS (Netherlands)

    Koch, Maximilian; Glatz, Juergen; Ermolayev, Vladimir; de Vries, Elisabeth G. E.; van Dam, Gooitzen M.; Englmeier, Karl-Hans; Ntziachristos, Vasilis

    Intraoperative fluorescence molecular imaging based on targeted fluorescence agents is an emerging approach to improve surgical and endoscopic imaging and guidance. Short exposure times per frame and implementation at video rates are necessary to provide continuous feedback to the physician and

  3. 5-ALA induced fluorescent image analysis of actinic keratosis (United States)

    Cho, Yong-Jin; Bae, Youngwoo; Choi, Eung-Ho; Jung, Byungjo


    In this study, we quantitatively analyzed 5-ALA induced fluorescent images of actinic keratosis using digital fluorescent color and hyperspectral imaging modalities. UV-A was utilized to induce fluorescent images and actinic keratosis (AK) lesions were demarcated from surrounding the normal region with different methods. Eight subjects with AK lesion were participated in this study. In the hyperspectral imaging modality, spectral analysis method was utilized for hyperspectral cube image and AK lesions were demarcated from the normal region. Before image acquisition, we designated biopsy position for histopathology of AK lesion and surrounding normal region. Erythema index (E.I.) values on both regions were calculated from the spectral cube data. Image analysis of subjects resulted in two different groups: the first group with the higher fluorescence signal and E.I. on AK lesion than the normal region; the second group with lower fluorescence signal and without big difference in E.I. between two regions. In fluorescent color image analysis of facial AK, E.I. images were calculated on both normal and AK lesions and compared with the results of hyperspectral imaging modality. The results might indicate that the different intensity of fluorescence and E.I. among the subjects with AK might be interpreted as different phases of morphological and metabolic changes of AK lesions.

  4. Fluorescence Imaging Study of Impinging Underexpanded Jets (United States)

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


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

  5. Semiconductor Nanocrystals for Biological Imaging

    Energy Technology Data Exchange (ETDEWEB)

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


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

  6. Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system. (United States)

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


    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 (

  7. Effects of Depilation-Induced Skin Pigmentation and Diet-Induced Fluorescence on In Vivo Fluorescence Imaging


    Kwon, Sunkuk; Sevick-Muraca, Eva M.


    Near-infrared fluorescence imaging (NIRFI) and far-red fluorescence imaging (FRFI) were used to investigate effects of depilation-induced skin pigmentation and diet-induced background fluorescence on fluorescent signal amplitude and lymphatic contraction frequency in C57BL6 mice. Far-red fluorescent signal amplitude, but not frequency, was affected by diet-induced fluorescence, which was removed by feeding the mice an alfalfa-free diet, and skin pigmentation further impacted the amplitude mea...

  8. Hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic imaging (United States)

    Chen, Zhenyue; Deán-Ben, Xosé Luís.; Gottschalk, Sven; Razansky, Daniel


    Fluorescence imaging is widely employed in all fields of cell and molecular biology due to its high sensitivity, high contrast and ease of implementation. However, the low spatial resolution and lack of depth information, especially in strongly-scattering samples, restrict its applicability for deep-tissue imaging applications. On the other hand, optoacoustic imaging is known to deliver a unique set of capabilities such as high spatial and temporal resolution in three dimensions, deep penetration and spectrally-enriched imaging contrast. Since fluorescent substances can generate contrast in both modalities, simultaneous fluorescence and optoacoustic readings can provide new capabilities for functional and molecular imaging of living organisms. Optoacoustic images can further serve as valuable anatomical references based on endogenous hemoglobin contrast. Herein, we propose a hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic tomography, both operating in reflection mode, which synergistically combines the advantages of stand-alone systems. Validation of the spatial resolution and sensitivity of the system were first carried out in tissue mimicking phantoms while in vivo imaging was further demonstrated by tracking perfusion of an optical contrast agent in a mouse brain in the hybrid imaging mode. Experimental results show that the proposed system effectively exploits the contrast mechanisms of both imaging modalities, making it especially useful for accurate monitoring of fluorescence-based signal dynamics in highly scattering samples.

  9. Dual PET and Near-Infrared Fluorescence Imaging Probes as Tools for Imaging in Oncology (United States)

    An, Fei-Fei; Chan, Mark; Kommidi, Harikrishna; Ting, Richard


    OBJECTIVE The purpose of this article is to summarize advances in PET fluorescence resolution, agent design, and preclinical imaging that make a growing case for clinical PET fluorescence imaging. CONCLUSION Existing SPECT, PET, fluorescence, and MRI contrast imaging techniques are already deeply integrated into the management of cancer, from initial diagnosis to the observation and management of metastases. Combined positron-emitting fluorescent contrast agents can convey new or substantial benefits that improve on these proven clinical contrast agents. PMID:27223168

  10. Development of ultrasound-assisted fluorescence imaging of indocyanine green. (United States)

    Morikawa, Hiroyasu; Toyota, Shin; Wada, Kenji; Uchida-Kobayashi, Sawako; Kawada, Norifumi; Horinaka, Hiromichi


    Indocyanine green (ICG) accumulation in hepatocellular carcinoma means tumors can be located by fluorescence. However, because of light scattering, it is difficult to detect ICG fluorescence from outside the body. We propose a new fluorescence imaging method that detects changes in the intensity of ICG fluorescence by ultrasound-induced temperature changes. ICG fluorescence intensity decreases as the temperature rises. Therefore, it should theoretically be possible to detect tissue distribution of ICG using ultrasound to heat tissue, moving the point of ultrasound transmission, and monitoring changes in fluorescence intensity. A new probe was adapted for clinical application. It consisted of excitation light from a laser, fluorescence sensing through a light pipe, and heating by ultrasound. We applied the probe to bovine liver to image the accumulation of ICG. ICG emits fluorescence (820 nm) upon light irradiation (783 nm). With a rise in temperature, the fluorescence intensity of ICG decreased by 0.85 %/°C. The distribution of fluorescent ICG was detected using an ultrasonic warming method in a new integrated probe. Modulating fluorescence by changing the temperature using ultrasound can determine where ICG accumulates at a depth, highlighting its potential as a means to locate hepatocellular carcinoma.

  11. Fluorescent Proteins for Investigating Biological Events in Acidic Environments

    Directory of Open Access Journals (Sweden)

    Hajime Shinoda


    Full Text Available The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles is an important platform for modification, transport and degradation of biomolecules as well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs. Due to the highly acidic luminal environment (pH ~ 4.5–6.0, most FPs and related sensors are apt to lose their fluorescence. To address the need to image in acidic environments, several research groups have developed acid-tolerant FPs in a wide color range. Furthermore, the engineering of pH insensitive sensors, and their concomitant use with pH sensitive sensors for the purpose of pH-calibration has enabled characterization of the role of luminal ions. In this short review, we summarize the recent development of acid-tolerant FPs and related functional sensors and discuss the future prospects for this field.

  12. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees


    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH

  13. Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis. (United States)

    Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith


    To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

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

  15. Quantification of tumor fluorescence during intraoperative optical cancer imaging. (United States)

    Judy, Ryan P; Keating, Jane J; DeJesus, Elizabeth M; Jiang, Jack X; Okusanya, Olugbenga T; Nie, Shuming; Holt, David E; Arlauckas, Sean P; Low, Phillip S; Delikatny, E James; Singhal, Sunil


    Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth.

  16. Fluorescent imaging of cancerous tissues for targeted surgery (United States)

    Bu, Lihong; Shen, Baozhong; Cheng, Zhen


    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

  17. Detection of rheumatoid arthritis in humans by fluorescence imaging (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


    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.

  18. Screening of biologically important Zn2 + by a chemosensor with fluorescent turn on-off mechanism (United States)

    Khan, Tanveer A.; Sheoran, Monika; Nikhil Raj M., Venkata; Jain, Surbhi; Gupta, Diksha; Naik, Sunil G.


    Reported herein the synthesis, characterization and biologically important zinc ion binding propensity of a weakly fluorescent chemosensor, 4-methyl-2,6-bis((E)-(2-(4-phenylthiazol-2-yl)hydrazono)methyl)phenol (1). 1H NMR spectroscopic titration experiment reveals the binding knack of 1 to the essential Zn2 +. The photo-physical studies of 1 exhibit an enhancement in the fluorescence by several folds upon binding with the zinc ions attributed to PET-off process, with a binding constant value of 5.22 × 103 M- 1. 1 exhibits an excellent detection range for Zn2 + with lower detection limit value of 2.31 × 10- 8 M. The selectivity of 1 was studied with various mono and divalent metal cations and it was observed that most cations either quenches the fluorescence or remains unchanged except for Cd2 +, which shows a slight enhancement in fluorescence intensity of 1. The ratiometric displacement of Cd2 + ions by Zn2 + ions shows an excellent selectivity towards in-situ detection of Zn2 + ions. Photo-physical studies also support the reversible binding of 1 to Zn2 + ions having on and off mechanism in presence of EDTA. Such recognition of the biologically important zinc ions finds potential application in live cell imaging.

  19. Recent development of fluorescent imaging for specific detection of tumors

    International Nuclear Information System (INIS)

    Nakata, Eiji; Morii, Takashi; Uto, Yoshihiro; Hori, Hitoshi


    Increasing recent studies on fluorescent imaging for specific detection of tumors are described here on strategies of molecular targeting, metabolic specificity and hypoxic circumstance. There is described an instance of a conjugate of antibody and pH-activable fluorescent ligand, which specifically binds to the tumor cells, is internalized in the cellular lysozomes where their pH is low, and then is activated to become fluorescent only in viable tumor cells. For the case of metabolic specificity, excessive loading of the precursor (5-aminolevulinic acid) of protoporphyrin IX (ppIX), due to their low activity to convert ppIX to heme B, results in making tumors observable in red as ppIX emits fluorescence (red, 585 nm) when excited by blue ray of 410 nm. Similarly, imaging with indocyanine green which is accumulated in hepatoma cells is reported in success in detection of small lesion and metastasis when the dye is administered during operation. Reductive reactions exceed in tumor hypoxic conditions, of which feature is usable for imaging. Conjugates of nitroimidazole and fluorescent dye are reported to successfully image tumors by nitro reduction. Authors' UTX-12 is a non-fluorescent nitroaromatic derivative of pH-sensitive fluorescent dye seminaphtharhodafluor (SNARF), and is designed for the nitro group, the hypoxia-responding sensor, to be reduced in tumor hypoxic conditions and then for the aromatic moiety to be cleaved to release free SNARF. Use of hypoxia-inducible factor-1 (HIF-1) for imaging has been also reported in many. As above, studies on fluorescent imaging for specific detection of tumors are mostly at fundamental step but its future is conceivably promising along with advances in other technology like fluorescent endoscopy and multimodal imaging. (author)

  20. Phenotyping of Arabidopsis Drought Stress Response Using Kinetic Chlorophyll Fluorescence and Multicolor Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Jieni Yao


    Full Text Available Plant responses to drought stress are complex due to various mechanisms of drought avoidance and tolerance to maintain growth. Traditional plant phenotyping methods are labor-intensive, time-consuming, and subjective. Plant phenotyping by integrating kinetic chlorophyll fluorescence with multicolor fluorescence imaging can acquire plant morphological, physiological, and pathological traits related to photosynthesis as well as its secondary metabolites, which will provide a new means to promote the progress of breeding for drought tolerant accessions and gain economic benefit for global agriculture production. Combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging proved to be efficient for the early detection of drought stress responses in the Arabidopsis ecotype Col-0 and one of its most affected mutants called reduced hyperosmolality-induced [Ca2+]i increase 1. Kinetic chlorophyll fluorescence curves were useful for understanding the drought tolerance mechanism of Arabidopsis. Conventional fluorescence parameters provided qualitative information related to drought stress responses in different genotypes, and the corresponding images showed spatial heterogeneities of drought stress responses within the leaf and the canopy levels. Fluorescence parameters selected by sequential forward selection presented high correlations with physiological traits but not morphological traits. The optimal fluorescence traits combined with the support vector machine resulted in good classification accuracies of 93.3 and 99.1% for classifying the control plants from the drought-stressed ones with 3 and 7 days treatments, respectively. The results demonstrated that the combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging with the machine learning technique was capable of providing comprehensive information of drought stress effects on the photosynthesis and the secondary metabolisms. It is a promising

  1. FY08 Annual Report for Nuclear Resonance Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Glen A.; Caggiano, Joseph A.


    FY08 annual report for project the "Nuclear Resonance Fluorescence Imaging" project. Reviews accomplishments of last 3 years, including U-235 signature search, comparison of different photon sources, and examination of NRF measurements using monochromatic photon source.

  2. BlobFinder, a tool for fluorescence microscopy image cytometry


    Allalou, Amin; Wählby, Carolina


    Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application ...

  3. Patch-based anisotropic diffusion scheme for fluorescence diffuse optical tomography--part 2: image reconstruction. (United States)

    Correia, Teresa; Koch, Maximilian; Ale, Angelique; Ntziachristos, Vasilis; Arridge, Simon


    Fluorescence diffuse optical tomography (fDOT) provides 3D images of fluorescence distributions in biological tissue, which represent molecular and cellular processes. The image reconstruction problem is highly ill-posed and requires regularisation techniques to stabilise and find meaningful solutions. Quadratic regularisation tends to either oversmooth or generate very noisy reconstructions, depending on the regularisation strength. Edge preserving methods, such as anisotropic diffusion regularisation (AD), can preserve important features in the fluorescence image and smooth out noise. However, AD has limited ability to distinguish an edge from noise. We propose a patch-based anisotropic diffusion regularisation (PAD), where regularisation strength is determined by a weighted average according to the similarity between patches around voxels within a search window, instead of a simple local neighbourhood strategy. However, this method has higher computational complexity and, hence, we wavelet compress the patches (PAD-WT) to speed it up, while simultaneously taking advantage of the denoising properties of wavelet thresholding. Furthermore, structural information can be incorporated into the image reconstruction with PAD-WT to improve image quality and resolution. In this case, the weights used to average voxels in the image are calculated using the structural image, instead of the fluorescence image. The regularisation strength depends on both structural and fluorescence images, which guarantees that the method can preserve fluorescence information even when it is not structurally visible in the anatomical images. In part 1, we tested the method using a denoising problem. Here, we use simulated and in vivo mouse fDOT data to assess the algorithm performance. Our results show that the proposed PAD-WT method provides high quality and noise free images, superior to those obtained using AD.

  4. Classifying apples by the means of fluorescence imaging


    Codrea, Marius C.; Nevalainen, Olli S.; Tyystjärvi, Esa; VAN DE VEN, Martin; VALCKE, Roland


    Classification of harvested apples when predicting their storage potential is an important task. This paper describes how chlorophyll a fluorescence images taken in blue light through a red filter, can be used to classify apples. In such an image, fluorescence appears as a relatively homogenous area broken by a number of small nonfluorescing spots, corresponding to normal corky tissue patches, lenticells, and to damaged areas that lower the quality of the apple. The damaged regions appear mor...

  5. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel


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

  6. Integrated ultrasonic particle positioning and low excitation light fluorescence imaging

    International Nuclear Information System (INIS)

    Bernassau, A. L.; Al-Rawhani, M.; Beeley, J.; Cumming, D. R. S.


    A compact hybrid system has been developed to position and detect fluorescent micro-particles by combining a Single Photon Avalanche Diode (SPAD) imager with an acoustic manipulator. The detector comprises a SPAD array, light-emitting diode (LED), lenses, and optical filters. The acoustic device is formed of multiple transducers surrounding an octagonal cavity. By stimulating pairs of transducers simultaneously, an acoustic landscape is created causing fluorescent micro-particles to agglomerate into lines. The fluorescent pattern is excited by a low power LED and detected by the SPAD imager. Our technique combines particle manipulation and visualization in a compact, low power, portable setup

  7. Chlorophyll Fluorescence Imaging Uncovers Photosynthetic Fingerprint of Citrus Huanglongbing

    Directory of Open Access Journals (Sweden)

    Haiyan Cen


    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.

  8. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone. (United States)

    Zhu, Hongying; Ozcan, Aydogan


    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.

  9. Calibration Curves for Biological Dosimetry by Fluorescence In situ Hybridisation

    International Nuclear Information System (INIS)

    Stonati, L.; Durante, M.; Gensabella, G.; Gialanella, G.; Grossi, G.F.; Pugliese, M.; Scampoli, P.; Sgura, A.; Testa, A.; Tanzarella, C.


    Dose-response curves were measured for the induction of chromosomal aberrations in peripheral blood lymphocytes after acute exposure in vitro to 60 Co γ rays. Blood was obtained from four different healthy donors, and chromosomes were either observed at metaphase, following colcemid accumulation, or prematurely condensed by calyculin A. Cells were analysed in three different Italian laboratories. Chromosomes 1, 2, and 4 were painted, and simple-type interchanges between painted and non-painted chromosomes were scored in cells exposed in the dose range 0.1-3.0 Gy. The chemical-induced premature chromosome condensation method was also used combined with chromosome painting (chromosome 4 only) to determine calibration curves for high dose exposures (up to 20 Gy X rays). Calibration curves described in this paper will be used in our laboratories for biological dosimetry by fluorescence in situ hybridisation. (author)

  10. Applications of two-photon fluorescence microscopy in deep-tissue imaging (United States)

    Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.


    Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

  11. Photobleaching correction in fluorescence microscopy images

    International Nuclear Information System (INIS)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H


    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

  12. Fluorescent porous silicon biological probes with high quantum efficiency and stability. (United States)

    Tu, Chang-Ching; Chou, Ying-Nien; Hung, Hsiang-Chieh; Wu, Jingda; Jiang, Shaoyi; Lin, Lih Y


    We demonstrate porous silicon biological probes as a stable and non-toxic alternative to organic dyes or cadmium-containing quantum dots for imaging and sensing applications. The fluorescent silicon quantum dots which are embedded on the porous silicon surface are passivated with carboxyl-terminated ligands through stable Si-C covalent bonds. The porous silicon bio-probes have shown photoluminescence quantum yield around 50% under near-UV excitation, with high photochemical and thermal stability. The bio-probes can be efficiently conjugated with antibodies, which is confirmed by a standard enzyme-linked immunosorbent assay (ELISA) method.

  13. X-ray fluorescence imaging with synchrotron radiation

    International Nuclear Information System (INIS)

    Rivers, M.L.


    The micro-distribution of trace elements is of great interest in fields such as geochemistry, biology and material science. The synchrotron x-ray fluorescence microprobe provides a technique to quantitatively measure trace element compositions at individual points and to construct semiquantitative two dimensional maps of trace element compositions. This paper describes an x-ray fluorescence system used at the National Synchrotron Light Source

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

    International Nuclear Information System (INIS)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

    Sakhalkar, H S [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, M [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Oliver, T [Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 (United States); Cao, Y [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Oldham, M [Department of Radiation Oncology Physics, and Biomedical Engineering, Duke University Medical Center, Durham, NC 27710 (United States)


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

  16. Fluorescence lifetime imaging of oxygen in dental biofilm (United States)

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


    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.

  17. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging. (United States)

    Nooshabadi, Fatemeh; Yang, Hee-Jeong; Bixler, Joel N; Kong, Ying; Cirillo, Jeffrey D; Maitland, Kristen C


    Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.

  18. Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging. (United States)

    Xiong, Xiaoqing; Song, Fengling; Wang, Jingyun; Zhang, Yukang; Xue, Yingying; Sun, Liangliang; Jiang, Na; Gao, Pan; Tian, Lu; Peng, Xiaojun


    Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

  19. Small-Animal Imaging Using Diffuse Fluorescence Tomography. (United States)

    Davis, Scott C; Tichauer, Kenneth M


    Diffuse fluorescence tomography (DFT) has been developed to image the spatial distribution of fluorescence-tagged tracers in living tissue. This capability facilitates the recovery of any number of functional parameters, including enzymatic activity, receptor density, blood flow, and gene expression. However, deploying DFT effectively is complex and often requires years of know-how, especially for newer mutlimodal systems that combine DFT with conventional imaging systems. In this chapter, we step through the process of using MRI-DFT imaging of a receptor-targeted tracer in small animals.

  20. 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


    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.

  1. Problems of fluorescent imaging and its solution using nanofluorophores. Part I: Advantages of fluorescent nanoparticles over conventional organic fluorophores

    International Nuclear Information System (INIS)

    Zhelev, Z.; Hadjidekov, G.; Zlateva, G.; Spasov, L.; Bakalova, R.


    The application of fluorescence in deep-tissue imaging is rapidly expanding in fast several years. The progress in fluorescent molecular probes and fluorescent imaging techniques gives an opportunity to detect single cells and even molecules in live organisms. The highly sensitive and high-speed fluorescent molecular sensors and detection devices allow the application of fluorescence in functional imaging. With development of novel bright fluorophores based on nano-technologies and fluorescence scanners with high spatial and temporal resolution, the fluorescent imaging has a potential to become an alternative of the other non-invasive imaging techniques as magnetic resonance imaging, positron-emission tomography, X-ray, computing tomography. This review outlines the current status and future trends of fluorescent nanoparticles - quantum dots (QDs), as a new generation of fluorophores in experimental and pre-clinical fluorescent imaging diagnostic. Part 1 focuses on the advantages of quantum dots over conventional organic fluorophores and defines the major requirements to the 'perfect' fluorophore for fluorescent deep-tissue imaging diagnostic. The analysis is based on the limitations of fluorescent imaging in vivo and overcome by using quantum dots

  2. Particle Image Velocimetry Applications of Fluorescent Dye-Doped Particles


    Petrosky, Brian Joseph


    Laser flare can often be a major issue in particle image velocimetry (PIV) involving solid boundaries in a flow or a gas-liquid interface. The use of fluorescent light from dye-doped particles has been demonstrated in water applications, but reproducing the technique in an airflow is more difficult due to particle size constraints and safety concerns. The following thesis is formatted in a hybrid manuscript style, including a full paper presenting the applications of fluorescent Kiton R...

  3. Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence (United States)

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


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

  4. Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence. (United States)

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


    Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp(2)-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.

  5. A portable fluorescence microscopic imaging system for cholecystectomy (United States)

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


    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.

  6. Enhanced speed in fluorescence imaging using beat frequency multiplexing (United States)

    Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke


    Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

  7. In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment. (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir


    Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. ©2014 American Association for Cancer Research.

  8. In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir


    Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

  9. Community detection for fluorescent lifetime microscopy image segmentation (United States)

    Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Achilefu, Samuel; Nussinov, Zohar


    Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.


    Directory of Open Access Journals (Sweden)

    L. Guidi


    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.

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

    NARCIS (Netherlands)

    Jalink, H.; Schoor, van der R.


    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.

  12. Snapshot imaging Fraunhofer line discriminator for detection of plant fluorescence (United States)

    Gupta Roy, S.; Kudenov, M. W.


    Non-invasive quantification of plant health is traditionally accomplished using reflectance based metrics, such as the normalized difference vegetative index (NDVI). However, measuring plant fluorescence (both active and passive) to determine photochemistry of plants has gained importance. Due to better cost efficiency, lower power requirements, and simpler scanning synchronization, detecting passive fluorescence is preferred over active fluorescence. In this paper, we propose a high speed imaging approach for measuring passive plant fluorescence, within the hydrogen alpha Fraunhofer line at ~656 nm, using a Snapshot Imaging Fraunhofer Line Discriminator (SIFOLD). For the first time, the advantage of snapshot imaging for high throughput Fraunhofer Line Discrimination (FLD) is cultivated by our system, which is based on a multiple-image Fourier transform spectrometer and a spatial heterodyne interferometer (SHI). The SHI is a Sagnac interferometer, which is dispersion compensated using blazed diffraction gratings. We present data and techniques for calibrating the SIFOLD to any particular wavelength. This technique can be applied to quantify plant fluorescence at low cost and reduced complexity of data collection.

  13. Mitigating fluorescence spectral overlap in wide-field endoscopic imaging (United States)

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


    Abstract. The number of molecular species suitable for multispectral fluorescence imaging is limited due to the overlap of the emission spectra of indicator fluorophores, e.g., dyes and nanoparticles. To remove fluorophore emission cross-talk in wide-field multispectral fluorescence molecular imaging, we evaluate three different solutions: (1) image stitching, (2) concurrent imaging with cross-talk ratio subtraction algorithm, and (3) frame-sequential imaging. A phantom with fluorophore emission cross-talk is fabricated, and a 1.2-mm ultrathin scanning fiber endoscope (SFE) is used to test and compare these approaches. Results show that fluorophore emission cross-talk could be successfully avoided or significantly reduced. Near term, the concurrent imaging method of wide-field multispectral fluorescence SFE is viable for early stage cancer detection and localization in vivo. Furthermore, a means to enhance exogenous fluorescence target-to-background ratio by the reduction of tissue autofluorescence background is demonstrated. PMID:23966226

  14. Fast globally optimal segmentation of cells in fluorescence microscopy images. (United States)

    Bergeest, Jan-Philip; Rohr, Karl


    Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

  15. Mesh adaptation technique for Fourier-domain fluorescence lifetime imaging

    International Nuclear Information System (INIS)

    Soloviev, Vadim Y.


    A novel adaptive mesh technique in the Fourier domain is introduced for problems in fluorescence lifetime imaging. A dynamical adaptation of the three-dimensional scheme based on the finite volume formulation reduces computational time and balances the ill-posed nature of the inverse problem. Light propagation in the medium is modeled by the telegraph equation, while the lifetime reconstruction algorithm is derived from the Fredholm integral equation of the first kind. Stability and computational efficiency of the method are demonstrated by image reconstruction of two spherical fluorescent objects embedded in a tissue phantom

  16. Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent. (United States)

    Schwenck, Johannes; Maier, Florian C; Kneilling, Manfred; Wiehr, Stefan; Fuchs, Kerstin


    This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models. Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

  17. Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology. (United States)

    Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A


    Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

  18. Submicron hard X-ray fluorescence imaging of synthetic elements. (United States)

    Jensen, Mark P; Aryal, Baikuntha P; Gorman-Lewis, Drew; Paunesku, Tatjana; Lai, Barry; Vogt, Stefan; Woloschak, Gayle E


    Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as μ-XANES (X-ray absorption near edge structure). We have used XFM to image and simultaneously quantify the transuranic element plutonium at the L(3) or L(2)-edge as well as Th and lighter biologically essential elements in individual rat pheochromocytoma (PC12) cells after exposure to the long-lived plutonium isotope (242)Pu. Elemental maps demonstrate that plutonium localizes principally in the cytoplasm of the cells and avoids the cell nucleus, which is marked by the highest concentrations of phosphorus and zinc, under the conditions of our experiments. The minimum detection limit under typical acquisition conditions with an incident X-ray energy of 18 keV for an average 202 μm(2) cell is 1.4 fg Pu or 2.9×10(-20) moles Pu μm(-2), which is similar to the detection limit of K-edge XFM of transition metals at 10 keV. Copper electron microscopy grids were used to avoid interference from gold X-ray emissions, but traces of strontium present in naturally occurring calcium can still interfere with plutonium detection using its L(α) X-ray emission. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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


    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. Biomarkers and Biological Spectral Imaging (United States)


    G. Sowa, H. H. Mantsch, National Research Council Canada; S. L. Zhang, Unilever Research (USA) 85 Brain tissue charcterization using spectral imaging...image registration and of the expert staff of Hill Top Research in Winnipeg for hosting the hydration study. Financial assistance from Unilever Research

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

    International Nuclear Information System (INIS)

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


    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.

  2. 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: [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)


    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.

  3. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging. (United States)

    Zhao, Ming; Li, Yu; Peng, Leilei


    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

  4. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes (United States)

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


    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

  5. Analysis of hyperspectral fluorescence images for poultry skin tumor inspection (United States)

    Kong, Seong G.; Chen, Yud-Ren; Kim, Intaek; Kim, Moon S.


    We present a hyperspectral fluorescence imaging system with a fuzzy inference scheme for detecting skin tumors on poultry carcasses. Hyperspectral images reveal spatial and spectral information useful for finding pathological lesions or contaminants on agricultural products. Skin tumors are not obvious because the visual signature appears as a shape distortion rather than a discoloration. Fluorescence imaging allows the visualization of poultry skin tumors more easily than reflectance. The hyperspectral image samples obtained for this poultry tumor inspection contain 65 spectral bands of fluorescence in the visible region of the spectrum at wavelengths ranging from 425 to 711 nm. The large amount of hyperspectral image data is compressed by use of a discrete wavelet transform in the spatial domain. Principal-component analysis provides an effective compressed representation of the spectral signal of each pixel in the spectral domain. A small number of significant features are extracted from two major spectral peaks of relative fluorescence intensity that have been identified as meaningful spectral bands for detecting tumors. A fuzzy inference scheme that uses a small number of fuzzy rules and Gaussian membership functions successfully detects skin tumors on poultry carcasses. Spatial-filtering techniques are used to significantly reduce false positives.

  6. Optofluidic fluorescent imaging cytometry on a cell phone. (United States)

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


    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

  7. Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields. (United States)

    Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles


    Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

  8. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, S.; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan


    Roč. 22, č. 2 (2016), s. 290-299 ISSN 1431-9276 R&D Projects: GA ČR(CZ) GAP305/11/2476; GA ČR(CZ) GPP501/12/P951 Institutional support: RVO:61389030 ; RVO:61388955 Keywords : raster image correlation spectroscopy * fluorescence recovery after photobleaching * auxin influx Subject RIV: EB - Genetics ; Molecular Biology; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 1.891, year: 2016

  9. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA. (United States)

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


    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Microholographic imaging of biological samples

    International Nuclear Information System (INIS)

    Haddad, W.S.; Cullen, D.; Solem, J.C.; Longworth, J.W.; McPherson, A.; Boyer, K.; Rhodes, C.K.


    A camera system suitable for x-ray microholography has been constructed. Visible light Fourier transform microholograms of biological samples and other test targets have been recorded and reconstructed digitally using a glycerol microdrop as a reference wave source. Current results give a resolution of ∼4 - 10 λ with λ = 514.5 nm. 11 refs., 1 fig

  11. Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy. (United States)

    Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro


    A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

  12. Detecting crop population growth using chlorophyll fluorescence imaging. (United States)

    Wang, Heng; Qian, Xiangjie; Zhang, Lan; Xu, Sailong; Li, Haifeng; Xia, Xiaojian; Dai, Liankui; Xu, Liang; Yu, Jingquan; Liu, Xu


    For both field and greenhouse crops, it is challenging to evaluate their growth information on a large area over a long time. In this work, we developed a chlorophyll fluorescence imaging-based system for crop population growth information detection. Modular design was used to make the system provide high-intensity uniform illumination. This system can perform modulated chlorophyll fluorescence induction kinetics measurement and chlorophyll fluorescence parameter imaging over a large area of up to 45  cm×34  cm. The system can provide different lighting intensity by modulating the duty cycle of its control signal. Results of continuous monitoring of cucumbers in nitrogen deficiency show the system can reduce the judge error of crop physiological status and improve monitoring efficiency. Meanwhile, the system is promising in high throughput application scenarios.

  13. Fluorescent Pluronic nanodots for in vivo two-photon imaging

    International Nuclear Information System (INIS)

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Stephan, Olivier; Marder, Seth R; Sanden, Boudewijn Van der


    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 μm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

  14. Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities (United States)

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


    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

  15. Fluorescent nanodiamonds for biomedical thermal imaging (United States)

    Ramirez Hernandez, Juan Enrique; Garcia Sole, Jose; Rafael Pedroza Montero, Martin; Barboza Flores, Marcelino; Jaque, Daniel; Martinez Maestro, Laura; Josefina Santacruz Gomez, Karla; Sanz Rodriguez, Francisco; Dong, Ningning; Calderon, Tomas


    The use of nanoparticles designed for a particular purpose in biological research has increased exponentially in the last ten years. Nanoscale particles have dimensions similar to those of most biological systems and present exceptional physical, chemical and optical properties. The optical absorption and emission of nanoparticles may be tuned by varying their shape, size and composition and recent advances in their synthesis and design suggest their potential use as probes in the detection and treatment of diseases such as cancer. One of these promising materials is nanodiamond which possesses excellent surface modification capacity and high biocompatibility. In addition to being compatible with the human body, nanodiamonds can be used as radiation sensors. In this report, the ability of nanodiamonds to be used as nanothermometers was studied by the obtention of a nanothermic scale (temperature dependence of nanodiamond's emission spectrum) to accurately measure temperature in small volumes.

  16. A low-cost method for visible fluorescence imaging. (United States)

    Tarver, Crissy L; Pusey, Marc


    A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

  17. Optimization of microsatellite DNA Gelred fluorescence imaging ...

    African Journals Online (AJOL)



    Oct 11, 2012 ... In order to explore the best microsatellite DNA Gelred imaging technology, this ... analysis and character identification breeding practice, because it is ... detection methods are agarose gel electrophoresis (AGE) with ethidium ... method (PG). Gelred 10000X stock reagent was diluted in the 1.5% agarose gel.

  18. Fluorescence lifetime imaging microscopy using near-infrared contrast agents. (United States)

    Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S


    Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

  19. A Review of Indocyanine Green Fluorescent Imaging in Surgery

    Directory of Open Access Journals (Sweden)

    Jarmo T. Alander


    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.

  20. Quantitative imaging of single upconversion nanoparticles in biological tissue.

    Directory of Open Access Journals (Sweden)

    Annemarie Nadort

    Full Text Available The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs, enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (∼1 cm depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 µm, unlike a hardly detectable single fluorescent (fluorescein dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement.

  1. A 3D imaging system integrating photoacoustic and fluorescence orthogonal projections for anatomical, functional and molecular assessment of rodent models (United States)

    Brecht, Hans P.; Ivanov, Vassili; Dumani, Diego S.; Emelianov, Stanislav Y.; Anastasio, Mark A.; Ermilov, Sergey A.


    We have developed a preclinical 3D imaging instrument integrating photoacoustic tomography and fluorescence (PAFT) addressing known deficiencies in sensitivity and spatial resolution of the individual imaging components. PAFT is designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct registration of the two imaging modalities. Orthogonal photoacoustic projections are utilized to reconstruct large (21 cm3 ) volumes showing vascularized anatomical structures and regions of induced optical contrast with spatial resolution exceeding 100 µm. The major advantage of orthogonal fluorescence projections is significant reduction of background noise associated with transmitted or backscattered photons. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the fluorescent biomarkers. PAFT performance characteristics were assessed by imaging optical and fluorescent contrast agents in tissue mimicking phantoms and in vivo. The proposed PAFT technology will enable functional and molecular volumetric imaging using fluorescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs.

  2. Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers (United States)

    Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.


    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 fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

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


    Elson, DS; Jo, JA; Marcu, L


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

  4. Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors (United States)

    Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.


    Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

  5. 2D/3D cryo x-ray fluorescence imaging at the bionanoprobe at the advanced photon source

    International Nuclear Information System (INIS)

    Chen, S.; Vine, D. J.; Lai, B.; Paunesku, T.; Yuan, Y.; Woloschak, G. E.; Deng, J.; Jin, Q.; Hong, Y. P.; Flachenecker, C.; Hornberger, B.; Brister, K.; Jacobsen, C.; Vogt, S.


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

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

    International Nuclear Information System (INIS)

    Li, Man; Jiang, Yuhua; Zhang, Di; Ding, Peigang; Wang, Zhenji; Ye, Yong; Zhao, Yufen


    A novel rhodamine-based fluorescent probe L2 with two “S” groups was synthesized and characterized. As expected, L2 exhibited high selectivity and sensitivity for Hg 2+ over other commonly coexistent metal ions. Its selectivity is excellent, and the detection limit was measured to be 1 ppm. The significant changes in the fluorescence color could be used for naked-eye detection. Furthermore, fluorescence imaging experiments of Hg 2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems. -- Highlights: • A new rhodamine-based chemodosimeter (L2) was synthesized and characterized. • L2 exhibited high selectivity and excellent sensitivity detection of Hg 2+ . • The significant changes in color could be used for naked-eye detection. • The fluorescence imaging experiments of Hg 2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems

  7. Multifunctional nanoparticles for MR and fluorescence imaging = (United States)

    Pinho, Sonia Luzia Claro de

    In the past few years a new generation of multifunctional nanoparticles (NPs) has been proposed for biomedical applications, whose structure is more complex than the structure of their predecessor monofunctional counterparts. The development of these novel NPs aims at enabling or improving the performance in imaging, diagnosis and therapeutic applications. The structure of such NPs comprises several components exhibiting various functionalities that enable the nanoparticles to perform multiple tasks simultaneously, such as active targeting of certain cells or compartmentalization, imaging and delivery of active drugs. This thesis presents two types of bimodal bio-imaging probes and describes their physical and chemical properties, namely their texture, structure, and 1H dynamics and relaxometry, in order to evaluate their potential as MRI contrast agents. The photoluminescence properties of these probes are studied, aiming at assessing their interest as optical contrast agents. These materials combine the properties of the trivalent lanthanide (Ln3+) complexes and nanoparticles, offering an excellent solution for bimodal imaging. The designed T1- type contrast agent are SiO2 APS/DTPA:Gd:Ln or SiO2 APS/PMN:Gd:Ln (Ln= Eu or Tb) systems, bearing the active magnetic center (Gd3+) and the optically-active ions (Eu3+ and Tb3+) on the surface of silica NPs. Concerning the relaxometry properties, moderate r1 increases and significant r2 increases are observed in the NPs presence, especially at high magnetic fields, due to susceptibility effects on r2. The Eu3+ ions reside in a single low-symmetry site, and the photoluminescence emission is not influenced by the simultaneous presence of Gd3+ and Eu3+. The presence of Tb3+, rather than Eu3+ ion, further increases r1 but decreases r2. The uptake of these NPs by living cells is fast and results in an intensity increase in the T1-weighted MRI images. The optical features of the NPs in cellular pellets are also studied and

  8. Contextual analysis of immunological response through whole-organ fluorescent imaging. (United States)

    Woodruff, Matthew C; Herndon, Caroline N; Heesters, B A; Carroll, Michael C


    As fluorescent microscopy has developed, significant insights have been gained into the establishment of immune response within secondary lymphoid organs, particularly in draining lymph nodes. While established techniques such as confocal imaging and intravital multi-photon microscopy have proven invaluable, they provide limited insight into the architectural and structural context in which these responses occur. To interrogate the role of the lymph node environment in immune response effectively, a new set of imaging tools taking into account broader architectural context must be implemented into emerging immunological questions. Using two different methods of whole-organ imaging, optical clearing and three-dimensional reconstruction of serially sectioned lymph nodes, fluorescent representations of whole lymph nodes can be acquired at cellular resolution. Using freely available post-processing tools, images of unlimited size and depth can be assembled into cohesive, contextual snapshots of immunological response. Through the implementation of robust iterative analysis techniques, these highly complex three-dimensional images can be objectified into sortable object data sets. These data can then be used to interrogate complex questions at the cellular level within the broader context of lymph node biology. By combining existing imaging technology with complex methods of sample preparation and capture, we have developed efficient systems for contextualizing immunological phenomena within lymphatic architecture. In combination with robust approaches to image analysis, these advances provide a path to integrating scientific understanding of basic lymphatic biology into the complex nature of immunological response.

  9. Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh. (United States)

    Rizk, Aurélien; Paul, Grégory; Incardona, Pietro; Bugarski, Milica; Mansouri, Maysam; Niemann, Axel; Ziegler, Urs; Berger, Philipp; Sbalzarini, Ivo F


    Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

  10. Bio-degradable highly fluorescent conjugated polymer nanoparticles for bio-medical imaging applications. (United States)

    Repenko, Tatjana; Rix, Anne; Ludwanowski, Simon; Go, Dennis; Kiessling, Fabian; Lederle, Wiltrud; Kuehne, Alexander J C


    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.

  11. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy (United States)

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


    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

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

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda


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

  13. qF-SSOP: real-time optical property corrected fluorescence imaging (United States)

    Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain


    Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

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

    Directory of Open Access Journals (Sweden)

    Christopher P. Irwin


    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.

  15. Fluorescence imaging of glutamate release in neurons

    International Nuclear Information System (INIS)

    Wang, Ziqiang; Yeung, Edward S.


    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 charge-coupled device (CCD) imaging is down to μM levels of glutamate with reasonable response time (∼30 s). The standard glutamate test shows a linear response over 3 orders of magnitude, from μM to 0.1 mM range. The in vitro monitoring of glutamate release from cultured neuron cells demonstrated excellent spatial and temporal resolution. (c) 1999 Society for Applied Spectroscopy

  16. Fluorescent supramolecular micelles for imaging-guided cancer therapy (United States)

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


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

  17. Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy. (United States)

    Matsuda, Atsushi; Schermelleh, Lothar; Hirano, Yasuhiro; Haraguchi, Tokuko; Hiraoka, Yasushi


    Correction of chromatic shift is necessary for precise registration of multicolor fluorescence images of biological specimens. New emerging technologies in fluorescence microscopy with increasing spatial resolution and penetration depth have prompted the need for more accurate methods to correct chromatic aberration. However, the amount of chromatic shift of the region of interest in biological samples often deviates from the theoretical prediction because of unknown dispersion in the biological samples. To measure and correct chromatic shift in biological samples, we developed a quadrisection phase correlation approach to computationally calculate translation, rotation, and magnification from reference images. Furthermore, to account for local chromatic shifts, images are split into smaller elements, for which the phase correlation between channels is measured individually and corrected accordingly. We implemented this method in an easy-to-use open-source software package, called Chromagnon, that is able to correct shifts with a 3D accuracy of approximately 15 nm. Applying this software, we quantified the level of uncertainty in chromatic shift correction, depending on the imaging modality used, and for different existing calibration methods, along with the proposed one. Finally, we provide guidelines to choose the optimal chromatic shift registration method for any given situation.

  18. Image-guided cancer surgery using near-infrared fluorescence (United States)

    Vahrmeijer, Alexander L.; Hutteman, Merlijn; van der Vorst, Joost R.; van de Velde, C.J.H.; Frangioni, John V.


    Paradigm shifts in surgery arise when surgeons are empowered to perform surgery faster, better, and/or less expensively. Optical imaging that exploits invisible near-infrared fluorescent light has the potential to improve cancer surgery outcomes while minimizing anesthesia time and lowering healthcare costs. Because of this, the last few years have witnessed an explosion of proof-of-concept clinical trials in the field. In this review, we introduce the concept of near-infrared fluorescence imaging for cancer surgery, review the clinical trial literature to date, outline the key issues pertaining to imaging system and contrast agent optimization, discuss limitations and leverage, and provide a framework for making the technology available for the routine care of cancer patients in the near future. PMID:23881033

  19. Portable Fluorescence Imaging System for Hypersonic Flow Facilities (United States)

    Wilkes, J. A.; Alderfer, D. W.; Jones, S. B.; Danehy, P. M.


    A portable fluorescence imaging system has been developed for use in NASA Langley s hypersonic wind tunnels. The system has been applied to a small-scale free jet flow. Two-dimensional images were taken of the flow out of a nozzle into a low-pressure test section using the portable planar laser-induced fluorescence system. Images were taken from the center of the jet at various test section pressures, showing the formation of a barrel shock at low pressures, transitioning to a turbulent jet at high pressures. A spanwise scan through the jet at constant pressure reveals the three-dimensional structure of the flow. Future capabilities of the system for making measurements in large-scale hypersonic wind tunnel facilities are discussed.

  20. The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging. (United States)

    Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie


    pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

  1. Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Wang Ching-Wei


    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.

  3. Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast. (United States)

    Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei


    Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

  4. Monosodium glutamate derived tricolor fluorescent carbon nanoparticles for cell-imaging application. (United States)

    Zheng, Nannan; Ding, Sha; Zhou, Xingping


    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.

  5. GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method (United States)

    Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae


    We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

  6. Clinical results of fluorescence lifetime imaging in ophthalmology (United States)

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


    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.

  7. An excited-state intramolecular photon transfer fluorescence probe for localizable live cell imaging of cysteine (United States)

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


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

  8. Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical Scaffolds. (United States)

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


    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

  9. Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy (United States)

    Ahmed, Syeed Ehsan

    Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

  10. Colorectal cancer detection by hyperspectral imaging using fluorescence excitation scanning (United States)

    Leavesley, Silas J.; Deal, Joshua; Hill, Shante; Martin, Will A.; Lall, Malvika; Lopez, Carmen; Rider, Paul F.; Rich, Thomas C.; Boudreaux, Carole W.


    Hyperspectral imaging technologies have shown great promise for biomedical applications. These techniques have been especially useful for detection of molecular events and characterization of cell, tissue, and biomaterial composition. Unfortunately, hyperspectral imaging technologies have been slow to translate to clinical devices - likely due to increased cost and complexity of the technology as well as long acquisition times often required to sample a spectral image. We have demonstrated that hyperspectral imaging approaches which scan the fluorescence excitation spectrum can provide increased signal strength and faster imaging, compared to traditional emission-scanning approaches. We have also demonstrated that excitation-scanning approaches may be able to detect spectral differences between colonic adenomas and adenocarcinomas and normal mucosa in flash-frozen tissues. Here, we report feasibility results from using excitation-scanning hyperspectral imaging to screen pairs of fresh tumoral and nontumoral colorectal tissues. Tissues were imaged using a novel hyperspectral imaging fluorescence excitation scanning microscope, sampling a wavelength range of 360-550 nm, at 5 nm increments. Image data were corrected to achieve a NIST-traceable flat spectral response. Image data were then analyzed using a range of supervised and unsupervised classification approaches within ENVI software (Harris Geospatial Solutions). Supervised classification resulted in >99% accuracy for single-patient image data, but only 64% accuracy for multi-patient classification (n=9 to date), with the drop in accuracy due to increased false-positive detection rates. Hence, initial data indicate that this approach may be a viable detection approach, but that larger patient sample sizes need to be evaluated and the effects of inter-patient variability studied.

  11. Study on excitation and fluorescence spectrums of Japanese citruses to construct machine vision systems for acquiring fluorescent images (United States)

    Momin, Md. Abdul; Kondo, Naoshi; Kuramoto, Makoto; Ogawa, Yuichi; Shigi, Tomoo


    Research was conducted to acquire knowledge of the ultraviolet and visible spectrums from 300 -800 nm of some common varieties of Japanese citrus, to investigate the best wave-lengths for fluorescence excitation and the resulting fluorescence wave-lengths and to provide a scientific background for the best quality fluorescent imaging technique for detecting surface defects of citrus. A Hitachi U-4000 PC-based microprocessor controlled spectrophotometer was used to measure the absorption spectrum and a Hitachi F-4500 spectrophotometer was used for the fluorescence and excitation spectrums. We analyzed the spectrums and the selected varieties of citrus were categorized into four groups of known fluorescence level, namely strong, medium, weak and no fluorescence.The level of fluorescence of each variety was also examined by using machine vision system. We found that around 340-380 nm LEDs or UV lamps are appropriate as lighting devices for acquiring the best quality fluorescent image of the citrus varieties to examine their fluorescence intensity. Therefore an image acquisition device was constructed with three different lighting panels with UV LED at peak 365 nm, Blacklight blue lamps (BLB) peak at 350 nm and UV-B lamps at peak 306 nm. The results from fluorescent images also revealed that the findings of the measured spectrums worked properly and can be used for practical applications such as for detecting rotten, injured or damaged parts of a wide variety of citrus.

  12. Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects (United States)

    Chen, Xucai; Wang, Jianjun; Versluis, Michel; de Jong, Nico; Villanueva, Flordeliza S.


    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.

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

    International Nuclear Information System (INIS)

    Ji Dongmei; Lv Wei; Huang Zhengxi; Xia Andong; Xu Min; Ma Weimin; Mi Hualing; Ogawa Teruo


    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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  15. Novel fluorescent carbonic nanomaterials for sensing and imaging

    International Nuclear Information System (INIS)

    Demchenko, Alexander P; Dekaliuk, Mariia O


    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. (topical review)

  16. Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy. (United States)

    Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D


    Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

  17. Fluorescence decay time imaging using an imaging photon detector with a radio frequency photon correlation system (United States)

    Morgan, Christopher G.; Mitchell, A. C.; Murray, J. G.


    An imaging photon detector has been modified to incorporate fast timing electronics coupled to a custom built photon correlator interfaced to a RISC computer. Using excitation with intensity- muodulated light, fluorescence images can be readily obtained where contrast is determined by the decay time of emission, rather than by intensity. This technology is readily extended to multifrequency phase/demodulation fluorescence imaging or to differential polarised phase fluorometry. The potential use of the correlator for confocal imaging with a laser scanner is also briefly discussed.

  18. High-contrast fluorescence imaging based on the polarization dependence of the fluorescence enhancement using an optical interference mirror slide. (United States)

    Yasuda, Mitsuru; Akimoto, Takuo


    High-contrast fluorescence imaging using an optical interference mirror (OIM) slide that enhances the fluorescence from a fluorophore located on top of the OIM surface is reported. To enhance the fluorescence and reduce the background light of the OIM, transverse-electric-polarized excitation light was used as incident light, and the transverse-magnetic-polarized fluorescence signal was detected. As a result, an approximate 100-fold improvement in the signal-to-noise ratio was achieved through a 13-fold enhancement of the fluorescence signal and an 8-fold reduction of the background light.

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

    International Nuclear Information System (INIS)

    Kosior, Ewelina


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

  20. Contribution of fluorescence in situ hybridization to biological dosimetry

    International Nuclear Information System (INIS)

    Sorokine-Durm, I.; Roy, L.; Durand, V.; Voisin, P.


    Fluorescence in situ hybridization with composite whole chromosome specific DNA probes for human chromosomes 2, 4 and 12 an α-satellite centromeric DNA probe labelled with biotin were used to measure symmetrical and terminal translocations (dose rate 0.5 Gy/min) and dicentrics (0.1 Gy/min) induced in vitro by 60 Co γ-irradiation (0-5 Gy). The suitability of fluorescence in situ hybridization (F.I.S.H.) technique for dicentrics detection is compared with the conventional technique. Dose-response curves for γ-rays ( 60 Co) for two dose rates are shown (dicentrics and translocations). (authors). 10 refs., 2 figs

  1. Silole-Based Red Fluorescent Organic Dots for Bright Two-Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging. (United States)

    Chen, Bin; Feng, Guangxue; He, Bairong; Goh, Chiching; Xu, Shidang; Ramos-Ortiz, Gabriel; Aparicio-Ixta, Laura; Zhou, Jian; Ng, Laiguan; Zhao, Zujin; Liu, Bin; Tang, Ben Zhong


    Robust luminescent dyes with efficient two-photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation-caused quenching. In this work, a red fluorescent silole, 2,5-bis[5-(dimesitylboranyl)thiophen-2-yl]-1-methyl-1,3,4-triphenylsilole ((MesB)2 DTTPS), is synthesized and characterized. (MesB)2 DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation-enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)2 DTTPS within lipid-PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two-photon absorption cross section of 3.43 × 10(5) GM, which yields a two-photon action cross section of 1.09 × 10(5) GM. These (MesB)2 DTTPS dots show good biocompatibility and are successfully applied to one-photon and two-photon fluorescence imaging of MCF-7 cells and two-photon in vivo visualization of the blood vascular of mouse muscle in a high-contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 μm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy (United States)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees


    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

  3. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging. (United States)

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


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

  4. In Vivo Dual Fluorescence Imaging to Detect Joint Destruction. (United States)

    Cho, Hongsik; Bhatti, Fazal-Ur-Rehman; Lee, Sangmin; Brand, David D; Yi, Ae-Kyung; Hasty, Karen A


    Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis. © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  5. Efficient processing of fluorescence images using directional multiscale representations. (United States)

    Labate, D; Laezza, F; Negi, P; Ozcan, B; Papadakis, M


    Recent advances in high-resolution fluorescence microscopy have enabled the systematic study of morphological changes in large populations of cells induced by chemical and genetic perturbations, facilitating the discovery of signaling pathways underlying diseases and the development of new pharmacological treatments. In these studies, though, due to the complexity of the data, quantification and analysis of morphological features are for the vast majority handled manually, slowing significantly data processing and limiting often the information gained to a descriptive level. Thus, there is an urgent need for developing highly efficient automated analysis and processing tools for fluorescent images. In this paper, we present the application of a method based on the shearlet representation for confocal image analysis of neurons. The shearlet representation is a newly emerged method designed to combine multiscale data analysis with superior directional sensitivity, making this approach particularly effective for the representation of objects defined over a wide range of scales and with highly anisotropic features. Here, we apply the shearlet representation to problems of soma detection of neurons in culture and extraction of geometrical features of neuronal processes in brain tissue, and propose it as a new framework for large-scale fluorescent image analysis of biomedical data.

  6. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography (United States)

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


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

  7. Direct comparison of soft x-ray images of organelles with optical fluorescence images

    International Nuclear Information System (INIS)

    Ishino, Masahiko; Kado, Masataka; Kishimoto, Maki; Nishikino, Masaharu; Ohba, Toshiyuki; Kaihori, Takeshi; Kawachi, Tetsuya; Tamotsu, Satoshi; Yasuda, Keiko; Mikata, Yuji; Shinohara, Kunio


    Soft x-ray microscopes operating in the water window region are capable of imaging living hydrated cells. Up to now, we have been able to take some soft x-ray images of living cells by the use of a contact x-ray microscope system with laser produced plasma soft x-ray source. Since the soft x-ray images are different from the optical images obtained with an ordinary microscope, it is very important to identify what is seen in the x-ray images. Hence, we have demonstrated the direct comparison between the images of organelles obtained with a fluorescence microscope and those with a soft x-ray microscope. Comparing the soft x-ray images to the fluorescence images, the fine structures of the organelles could be identified and observed. (author)

  8. Compact instrument for fluorescence image-guided surgery (United States)

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


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

  9. Particle Image Velocimetry Applications Using Fluorescent Dye-Doped Particles (United States)

    Petrosky, Brian J.; Maisto, Pietro; Lowe, K. Todd; Andre, Matthieu A.; Bardet, Philippe M.; Tiemsin, Patsy I.; Wohl, Christopher J.; Danehy, Paul M.


    Polystyrene latex sphere particles are widely used to seed flows for velocimetry techniques such as Particle Image Velocimetry (PIV) and Laser Doppler Velocimetry (LDV). These particles may be doped with fluorescent dyes such that signals spectrally shifted from the incident laser wavelength may be detected via Laser Induced Fluorescence (LIF). An attractive application of the LIF signal is achieving velocimetry in the presence of strong interference from laser scatter, opening up new research possibilities very near solid surfaces or at liquid/gas interfaces. Additionally, LIF signals can be used to tag different fluid streams to study mixing. While fluorescence-based PIV has been performed by many researchers for particles dispersed in water flows, the current work is among the first in applying the technique to micron-scale particles dispersed in a gas. A key requirement for such an application is addressing potential health hazards from fluorescent dyes; successful doping of Kiton Red 620 (KR620) has enabled the use of this relatively safe dye for fluorescence PIV for the first time. In this paper, basic applications proving the concept of PIV using the LIF signal from KR620-doped particles are exhibited for a free jet and a twophase flow apparatus. Results indicate that while the fluorescence PIV techniques are roughly 2 orders of magnitude weaker than Mie scattering, they provide a viable method for obtaining data in flow regions previously inaccessible via standard PIV. These techniques have the potential to also complement Mie scattering signals, for example in multi-stream and/or multi-phase experiments.

  10. Imaging systems and algorithms to analyze biological samples in real-time using mobile phone microscopy. (United States)

    Shanmugam, Akshaya; Usmani, Mohammad; Mayberry, Addison; Perkins, David L; Holcomb, Daniel E


    Miniaturized imaging devices have pushed the boundaries of point-of-care imaging, but existing mobile-phone-based imaging systems do not exploit the full potential of smart phones. This work demonstrates the use of simple imaging configurations to deliver superior image quality and the ability to handle a wide range of biological samples. Results presented in this work are from analysis of fluorescent beads under fluorescence imaging, as well as helminth eggs and freshwater mussel larvae under white light imaging. To demonstrate versatility of the systems, real time analysis and post-processing results of the sample count and sample size are presented in both still images and videos of flowing samples.

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

    Directory of Open Access Journals (Sweden)

    Sasano Masahiko


    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.

  12. Dual-emissive quantum dots for multispectral intraoperative fluorescence imaging. (United States)

    Chin, Patrick T K; Buckle, Tessa; Aguirre de Miguel, Arantxa; Meskers, Stefan C J; Janssen, René A J; van Leeuwen, Fijs W B


    Fluorescence molecular imaging is rapidly increasing its popularity in image guided surgery applications. To help develop its full surgical potential it remains a challenge to generate dual-emissive imaging agents that allow for combined visible assessment and sensitive camera based imaging. To this end, we now describe multispectral InP/ZnS quantum dots (QDs) that exhibit a bright visible green/yellow exciton emission combined with a long-lived far red defect emission. The intensity of the latter emission was enhanced by X-ray irradiation and allows for: 1) inverted QD density dependent defect emission intensity, showing improved efficacies at lower QD densities, and 2) detection without direct illumination and interference from autofluorescence. Copyright 2010 Elsevier Ltd. All rights reserved.

  13. Fluorescence Imaging Study of Transition in Underexpanded Free Jets (United States)

    Wilkes, Jennifer A.; Danehy, Paul M.; Nowak, Robert J.


    Planar laser-induced fluorescence (PLIF) is demonstrated to be a valuable tool for studying the onset of transition to turbulence. For this study, we have used PLIF of nitric oxide (NO) to image underexpanded axisymmetric free jets issuing into a low-pressure chamber through a smooth converging nozzle with a sonic orifice. Flows were studied over a range of Reynolds numbers and nozzle-exit-to-ambient pressure ratios with the aim of empirically determining criteria governing the onset of turbulence. We have developed an image processing technique, involving calculation of the standard deviation of the intensity in PLIF images, in order to aid in the identification of turbulence. We have used the resulting images to identify laminar, transitional and turbulent flow regimes. Jet scaling parameters were used to define a rescaled Reynolds number that incorporates the influence of a varying pressure ratio. An empirical correlation was found between transition length and this rescaled Reynolds number for highly underexpanded jets.

  14. Pathological diagnosis of bladder cancer by image analysis of hypericin induced fluorescence cystoscopic images (United States)

    Kah, James C. Y.; Olivo, Malini C.; Lau, Weber K. O.; Sheppard, Colin J. R.


    Photodynamic diagnosis of bladder carcinoma based on hypericin fluorescence cystoscopy has shown to have a higher degree of sensitivity for the detection of flat bladder carcinoma compared to white light cystoscopy. The potential of the photosensitizer hypericin-induced fluorescence in performing non-invasive optical biopsy to grade bladder cancer in vivo using fluorescence cystoscopic image analysis without surgical resection for tissue biopsy is investigated in this study. The correlation between tissue fluorescence and histopathology of diseased tissue was explored and a diagnostic algorithm based on fluorescence image analysis was developed to classify the bladder cancer without surgical resection for tissue biopsy. Preliminary results suggest a correlation between tissue fluorescence and bladder cancer grade. By combining both the red-to-blue and red-to-green intensity ratios into a 2D scatter plot yields an average sensitivity and specificity of around 70% and 85% respectively for pathological cancer grading of the three different grades of bladder cancer. Therefore, the diagnostic algorithm based on colorimetric intensity ratio analysis of hypericin fluorescence cystoscopic images developed in this preliminary study shows promising potential to optically diagnose and grade bladder cancer in vivo.

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

    Zeller, Perrine; Ploux, Olivier; Méjean, Annick


    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.

  16. An off-on fluorescence probe targeting mitochondria based on oxidation-reduction response for tumor cell and tissue imaging (United States)

    Yao, Hanchun; Cao, Li; Zhao, Weiwei; Zhang, Suge; Zeng, Man; Du, Bin


    In this study, a tumor-targeting poly( d, l-lactic-co-glycolic acid) (PLGA) loaded "off-on" fluorescent probe nanoparticle (PFN) delivery system was developed to evaluate the region of tumor by off-on fluorescence. The biodegradability of the nanosize PFN delivery system readily released the probe under tumor acidic conditions. The probe with good biocompatibility was used to monitor the intracellular glutathione (GSH) of cancer cells and selectively localize to mitochondria for tumor imaging. The incorporated tumor-targeting probe was based on the molecular photoinduced electron transfer (PET) mechanism preventing fluorescence ("off" state) and could be easily released under tumor acidic conditions. However, the released tumor-targeting fluorescence probe molecule was selective towards GSH with high selectivity and an ultra-sensitivity for the mitochondria of cancer cells and tissues significantly increasing the probe molecule fluorescence signal ("on" state). The tumor-targeting fluorescence probe showed sensitivity to GSH avoiding interference from cysteine and homocysteine. The PFNs could enable fluorescence-guided cancer imaging during cancer therapy. This work may expand the biological applications of PFNs as a diagnostic reagent, which will be beneficial for fundamental research in tumor imaging. [Figure not available: see fulltext.

  17. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization (United States)

    Moffitt, Jeffrey R.; Zhuang, Xiaowei


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

  18. A portable microscopy system for fluorescence, polarized, and brightfield imaging (United States)

    Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard


    The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

  19. Detection of biological warfare agents using ultra violet-laser induced fluorescence LIDAR. (United States)

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


    This review has been written to highlight the threat of biological warfare agents, their types and detection. Bacterial biological agent Bacillus anthracis (bacteria causing the disease anthrax) which is most likely to be employed in biological warfare is being discussed in detail. Standoff detection of biological warfare agents in aerosol form using Ultra violet-Laser Induced Fluorescence (UV-LIF) spectroscopy method has been studied. Range-resolved detection and identification of biological aerosols by both nano-second and non-linear femto-second LIDAR is also discussed. Calculated received fluorescence signal for a cloud of typical biological agent Bacillus globigii (Simulants of B. anthracis) at a location of ~5.0 km at different concentrations in presence of solar background radiation has been described. Overview of current research efforts in internationally available working UV-LIF LIDAR systems are also mentioned briefly. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Imaging atoms from resonance fluorescence spectrum beyond the diffraction limit (United States)

    Liao, Zeyang; Al-Amri, Mohammad; Zubairy, M. Suhail


    We calculate the resonance fluorescence spectrum of a linear chain of two-level atoms driven by a gradient coherent laser field. The result shows that we can determine the positions of atoms from the spectrum even when the atoms locate within subwavelength range and the dipole-dipole interaction is significant. This far-field resonance fluorescence localization microscopy method does not require point-by-point scanning and it may be more time-efficient. We also give a possible scheme to extract the position information in an extended region without requiring more peak power of laser. We also briefly discuss how to do a 2D imaging based on our scheme. This work is supported by grants from the King Abdulaziz City for Science and Technology (KACST) and the Qatar National Research Fund (QNRF) under the NPRP project.

  1. Multispectral fluorescence imaging technique for discrimination of cucumber (Cucumis Sativus) seed viability (United States)

    In this study, we developed a nondestructive method for discriminating viable cucumber (Cucumis sativus) seeds based on hyperspectral fluorescence imaging. The fluorescence spectra of cucumber seeds in the 420–700 nm range were extracted from hyperspectral fluorescence images obtained using 365 nm u...

  2. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging

    International Nuclear Information System (INIS)

    Ntziachristos, Vasilis; Bremer, Christoph; Weissleder, Ralph


    A recent development in biomedical imaging is the non-invasive mapping of molecular events in intact tissues using fluorescence. Underpinning to this development is the discovery of bio-compatible, specific fluorescent probes and proteins and the development of highly sensitive imaging technologies for in vivo fluorescent detection. Of particular interest are fluorochromes that emit in the near infrared (NIR), a spectral window, whereas hemoglobin and water absorb minimally so as to allow photons to penetrate for several centimetres in tissue. In this review article we concentrate on optical imaging technologies used for non-invasive imaging of the distribution of such probes. We illuminate the advantages and limitations of simple photographic methods and turn our attention to fluorescence-mediated molecular tomography (FMT), a technique that can three-dimensionally image gene expression by resolving fluorescence activation in deep tissues. We describe theoretical specifics, and we provide insight into its in vivo capacity and the sensitivity achieved. Finally, we discuss its clinical feasibility. (orig.)

  3. In situ Analysis of Coral Recruits Using Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Adi Zweifler


    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.

  4. Mechanical Damage Detection of Indonesia Local Citrus Based on Fluorescence Imaging (United States)

    Siregar, T. H.; Ahmad, U.; Sutrisno; Maddu, A.


    Citrus experienced physical damage in peel will produce essential oils that contain polymethoxylated flavone. Polymethoxylated flavone is fluorescence substance; thus can be detected by fluorescence imaging. This study aims to study the fluorescence spectra characteristic and to determine the damage region in citrus peel based on fluorescence image. Pulung citrus from Batu district, East Java, as a famous citrus production area in Indonesia, was used in the experiment. It was observed that the image processing could detect the mechanical damage region. Fluorescence imaging can be used to classify the citrus into two categories, sound and defect citruses.

  5. A Fast Global Fitting Algorithm for Fluorescence Lifetime Imaging Microscopy Based on Image Segmentation


    Pelet, S.; Previte, M.J.R.; Laiho, L.H.; So, P.T. C.


    Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained ana...

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  7. Laser scanning endoscope via an imaging fiber bundle for fluorescence imaging (United States)

    Yeboah, Lorenz D.; Nestler, Dirk; Steiner, Rudolf W.


    Based on a laser scanning endoscope via an imaging fiber bundle, a new approach for a tumor diagnostic system has been developed to assist physicians in the diagnosis before the actual PDT is carried out. Laser induced, spatially resolved fluorescence images of diseased tissue can be compared with images received by video endoscopy using a white light source. The set- up is required to produce a better contrast between infected and healthy tissue and might serve as a constructive diagnostic help for surgeons. The fundamental idea is to scan a low-power laser beam on an imaging fiber bundle and to achieve a spatially resolved projection on the tissue surface. A sufficiently high laser intensity from the diode laser is concentrated on each single spot of the tissue exciting fluorescence when a dye has previously been accumulated. Subsequently, video image of the tissue is recorded and stored. With an image processing unit, video and fluorescence images are overlaid producing a picture of the fluorescence intensity in the environment of the observed tissue.

  8. Quantum dots versus organic fluorophores in fluorescent deep-tissue imaging--merits and demerits. (United States)

    Bakalova, Rumiana; Zhelev, Zhivko; Gadjeva, Veselina


    The use of fluorescence in deep-tissue imaging is rapidly expanding in last several years. The progress in fluorescent molecular probes and fluorescent imaging techniques gives an opportunity to detect single cells and even molecular targets in live organisms. The highly sensitive and high-speed fluorescent molecular sensors and detection devices allow the application of fluorescence in functional imaging. With the development of novel bright fluorophores based on nanotechnologies and 3D fluorescence scanners with high spatial and temporal resolution, the fluorescent imaging has a potential to become an alternative of the other non-invasive imaging techniques as magnetic resonance imaging, positron-emission tomography, X-ray, computing tomography. The fluorescent imaging has also a potential to give a real map of human anatomy and physiology. The current review outlines the advantages of fluorescent nanoparticles over conventional organic dyes in deep-tissue imaging in vivo and defines the major requirements to the "perfect fluorophore". The analysis proceeds from the basic principles of fluorescence and major characteristics of fluorophores, light-tissue interactions, and major limitations of fluorescent deep-tissue imaging. The article is addressed to a broad readership - from specialists in this field to university students.

  9. Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging. (United States)

    Braaf, Boy; de Boer, Johannes F


    Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

  10. Scintillating Optical Fiber Imagers for biology

    International Nuclear Information System (INIS)

    Mastrippolito, R.


    S.O.F.I (Scintillating Optical Fiber Imager) is a detector developed to replace the autoradiographic films used in molecular biology for the location of radiolabelled ( 32 P) DNA molecules in blotting experiments. It analyses samples on a 25 x 25 cm 2 square area still 25 times faster than autoradiographic films, with a 1.75 and 3 mm resolution for two orthogonal directions. This device performs numerised images with a dynamic upper than 100 which allows the direct quantitation of the analysed samples. First, this thesis describes the S.O.F.I. development (Scintillating Optical Fibers, coding of these fibers and specific electronic for the treatment of the Multi-Anode Photo-Multiplier signals) and experiments made in collaboration with molecular biology laboratories. In a second place, we prove the feasibility of an automatic DNA sequencer issued from S.O.F.I [fr

  11. A CMOS In-Pixel CTIA High Sensitivity Fluorescence Imager. (United States)

    Murari, Kartikeya; Etienne-Cummings, Ralph; Thakor, Nitish; Cauwenberghs, Gert


    Traditionally, charge coupled device (CCD) based image sensors have held sway over the field of biomedical imaging. Complementary metal oxide semiconductor (CMOS) based imagers so far lack sensitivity leading to poor low-light imaging. Certain applications including our work on animal-mountable systems for imaging in awake and unrestrained rodents require the high sensitivity and image quality of CCDs and the low power consumption, flexibility and compactness of CMOS imagers. We present a 132×124 high sensitivity imager array with a 20.1 μm pixel pitch fabricated in a standard 0.5 μ CMOS process. The chip incorporates n-well/p-sub photodiodes, capacitive transimpedance amplifier (CTIA) based in-pixel amplification, pixel scanners and delta differencing circuits. The 5-transistor all-nMOS pixel interfaces with peripheral pMOS transistors for column-parallel CTIA. At 70 fps, the array has a minimum detectable signal of 4 nW/cm(2) at a wavelength of 450 nm while consuming 718 μA from a 3.3 V supply. Peak signal to noise ratio (SNR) was 44 dB at an incident intensity of 1 μW/cm(2). Implementing 4×4 binning allowed the frame rate to be increased to 675 fps. Alternately, sensitivity could be increased to detect about 0.8 nW/cm(2) while maintaining 70 fps. The chip was used to image single cell fluorescence at 28 fps with an average SNR of 32 dB. For comparison, a cooled CCD camera imaged the same cell at 20 fps with an average SNR of 33.2 dB under the same illumination while consuming over a watt.

  12. Microbial biofilm detection on food contact surfaces by macro-scale fluorescence imaging (United States)

    Hyperspectral fluorescence imaging methods were utilized to evaluate the potential of multispectral fluorescence methods for detection of pathogenic biofilm formations on four types of food contact surface materials: stainless steel, high density polyethylene (HDPE) commonly used for cutting boards,...

  13. Synthesis of Amphiphilic Hyperbranched AIE-active Fluorescent Organic Nanoparticles and Their Application in Biological Application. (United States)

    Lv, Qiulan; Wang, Ke; Xu, Dazhuang; Liu, Meiying; Wan, Qing; Huang, Hongye; Liang, Shangdong; Zhang, Xiaoyong; Wei, Yen


    Aggregation-induced emission (AIE) dyes have recently attracted much attention for biomedical applications for their remarkable AIE properties. However, the hydrophobic nature of AIE dyes made them difficult to be dispersed in physiological solution and problematic for biomedical application directly. Great efforts have been made to overcome this problem, and different strategies for preparation of water dispersible AIE based nanoprobes had been explored previously. However, a facile and effective strategy is still highly desirable and of great importance for the biomedical applications of AIE dye based on nanoprobes. In this work, the fabrication of amphiphilic hyperbranched fluorescent organic nanoparticles with a core-shell structure based on an AIE dye [tetraphenylethene acrylate (TPE-O-E)] and a hyperbranched polyamino compound [polyethylene imine (PEI)] through Michael addition reaction is described for the first time. The AIE dye as well as the final product PEI-TPE-O-E was characterized in detail by a number of techniques. To test their biomedical application potential, the cell viability as well as cell imaging properties of the PEI-TPE-O-E was also examined. The results showed that the PEI-TPE-O-E organic nanoparticles presented high water dispersiblity, ultrabright fluroerescence, low cytotoxicity and excellent biocompatibility, making them promising for biological imaging and gene delivery applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. 1-Million droplet array with wide-field fluorescence imaging for digital PCR. (United States)

    Hatch, Andrew C; Fisher, Jeffrey S; Tovar, Armando R; Hsieh, Albert T; Lin, Robert; Pentoney, Stephen L; Yang, David L; Lee, Abraham P


    Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

  15. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi


    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.

  16. Improved fluorescent X-ray image intensifying screen

    International Nuclear Information System (INIS)

    Landeghem, W.K. van; Suys, A.R.


    An X-ray image intensifying screen is described, which includes at least one fluorescent layer comprising phosphor particles dispersed in a binder and on top of such layer a protective layer containing a crosslinked polymer mass obtained by an acid-catalyzed reaction of a polymer or mixture of polymers containing reactive hydrogen atoms and a cross-linking agent, the cross-linking agent being an organic compound containing a plurality of etherified N-methylol groups. Examples are given of appropriate polymers and cross-linking agents. (author)

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

    Directory of Open Access Journals (Sweden)

    Patrick eKaifosh


    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

  18. Fluorescence-Doped Particles for Simultaneous Temperature and Velocity Imaging (United States)

    Danehy, Paul M.; Tiemsin, Pacita I.; Wohl, Chrostopher J.; Verkamp, Max; Lowe, T.; Maisto, P.; Byun, G.; Simpson, R.


    Polystyrene latex microspheres (PSLs) have been used for particle image velocimetry (PIV) and laser Doppler velocimetry (LDV) measurements for several decades. With advances in laser technologies, instrumentation, and data processing, the capability to collect more information about fluid flow beyond velocity is possible using new seed materials. To provide additional measurement capability, PSLs were synthesized with temperature-sensitive fluorescent dyes incorporated within the particle. These multifunctional PSLs would have the greatest impact if they could be used in large scale facilities with minimal modification to the facilities or the existing instrumentation. Consequently, several potential dyes were identified that were amenable to existing laser systems currently utilized in wind tunnels at NASA Langley Research Center as well as other wind and fluid (water) tunnels. PSLs incorporated with Rhodamine B, dichlorofluorescein (DCF, also known as fluorescein 548 or fluorescein 27) and other dyes were synthesized and characterized for morphology and spectral properties. The resulting particles were demonstrated to exhibit fluorescent emission, which would enable determination of both fluid velocity and temperature. They also would allow near-wall velocity measurements whereas laser scatter from surfaces currently prevents near-wall measurements using undoped seed materials. Preliminary results in a wind tunnel facility located at Virginia Polytechnic Institute and State University (Virginia Tech) have verified fluorescent signal detection and temperature sensitivity of fluorophore-doped PSLs.

  19. Modelling of microcracks image treated with fluorescent dye (United States)

    Glebov, Victor; Lashmanov, Oleg U.


    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

  20. Imaging morphogenesis: technological advances and biological insights. (United States)

    Keller, Philipp J


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

  1. Fluorescence imaging as a diagnostic of M-band x-ray drive condition in hohlraum with fluorescent Si targets

    International Nuclear Information System (INIS)

    Li, Qi; Hu, Zhimin; Yao, Li; Huang, Chengwu; Yuan, Zheng; Zhao, Yang; Xiong, Gang; Qing, Bo; Lv, Min; Zhu, Tuo; Deng, Bo; Li, Jin; Wei, Minxi; Zhan, Xiayu; Li, Jun; Yang, Yimeng; Su, Chunxiao; Yang, Guohong; Zhang, Jiyan; Li, Sanwei


    Fluorescence imaging of surrogate Si-doped CH targets has been used to provide a measurement for drive condition of high-energy x-ray (i.e. M-band x-ray) drive symmetry upon the capsule in hohlraum on Shenguang-II laser facility. A series of experiments dedicated to the study of photo-pumping and fluorescence effect in Si-plasma are presented. To investigate the feasibility of fluorescence imaging in Si-plasma, an silicon plasma in Si-foil target is pre-formed at ground state by the soft x-ray from a half-hohlraum, which is then photo-pumped by the K-shell lines from a spatially distinct laser-produced Si-plasma. The resonant Si photon pump is used to improve the fluorescence signal and cause visible image in the Si-foil. Preliminary fluorescence imaging of Si-ball target is performed in both Si-doped and pure Au hohlraum. The usual capsule at the center of the hohlraum is replaced with a solid Si-doped CH-ball (Si-ball). Since the fluorescence is proportional to the photon pump upon the Si-plasma, high-energy x-ray drive symmetry is equal to the fluorescence distribution of the Si-ball. (paper)

  2. Rapid analysis and exploration of fluorescence microscopy images. (United States)

    Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J


    Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

  3. Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging. (United States)

    Gilad, Assaf A; Shapiro, Mikhail G


    Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and industrial applications. This approach promises new treatments, imaging tools, and diagnostics for diseases ranging from gastrointestinal inflammatory syndromes to cancer, diabetes, and neurodegeneration. As these cellular technologies undergo pre-clinical and clinical development, it is becoming essential to monitor their location and function in vivo, necessitating appropriate molecular imaging strategies, and therefore, we have created an interest group within the World Molecular Imaging Society focusing on synthetic biology and reporter gene technologies. Here, we highlight recent advances in biomedical synthetic biology, including bacterial therapy, immunotherapy, and regenerative medicine. We then discuss emerging molecular imaging approaches to facilitate in vivo applications, focusing on reporter genes for noninvasive modalities such as magnetic resonance, ultrasound, photoacoustic imaging, bioluminescence, and radionuclear imaging. Because reporter genes can be incorporated directly into engineered genetic circuits, they are particularly well suited to imaging synthetic biological constructs, and developing them provides opportunities for creative molecular and genetic engineering.

  4. Potential applications of near infrared auto-fluorescence spectral polarized imaging for assessment of food quality (United States)

    Zhou, Kenneth J.; Chen, Jun


    The current growing of food industry for low production costs and high efficiency needs for maintenance of high-quality standards and assurance of food safety while avoiding liability issues. Quality and safety of food depend on physical (texture, color, tenderness etc.), chemical (fat content, moisture, protein content, pH, etc.), and biological (total bacterial count etc.) features. There is a need for a rapid (less than a few minutes) and accurate detection system in order to optimize quality and assure safety of food. However, the fluorescence ranges for known fluorophores are limited to ultraviolet emission bands, which are not in the tissue near infrared (NIR) "optical window". Biological tissues excited by far-red or NIR light would exhibit strong emission in spectral range of 650-1,100 nm although no characteristic peaks show the emission from which known fluorophores. The characteristics of the auto-fluorescence emission of different types of tissues were found to be different between different tissue components such as fat, high quality muscle food. In this paper, NIR auto-fluorescence emission from different types of muscle food and fat was measured. The differences of fluorescence intensities of the different types of muscle food and fat emissions were observed. These can be explained by the change of the microscopic structure of physical, chemical, and biological features in meat. The difference of emission intensities of fat and lean meat tissues was applied to monitor food quality and safety using spectral polarized imaging, which can be detect deep depth fat under the muscle food up to several centimeter.

  5. Organic fluorescent dye-based nanomaterials: Advances in the rational design for imaging and sensing applications. (United States)

    Svechkarev, Denis; Mohs, Aaron M


    Self-assembled fluorescent nanomaterials based on small-molecule organic dyes are gaining increasing popularity in imaging and sensing applications over the past decade. This is primarily due to their ability to combine spectral property tunability and biocompatibility of small molecule organic fluorophores with brightness, chemical, and colloidal stability of inorganic materials. Such a unique combination of features comes with rich versatility of dye-based nanomaterials: from aggregates of small molecules to sophisticated core-shell nanoarchitectures involving hyperbranched polymers. Along with the ongoing discovery of new materials and better ways of their synthesis, it is very important to continue systematic studies of fundamental factors that regulate the key properties of fluorescent nanomaterials: their size, polydispersity, colloidal stability, chemical stability, absorption and emission maxima, biocompatibility, and interactions with biological interfaces. In this review, we focus on the systematic description of various types of organic fluorescent nanomaterials, approaches to their synthesis, and ways to optimize and control their characteristics. The discussion is built on examples from reports on recent advances in design and applications of such materials. Conclusions made from this analysis allow a perspective on future development of fluorescent nanomaterials design for biomedical and related applications. Copyright© Bentham Science Publishers; For any queries, please email at

  6. Quantitative analysis of dynamic association in live biological fluorescent samples.

    Directory of Open Access Journals (Sweden)

    Pekka Ruusuvuori

    Full Text Available Determining vesicle localization and association in live microscopy may be challenging due to non-simultaneous imaging of rapidly moving objects with two excitation channels. Besides errors due to movement of objects, imaging may also introduce shifting between the image channels, and traditional colocalization methods cannot handle such situations. Our approach to quantifying the association between tagged proteins is to use an object-based method where the exact match of object locations is not assumed. Point-pattern matching provides a measure of correspondence between two point-sets under various changes between the sets. Thus, it can be used for robust quantitative analysis of vesicle association between image channels. Results for a large set of synthetic images shows that the novel association method based on point-pattern matching demonstrates robust capability to detect association of closely located vesicles in live cell-microscopy where traditional colocalization methods fail to produce results. In addition, the method outperforms compared Iterated Closest Points registration method. Results for fixed and live experimental data shows the association method to perform comparably to traditional methods in colocalization studies for fixed cells and to perform favorably in association studies for live cells.

  7. Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue (United States)

    Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.


    Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

  8. Economic and biological costs of cardiac imaging

    Directory of Open Access Journals (Sweden)

    Picano Eugenio


    Full Text Available Abstract Medical imaging market consists of several billion tests per year worldwide. Out of these, at least one third are cardiovascular procedures. Keeping in mind that each test represents a cost, often a risk, and a diagnostic hypothesis, we can agree that every unnecessary and unjustifiable test is one test too many. Small individual costs, risks, and wastes multiplied by billions of examinations per year represent an important population, society and environmental burden. Unfortunately, the appropriateness of cardiac imaging is extra-ordinarily low and there is little awareness in patients and physicians of differential costs, radiological doses, and long term risks of different imaging modalities. For a resting cardiac imaging test, being the average cost (not charges of an echocardiogram equal to 1 (as a cost comparator, the cost of a CT is 3.1x, of a SPECT 3.27x, of a Cardiovascular Magnetic Resonance imaging 5.51x, of a PET 14.03x, and of a right and left heart catheterization 19.96x. For stress cardiac imaging, compared with the treadmill exercise test equal to 1 (as a cost comparator, the cost of stress echocardiography is 2.1x and of a stress SPECT scintigraphy is 5.7x. Biohazards and downstream long-term costs linked to radiation-induced oncogenesis should also be considered. The radiation exposure is absent in echo and magnetic resonance, and corresponds to 500 chest x rays for a sestamibi cardiac stress scan and to 1150 chest x rays for a thallium scan. The corresponding extra-risk in a lifetime of fatal cancer is 1 in 2000 exposed patients for a sestamibi stress and 1 in 1000 for a thallium scan. Increased awareness of economic, biologic, and environmental costs of cardiac imaging will hopefully lead to greater appropriateness, wisdom and prudence from both the prescriber and the practitioner. In this way, the sustainability of cardiac imaging will eventually improve.

  9. IRDye78 Conjugates for Near-Infrared Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Atif Zaheer


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

  10. NATO Advanced Research Workshop on Fluorescence and other Optical Properties of Biological Particles for Biological Warfare Agent Sensors

    CERN Document Server

    Hoekstra, Alfons; Videen, Gorden; Optics of Biological Particles


    This book covers the optics of single biological particles, both theory and experiment, with emphasis on Elastic Light Scattering and Fluorescence. It deals with the optics of bacteria (bio-aerosols), marine particles (selected phytoplankton communities) and red and white blood cells. Moreover, there are dedicated chapters on a general theory for scattering by a cell, and modelling and simulation of scattering by inhomogeneous biological cells. Finally, one chapter is dedicated to astro-biological signatures, discussing the possibilities for detecting non-terrestrial biological material. The volume has up-to-date discussions on new experimental and numerical techniques, and many examples of applications of these techniques in real-life systems, as used to detect and characterize e.g. biological warfare agents or human blood cells.

  11. Semiconductor quantum dots as fluorescent probes for in vitro and in vivo bio-molecular and cellular imaging

    Directory of Open Access Journals (Sweden)

    Sarwat B. Rizvi


    Full Text Available Over the years, biological imaging has seen many advances, allowing scientists to unfold many of the mysteries surrounding biological processes. The ideal imaging resolution would be in nanometres, as most biological processes occur at this scale. Nanotechnology has made this possible with functionalised nanoparticles that can bind to specific targets and trace processes at the cellular and molecular level. Quantum dots (QDs or semiconductor nanocrystals are luminescent particles that have the potential to be the next generation fluorophores. This paper is an overview of the basics of QDs and their role as fluorescent probes for various biological imaging applications. Their potential clinical applications and the limitations that need to be overcome have also been discussed.

  12. Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm. (United States)

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


    Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Ns-scaled time-gated fluorescence lifetime imaging for forensic document examination (United States)

    Zhong, Xin; Wang, Xinwei; Zhou, Yan


    A method of ns-scaled time-gated fluorescence lifetime imaging (TFLI) is proposed to distinguish different fluorescent substances in forensic document examination. Compared with Video Spectral Comparator (VSC) which can examine fluorescence intensity images only, TFLI can detect questioned documents like falsification or alteration. TFLI system can enhance weak signal by accumulation method. The two fluorescence intensity images of the interval delay time tg are acquired by ICCD and fitted into fluorescence lifetime image. The lifetimes of fluorescence substances are represented by different colors, which make it easy to detect the fluorescent substances and the sequence of handwritings. It proves that TFLI is a powerful tool for forensic document examination. Furthermore, the advantages of TFLI system are ns-scaled precision preservation and powerful capture capability.

  14. Hyperspectral fluorescence imaging coupled with multivariate image analysis techniques for contaminant screening of leafy greens (United States)

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


    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.

  15. Single molecule localization imaging of telomeres and centromeres using fluorescence in situ hybridization and semiconductor quantum dots. (United States)

    Wang, Le; Zong, Shenfei; Wang, Zhuyuan; Lu, Ju; Chen, Chen; Zhang, Ruohu; Cui, Yiping


    Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores' blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.

  16. Intraoperative near-infrared fluorescent imaging during robotic operations. (United States)

    Macedo, Antonio Luiz de Vasconcellos; Schraibman, Vladimir


    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

  17. Selective and sensitive fluorescent sensor for Pd2+ using coumarin 460 for real-time and biological applications. (United States)

    Ashwin, Bosco Christin Maria Arputham; Sivaraman, Gandhi; Stalin, Thambusamy; Yuvakkumar, Rathinam; Muthu Mareeswaran, Paulpandian


    The efficient fluorescent property of coumarin 460 (C460) is utilized to sense the Pd 2+ selectively and sensitively. Fabrication of a sensor strip using commercial adhesive tape is achieved and the detection of Pd 2+ is attempted using a handy UV torch. The naked eye detection in solution state using UV chamber is also attempted. The calculated high binding constant values support the strong stable complex formation of Pd 2+ with C460. The detection limit up to 2.5 × 10 -7  M is achieved using fluorescence spectrometer, which is considerably low from the WHO's recommendation. The response of coumarin 460 with various cations also studied. The quenching is further studied by the lifetime measurements. The binding mechanism is clearly explained by the 1 H NMR titration. The sensing mechanism is established as ICT. C460 strip's Pd 2+ quenching detection is further confirmed by solid-state PL study. The in-vitro response of Pd 2+ in a living cell is also studied using fluorescent imaging studies by means of HeLa cell lines and this probe is very compatible with biological environments. It could be applicable to sense trace amounts of a Pd 2+ ion from various industries. Compared with previous reports, this one is very cheap, sensitive, selective and suitable for biological systems. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Comparison of segmentation algorithms for fluorescence microscopy images of cells. (United States)

    Dima, Alden A; Elliott, John T; Filliben, James J; Halter, Michael; Peskin, Adele; Bernal, Javier; Kociolek, Marcin; Brady, Mary C; Tang, Hai C; Plant, Anne L


    The analysis of fluorescence microscopy of cells often requires the determination of cell edges. This is typically done using segmentation techniques that separate the cell objects in an image from the surrounding background. This study compares segmentation results from nine different segmentation techniques applied to two different cell lines and five different sets of imaging conditions. Significant variability in the results of segmentation was observed that was due solely to differences in imaging conditions or applications of different algorithms. We quantified and compared the results with a novel bivariate similarity index metric that evaluates the degree of underestimating or overestimating a cell object. The results show that commonly used threshold-based segmentation techniques are less accurate than k-means clustering with multiple clusters. Segmentation accuracy varies with imaging conditions that determine the sharpness of cell edges and with geometric features of a cell. Based on this observation, we propose a method that quantifies cell edge character to provide an estimate of how accurately an algorithm will perform. The results of this study will assist the development of criteria for evaluating interlaboratory comparability. Published 2011 Wiley-Liss, Inc.

  19. Fluorescence Imaging of the Cytoskeleton in Plant Roots. (United States)

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


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

  20. Strategies of molecular imprinting-based fluorescence sensors for chemical and biological analysis. (United States)

    Yang, Qian; Li, Jinhua; Wang, Xiaoyan; Peng, Hailong; Xiong, Hua; Chen, Lingxin


    One pressing concern today is to construct sensors that can withstand various disturbances for highly selective and sensitive detecting trace analytes in complicated samples. Molecularly imprinted polymers (MIPs) with tailor-made binding sites are preferred to be recognition elements in sensors for effective targets detection, and fluorescence measurement assists in highly sensitive detection and user-friendly control. Accordingly, molecular imprinting-based fluorescence sensors (MI-FL sensors) have attracted great research interest in many fields such as chemical and biological analysis. Herein, we comprehensively review the recent advances in MI-FL sensors construction and applications, giving insights on sensing principles and signal transduction mechanisms, focusing on general construction strategies for intrinsically fluorescent or nonfluorescent analytes and improvement strategies in sensing performance, particularly in sensitivity. Construction strategies are well overviewed, mainly including the traditional indirect methods of competitive binding against pre-bound fluorescent indicators, employment of fluorescent functional monomers and embedding of fluorescence substances, and novel rational designs of hierarchical architecture (core-shell/hollow and mesoporous structures), post-imprinting modification, and ratiometric fluorescence detection. Furthermore, MI-FL sensor based microdevices are discussed, involving micromotors, test strips and microfluidics, which are more portable for rapid point-of-care detection and in-field diagnosing. Finally, the current challenges and future perspectives of MI-FL sensors are proposed. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Water-Soluble Triarylborane Chromophores for One- and Two-Photon Excited Fluorescence Imaging of Mitochondria in Cells. (United States)

    Griesbeck, Stefanie; Zhang, Zuolun; Gutmann, Marcus; Lühmann, Tessa; Edkins, Robert M; Clermont, Guillaume; Lazar, Adina N; Haehnel, Martin; Edkins, Katharina; Eichhorn, Antonius; Blanchard-Desce, Mireille; Meinel, Lorenz; Marder, Todd B


    Three water-soluble tetracationic quadrupolar chromophores comprising two three-coordinate boron π-acceptor groups bridged by thiophene-containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5-(3,5-Me2 C6 H2 )-2,2'-(C4 H2 S)2 -5'-(3,5-Me2 C6 H2 ) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one- and two-photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one- and two-photon-excited fluorescence imaging of mitochondrial function in cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Total reflection X-ray fluorescence with synchrotron radiation applied to biological and environmental samples

    International Nuclear Information System (INIS)

    Simabuco, S.M.; Matsumoto, E.; Jesus, E.F.O.; Lopes, R.T.; Perez, C.; Nascimento Filho, V.F.; Costa, R.S.S.; Tavares do Carmo, M.G.; Saunders, C.


    Full text: The Total Reflection X-ray Fluorescence has been applied for trace elements in water and aqueous solutions, environmental samples and biological materials after sample preparation and to surface analysis of silicon wafers. The present paper shows some results of applications for rainwater, atmospheric particulate material, colostrum and nuclear samples. (author)

  3. Biological Interaction of Molybdenocene Dichloride with Bovine Serum Albumin Using Fluorescence Spectroscopy (United States)

    Domínguez, Moralba; Cortes-Figueroa, Jose´ E.; Meléndez, Enrique


    Bioinorganic topics are ubiquitous in the inorganic chemistry curriculum; however, experiments to enhance understanding of related topics are scarce. In this proposed laboratory, upper undergraduate students assess the biological interaction of molybdenocene dichloride (Cp2MoCl2) with bovine serum albumin (BSA) by fluorescence spectroscopy.…

  4. Accumulative difference image protocol for particle tracking in fluorescence microscopy tested in mouse lymphonodes. (United States)

    Villa, Carlo E; Caccia, Michele; Sironi, Laura; D'Alfonso, Laura; Collini, Maddalena; Rivolta, Ilaria; Miserocchi, Giuseppe; Gorletta, Tatiana; Zanoni, Ivan; Granucci, Francesca; Chirico, Giuseppe


    The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

  5. Accumulative difference image protocol for particle tracking in fluorescence microscopy tested in mouse lymphonodes.

    Directory of Open Access Journals (Sweden)

    Carlo E Villa

    Full Text Available The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

  6. FLEX: an imaging spectrometer for measurement of vegetation fluorescence (United States)

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


    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.

  7. Full Field X-Ray Fluorescence Imaging Using Micro Pore Optics for Planetary Surface Exploration (United States)

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


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

  8. Combined fluorescence and phase contrast imaging at the Advanced Photon Source

    International Nuclear Information System (INIS)

    Hornberger, B.; Feser, M.; Jacobsen, C.; Vogt, S.; Legnini, D.; Paterson, D.; Rehak, P.; DeGeronimo, G.; Palmer, B.M.; Experimental Facilities Division; State Univ. of New York at Stony Brook Univ.; BNL; Univ. of Vermont


    X-ray fluorescence microprobes excel at detecting and quantifying trace metals in biological and environmental science samples, but typically do not detect low Z elements such as carbon and nitrogen. Therefore, it is hard to put the trace metals into context with their natural environment. We are implementing phase contrast capabilities with a segmented detector into several microprobes at the Advanced Photon Source (APS) to address this problem. Qualitative differential phase contrast images from a modified soft x-ray detector already provide very useful information for general users. We are also implementing a quantitative method to recover the absolute phase shift by Fourier filtering detector images. New detectors are under development which are optimized for the signal levels present at the APS. In this paper, we concentrate on fundamental signal to noise considerations comparing absorption and differential phase contrast

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

    International Nuclear Information System (INIS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei


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

  10. Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence. (United States)

    Shrestha, Sebina; Serafino, Michael J; Rico-Jimenez, Jesus; Park, Jesung; Chen, Xi; Zhaorigetu, Siqin; Walton, Brian L; Jo, Javier A; Applegate, Brian E


    Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

  11. Inspection of fecal contamination on strawberries using fluorescence imaging (United States)

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


    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. Aptamer-assembled nanomaterials for fluorescent sensing and imaging (United States)

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


    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.

  13. Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function (United States)

    Moerner, W. E.


    Since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. {62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS {103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature {388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB (Nat. Meth. {5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. {12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS {132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on

  14. 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: [Department of Biomedical Engineering, University of Arkansas, 120 Engineering Hall, Fayetteville, Arkansas 72701 (United States)


    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}.

  15. A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation. (United States)

    Pelet, S; Previte, M J R; Laiho, L H; So, P T C


    Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits. Copyright 2004 Biophysical Society

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

    Directory of Open Access Journals (Sweden)

    Robert K. Henderson


    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.

  17. Microbubble embedded with upconversion nanoparticles as a bimodal contrast agent for fluorescence and ultrasound imaging

    International Nuclear Information System (INIS)

    Jin, Birui; Lin, Min; You, Minli; Xu, Feng; Lu, Tianjian; Zong, Yujin; Wan, Mingxi; Duan, Zhenfeng


    Bimodal imaging offers additional imaging signal thus finds wide spread application in clinical diagnostic imaging. Fluorescence/ultrasound bimodal imaging contrast agent using fluorescent dyes or quantum dots for fluorescence signal has emerged as a promising method, which however requires visible light or UV irradiation resulting in photobleaching, photoblinking, auto-fluorescence and limited tissue penetration depth. To surmount these problems, we developed a novel bimodal contrast agent using layer-by-layer assembly of upconversion nanoparticles onto the surface of microbubbles. The resulting microbubbles with average size of 2 μm provide enhanced ultrasound echo for ultrasound imaging and upconversion emission upon near infrared irradiation for fluorescence imaging. The developed bimodal contrast agent holds great potential to be applied in ultrasound target technique for targeted diseases diagnostics and therapy. (paper)

  18. Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues (United States)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Teixeira Rosa, Ramon Gabriel; Salvio, Ana Gabriela; Pratavieira, Sebastião; Kurachi, Cristina


    Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics.

  19. Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues. (United States)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Teixeira Rosa, Ramon Gabriel; Salvio, Ana Gabriela; Pratavieira, Sebastião; Kurachi, Cristina


    Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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


    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.

  1. Fluorescence Lifetime Imaging in Stargardt Disease: Potential Marker for Disease Progression


    Dysli Chantal; Wolf Sebastian; Hatz Katja; Zinkernagel Martin


    PURPOSE The purpose of this study was to describe autofluorescence lifetime characteristics in Stargardt disease (STGD) using fluorescence lifetime imaging ophthalmoscopy (FLIO) and to investigate potential prognostic markers for disease activity and progression. METHODS Fluorescence lifetime data of 16 patients with STGD (mean age, 40 years; range, 22-56 years) and 15 age-matched controls were acquired using a fluorescence lifetime imaging ophthalmoscope based on a Heidelberg Eng...

  2. Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging. (United States)

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul


    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. 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


    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....

  4. Detection of fecal residue on poultry carcasses by laser induced fluorescence imaging techniques (United States)

    The potential use of laser-induced fluorescence imaging techniques was investigated for the detection of diluted fecal matters from various parts of the digestive tract, including colon, ceca, small intestine, and duodenum, on poultry carcasses. One of the challenges for using fluorescence imaging f...

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

    NARCIS (Netherlands)

    Uzunbajakava, N.; Otto, Cornelis


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

  6. Fluorescence imaging in the upper gastrointestinal tract for the detection of dysplasic changes (United States)

    Sukowski, Uwe; Ebert, Bernd; Ortner, Marianne; Mueller, Karsten; Voderholzer, W.; Weber-Eibel, J.; Dietel, M.; Lochs, Herbert; Rinneberg, Herbert H.


    During endoscopy of the esophagus fluorescence images were recorded at a delay of 20 ns after pulsed laser excitation simultaneously with conventional reflected white light images. To label malignant cells (dysplasia, tumor) 5-aminolaevulinic acid was applied prior to fluorescence guided bi-opsy. In this way pre-malignant and malignant lesions were detected not seen previously during routine endoscopy.

  7. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue. (United States)

    Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf


    High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

  8. A novel colorimetric and off–on fluorescent sensor for Hg{sup 2+} and its application in live cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Li, Man [Phosphorus Chemical Engineering Research Center of Henan Province, The College of Chemistry and Molecular Engineering, Zhengzhou University, Daxue Road 75, Zhengzhou 450052, Henan (China); Jiang, Yuhua [School of Pharmaceutical Science, Zhengzhou University, Zhengzhou (China); Zhang, Di; Ding, Peigang [Phosphorus Chemical Engineering Research Center of Henan Province, The College of Chemistry and Molecular Engineering, Zhengzhou University, Daxue Road 75, Zhengzhou 450052, Henan (China); Wang, Zhenji [School of Pharmaceutical Science, Zhengzhou University, Zhengzhou (China); Ye, Yong, E-mail: [Phosphorus Chemical Engineering Research Center of Henan Province, The College of Chemistry and Molecular Engineering, Zhengzhou University, Daxue Road 75, Zhengzhou 450052, Henan (China); Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing (China); Zhao, Yufen [Phosphorus Chemical Engineering Research Center of Henan Province, The College of Chemistry and Molecular Engineering, Zhengzhou University, Daxue Road 75, Zhengzhou 450052, Henan (China); Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing (China)


    A novel rhodamine-based fluorescent probe L2 with two “S” groups was synthesized and characterized. As expected, L2 exhibited high selectivity and sensitivity for Hg{sup 2+} over other commonly coexistent metal ions. Its selectivity is excellent, and the detection limit was measured to be 1 ppm. The significant changes in the fluorescence color could be used for naked-eye detection. Furthermore, fluorescence imaging experiments of Hg{sup 2+} ions in living EC-109 cells demonstrated its value of practical applications in biological systems. -- Highlights: • A new rhodamine-based chemodosimeter (L2) was synthesized and characterized. • L2 exhibited high selectivity and excellent sensitivity detection of Hg{sup 2+}. • The significant changes in color could be used for naked-eye detection. • The fluorescence imaging experiments of Hg{sup 2+} ions in living EC-109 cells demonstrated its value of practical applications in biological systems.

  9. Segmentation of fluorescence microscopy cell images using unsupervised mining. (United States)

    Du, Xian; Dua, Sumeet


    The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

  10. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source (United States)

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


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

  11. A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time

    Directory of Open Access Journals (Sweden)

    Michael J. McFadden


    Full Text Available Zika virus (ZIKV is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3 to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

  12. An image analysis system for near-infrared (NIR) fluorescence lymph imaging (United States)

    Zhang, Jingdan; Zhou, Shaohua Kevin; Xiang, Xiaoyan; Rasmussen, John C.; Sevick-Muraca, Eva M.


    Quantitative analysis of lymphatic function is crucial for understanding the lymphatic system and diagnosing the associated diseases. Recently, a near-infrared (NIR) fluorescence imaging system is developed for real-time imaging lymphatic propulsion by intradermal injection of microdose of a NIR fluorophore distal to the lymphatics of interest. However, the previous analysis software3, 4 is underdeveloped, requiring extensive time and effort to analyze a NIR image sequence. In this paper, we develop a number of image processing techniques to automate the data analysis workflow, including an object tracking algorithm to stabilize the subject and remove the motion artifacts, an image representation named flow map to characterize lymphatic flow more reliably, and an automatic algorithm to compute lymph velocity and frequency of propulsion. By integrating all these techniques to a system, the analysis workflow significantly reduces the amount of required user interaction and improves the reliability of the measurement.

  13. Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe. (United States)

    Jin, Qiang; Feng, Lei; Zhang, Shui-Jun; Wang, Dan-Dan; Wang, Fang-Jun; Zhang, Yi; Cui, Jing-Nan; Guo, Wen-Zhi; Ge, Guang-Bo; Yang, Ling


    In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.

  14. Fluorescence guided lymph node biopsy in large animals using direct image projection device (United States)

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


    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.

  15. Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging (United States)

    Sinefeld, David; Paudel, Hari P.; Wang, Tianyu; Wang, Mengran; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris


    Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.

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

    Directory of Open Access Journals (Sweden)

    Suresh K. Pandey


    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

  17. Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor (United States)

    Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming


    Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

  18. Radionuclide Imaging Technologies for Biological Systems

    Energy Technology Data Exchange (ETDEWEB)

    Howell, Calvin R. [Duke Univ., Durham, NC (United States); Reid, Chantal D. [Duke Univ., Durham, NC (United States); Weisenberger, Andrew G. [Thomas Jefferson National Accelerator Facility (TJNAF), Newport News, VA (United States)


    The main objective of this project is to develop technologies and experimental techniques for studying the dynamics of physiological responses of plants to changes in their interface with the local environment and to educate a new generation of scientists in an interdisciplinary environment of biology, physics and engineering. Also an important goal is to perform measurements to demonstrate the new data that can be produced and made available to the plant-biology community using the imaging technologies and experimental techniques developed in this project. The study of the plant-environment interface includes a wide range of topics in plant physiology, e.g., the root-soil interface, resource availability, impact of herbivores, influence of microbes on root surface, and responses to toxins in the air and soil. The initial scientific motivation for our work is to improve understanding of the mechanisms for physiological responses to abrupt changes in the local environment, in particular, the responses that result in short-term adjustments in resource (e.g., sugars, nutrients and water) allocations. Data of time-dependent responses of plants to environmental changes are essential in developing mechanistic models for substance intake and resource allocation. Our approach is to use radioisotope tracing techniques to study whole-plant and plant organ (e.g., leaves, stems, roots) dynamical responses to abrupt changes in environmental conditions such as concentration of CO2 in the atmosphere, nutrient availability and lighting. To this aim we are collaborating with the Radiation Detector and Imaging Group at the Thomas Jefferson National Laboratory Facility (JLab) to develop gamma-ray and beta particle imaging systems optimized for plant studies. The radioisotope tracing measurements are conducted at the Phytotron facility at Duke University. The Phytotron is a controlled environment plant research facility with a variety of plant growth chambers. One chamber

  19. Microscopic time-resolved imaging of singlet oxygen by delayed fluorescence in living cells. (United States)

    Scholz, Marek; Dědic, Roman; Hála, Jan


    Singlet oxygen is a highly reactive species which is involved in a number of processes, including photodynamic therapy of cancer. Its very weak near-infrared emission makes imaging of singlet oxygen in biological systems a long-term challenge. We address this challenge by introducing Singlet Oxygen Feedback Delayed Fluorescence (SOFDF) as a novel modality for semi-direct microscopic time-resolved wide-field imaging of singlet oxygen in biological systems. SOFDF has been investigated in individual fibroblast cells incubated with a well-known photosensitizer aluminium phthalocyanine tetrasulfonate. The SOFDF emission from the cells is several orders of magnitude stronger and much more readily detectable than the very weak near-infrared phosphorescence of singlet oxygen. Moreover, the analysis of SOFDF kinetics enables us to estimate the lifetimes of the involved excited states. Real-time SOFDF images with micrometer spatial resolution and submicrosecond temporal-resolution have been recorded. Interestingly, a steep decrease in the SOFDF intensity after the photodynamically induced release of a photosensitizer from lysosomes has been demonstrated. This effect could be potentially employed as a valuable diagnostic tool for monitoring and dosimetry in photodynamic therapy.

  20. Focused fluorescence excitation with time-reversed ultrasonically encoded light and imaging in thick scattering media

    International Nuclear Information System (INIS)

    Lai, Puxiang; Suzuki, Yuta; Xu, Xiao; Wang, Lihong V


    Scattering dominates light propagation in biological tissue, and therefore restricts both resolution and penetration depth in optical imaging within thick tissue. As photons travel into the diffusive regime, typically 1 mm beneath human skin, their trajectories transition from ballistic to diffusive due to the increased number of scattering events, which makes it impossible to focus, much less track, photon paths. Consequently, imaging methods that rely on controlled light illumination are ineffective in deep tissue. This problem has recently been addressed by a novel method capable of dynamically focusing light in thick scattering media via time reversal of ultrasonically encoded (TRUE) diffused light. Here, using photorefractive materials as phase conjugate mirrors, we show a direct visualization and dynamic control of optical focusing with this light delivery method, and demonstrate its application for focused fluorescence excitation and imaging in thick turbid media. These abilities are increasingly critical for understanding the dynamic interactions of light with biological matter and processes at different system levels, as well as their applications for biomedical diagnosis and therapy. (letter)

  1. Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review

    International Nuclear Information System (INIS)

    Sednev, Maksim V; Belov, Vladimir N; Hell, Stefan W


    The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe’s diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20–40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH. (topical review)

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

    Directory of Open Access Journals (Sweden)



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

  3. Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy. (United States)

    Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike


    An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

  4. Cryo-imaging of fluorescently labeled single cells in a mouse (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.


    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  5. Fluorescent screens and image processing for the APS linac test stand

    International Nuclear Information System (INIS)

    Berg, W.; Ko, K.


    A fluorescent screen was used to monitor relative beam position and spot size of a 56-MeV electron beam in the linac test stand. A chromium doped alumina ceramic screen inserted into the beam was monitored by a video camera. The resulting image was captured using a frame grabber and stored into memory. Reconstruction and analysis of the stored image was performed using PV-WAVE. This paper will discuss the hardware and software implementation of the fluorescent screen and imaging system. Proposed improvements for the APS linac fluorescent screens and image

  6. Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles. (United States)

    Schwartz, Shmulik; Fixler, Dror; Popovtzer, Rachela; Shefi, Orit


    Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. A novel peptide-based fluorescence chemosensor for selective imaging of hydrogen sulfide both in living cells and zebrafish. (United States)

    Wang, Peng; Wu, Jiang; Di, Cuixia; Zhou, Rong; Zhang, Hong; Su, Pingru; Xu, Cong; Zhou, Panpan; Ge, Yushu; Liu, Dan; Liu, Weisheng; Tang, Yu


    Hydrogen sulfide (H 2 S) plays an important role as a signaling compound (gasotransmitter) in living systems. However, the development of an efficient imaging chemosensor of H 2 S in live animals is a challenging field for chemists. Herein, a novel peptide-based fluorescence chemosensor L-Cu was designed and synthesized on the basis of the copper chelating with the peptide ligand (FITC-Ahx-Ser-Pro-Gly-His-NH 2 , L), and its H 2 S sensing ability has been evaluated both in living cells and zebrafish. The peptide backbone and Cu 2+ -removal sensing mechanism are used to deliver rapid response time, high sensitivity, and good biocompatibility. After a fast fluorescence quench by Cu 2+ coordinated with L, the fluorescence of L is recovered by adding S 2- to form insoluble copper sulfide in aqueous solution with a detection limit for hydrogen sulfide measured to be 31nM. Furthermore, the fluorescence chemosensor L-Cu showed excellent cell permeation and low biotoxicity to realize the intracellular biosensing, L-Cu has also been applied to image hydrogen sulfide in live zebrafish larvae. We expect that this peptide-based fluorescence chemosensor L-Cu can be used to study H 2 S-related chemical biology in physiological and pathological events. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Necemer, Marijan; Kump, Peter; Scancar, Janez; Jacimovic, Radojko; Simcic, Jurij; Pelicon, Primoz; Budnar, Milos; Jeran, Zvonka; Pongrac, Paula; Regvar, Marjana; Vogel-Mikus, Katarina


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

  9. A review of imaging techniques for systems biology

    Directory of Open Access Journals (Sweden)

    Po Ming J


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

  10. A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems (United States)

    Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.


    Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology. PMID:25252596

  11. Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system. (United States)

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


    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.

  12. Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules


    Elliott, Jonathan T.; Dsouza, Alisha V.; Marra, Kayla; Pogue, Brian W.; Roberts, David W.; Paulsen, Keith D.


    Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system spe...

  13. 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


    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.

  14. Visualization of subcapsular hepatic malignancy by indocyanine-green fluorescence imaging during laparoscopic hepatectomy. (United States)

    Kudo, Hiroki; Ishizawa, Takeaki; Tani, Keigo; Harada, Nobuhiro; Ichida, Akihiko; Shimizu, Atsushi; Kaneko, Junichi; Aoki, Taku; Sakamoto, Yoshihiro; Sugawara, Yasuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro


    Although laparoscopic hepatectomy has increasingly been used to treat cancers in the liver, the accuracy of intraoperative diagnosis may be inferior to that of open surgery because the ability to visualize and palpate the liver surface during laparoscopy is relatively limited. Fluorescence imaging has the potential to provide a simple compensatory diagnostic tool for identification of cancers in the liver during laparoscopic hepatectomy. In 17 patients who were to undergo laparoscopic hepatectomy, 0.5 mg/kg body weight of indocyanine green (ICG) was administered intravenously within the 2 weeks prior to surgery. Intraoperatively, a laparoscopic fluorescence imaging system obtained fluorescence images of its surfaces during mobilization of the liver. In all, 16 hepatocellular carcinomas (HCCs) and 16 liver metastases (LMs) were resected. Of these, laparoscopic ICG fluorescence imaging identified 12 HCCs (75%) and 11 LMs (69%) on the liver surfaces distributed over Couinaud's segments 1-8, including the 17 tumors that had not been identified by visual inspections of normal color images. The 23 tumors that were identified by fluorescence imaging were located closer to the liver surfaces than another nine tumors that were not identified by fluorescence imaging (median [range] depth 1 [0-5] vs. 11 [8-30] mm; p fluorescence imaging enables real-time identification of subcapsular liver cancers, thus facilitating estimation of the required extent of hepatic mobilization and determination of the location of an appropriate hepatic transection line.

  15. Fully time-resolved near-field scanning optical microscopy fluorescence imaging

    International Nuclear Information System (INIS)

    Kwak, Eun-Soo; Vanden Bout, David A.


    Time-correlated single photon counting has been coupled with near-field scanning optical microscopy (NSOM) to record complete fluorescence lifetime decays at each pixel in an NSOM image. The resulting three-dimensional data sets can be binned in the time dimension to create images of photons at particular time delays or images of the fluorescence lifetime. Alternatively, regions of interest identified in the topography and fluorescence images can be used to bin the data in the spatial dimensions resulting in high signal to noise fluorescence decays of particular regions of the sample. The technique has been demonstrated on films of poly(vinylalcohol), doped with the fluorescent dye, cascade blue (CB). The CB segregates into small circular regions of high concentration within the films during the drying process. The lifetime imaging shows that the spots have slightly faster excited state decays due to quenching of the luminescence as a result of the higher concentration. The technique is also used to image the fluorescence lifetime of an annealed film of poly(dihexylfluorene). The samples show high contrast in the total intensity fluorescence image, but the lifetime image reveals the sample to be extremely uniform

  16. Whole-slide imaging is a robust alternative to traditional fluorescent microscopy for fluorescence in situ hybridization imaging using break-apart DNA probes. (United States)

    Laurent, Camille; Guérin, Maxime; Frenois, François-Xavier; Thuries, Valérie; Jalabert, Laurence; Brousset, Pierre; Valmary-Degano, Séverine


    Fluorescence in situ hybridization is an indispensable technique used in routine pathology and for theranostic purposes. Because fluorescence in situ hybridization techniques require sophisticated microscopic workstations and long procedures of image acquisition with sometimes subjective and poorly reproducible results, we decided to test a whole-slide imaging system as an alternative approach. In this study, we used the latest generation of Pannoramic 250 Flash digital microscopes (P250 Flash digital microscopes; 3DHISTECH, Budapest, Hungary) to digitize fluorescence in situ hybridization slides of diffuse large B cells lymphoma cases for detecting MYC rearrangement. The P250 Flash digital microscope was found to be precise with better definition of split signals in cells containing MYC rearrangement with fewer truncated signals as compared to traditional fluorescence microscopy. This digital technique is easier thanks to the preview function, which allows almost immediate identification of the tumor area, and the panning and zooming functionalities as well as a shorter acquisition time. Moreover, fluorescence in situ hybridization analyses using the digital technique appeared to be more reproducible between pathologists. Finally, the digital technique also allowed prolonged conservation of photos. In conclusion, whole-slide imaging technologies represent rapid, robust, and highly sensitive methods for interpreting fluorescence in situ hybridization slides with break-apart probes. In addition, these techniques offer an easier way to interpret the signals and allow definitive storage of the images for pathology expert networks or e-learning databases. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. A support vector machine approach to the automatic identification of fluorescence spectra emitted by biological agents (United States)

    Gelfusa, M.; Murari, A.; Lungaroni, M.; Malizia, A.; Parracino, S.; Peluso, E.; Cenciarelli, O.; Carestia, M.; Pizzoferrato, R.; Vega, J.; Gaudio, P.


    Two of the major new concerns of modern societies are biosecurity and biosafety. Several biological agents (BAs) such as toxins, bacteria, viruses, fungi and parasites are able to cause damage to living systems either humans, animals or plants. Optical techniques, in particular LIght Detection And Ranging (LIDAR), based on the transmission of laser pulses and analysis of the return signals, can be successfully applied to monitoring the release of biological agents into the atmosphere. It is well known that most of biological agents tend to emit specific fluorescence spectra, which in principle allow their detection and identification, if excited by light of the appropriate wavelength. For these reasons, the detection of the UVLight Induced Fluorescence (UV-LIF) emitted by BAs is particularly promising. On the other hand, the stand-off detection of BAs poses a series of challenging issues; one of the most severe is the automatic discrimination between various agents which emit very similar fluorescence spectra. In this paper, a new data analysis method, based on a combination of advanced filtering techniques and Support Vector Machines, is described. The proposed approach covers all the aspects of the data analysis process, from filtering and denoising to automatic recognition of the agents. A systematic series of numerical tests has been performed to assess the potential and limits of the proposed methodology. The first investigations of experimental data have already given very encouraging results.

  18. Spirally-patterned pinhole arrays for long-term fluorescence cell imaging. (United States)

    Koo, Bon Ung; Kang, YooNa; Moon, SangJun; Lee, Won Gu


    Fluorescence cell imaging using a fluorescence microscope is an extensively used technique to examine the cell nucleus, internal structures, and other cellular molecules with fluorescence response time and intensity. However, it is difficult to perform high resolution cell imaging for a long period of time with this technique due to necrosis and apoptosis depending on the type and subcellular location of the damage caused by phototoxicity. A large number of studies have been performed to resolve this problem, but researchers have struggled to meet the challenge between cellular viability and image resolution. In this study, we employ a specially designed disc to reduce cell damage by controlling total fluorescence exposure time without deterioration of the image resolution. This approach has many advantages such as, the apparatus is simple, cost-effective, and easily integrated into the optical pathway through a conventional fluorescence microscope.

  19. Smart optical probes for near-infrared fluorescence imaging of Alzheimer's disease pathology

    International Nuclear Information System (INIS)

    Raymond, Scott B.; Bacskai, Brian J.; Skoch, Jesse; Hills, Ivory D.; Swager, Timothy M.; Nesterov, Evgueni E.


    Near-infrared fluorescent probes for amyloid-beta (Aβ) are an exciting option for molecular imaging in Alzheimer's disease research and may translate to clinical diagnostics. However, Aβ-targeted optical probes often suffer from poor specificity and slow clearance from the brain. We are designing smart optical probes that emit characteristic fluorescence signal only when bound to Aβ. We synthesized a family of dyes and tested Aβ-binding sensitivity with fluorescence spectroscopy and tissue-staining. Select compounds exhibited Aβ-dependent changes in fluorescence quantum yield, lifetime, and emission spectra that may be imaged microscopically or in vivo using new lifetime and spectral fluorescence imaging techniques. Smart optical probes that turn on when bound to Aβ will improve amyloid detection and may enable quantitative molecular imaging in vivo. (orig.)

  20. Mechanistic background and clinical applications of indocyanine green fluorescence imaging of hepatocellular carcinoma. (United States)

    Ishizawa, Takeaki; Masuda, Koichi; Urano, Yasuteru; Kawaguchi, Yoshikuni; Satou, Shouichi; Kaneko, Junichi; Hasegawa, Kiyoshi; Shibahara, Junji; Fukayama, Masashi; Tsuji, Shingo; Midorikawa, Yutaka; Aburatani, Hiroyuki; Kokudo, Norihiro


    Although clinical applications of intraoperative fluorescence imaging of liver cancer using indocyanine green (ICG) have begun, the mechanistic background of ICG accumulation in the cancerous tissues remains unclear. In 170 patients with hepatocellular carcinoma cells (HCC), the liver surfaces and resected specimens were intraoperatively examined by using a near-infrared fluorescence imaging system after preoperative administration of ICG (0.5 mg/kg i.v.). Microscopic examinations, gene expression profile analysis, and immunohistochemical staining were performed for HCCs, which showed ICG fluorescence in the cancerous tissues (cancerous-type fluorescence), and HCCs showed fluorescence only in the surrounding non-cancerous liver parenchyma (rim-type fluorescence). ICG fluorescence imaging enabled identification of 273 of 276 (99%) HCCs in the resected specimens. HCCs showed that cancerous-type fluorescence was associated with higher cancer cell differentiation as compared with rim-type HCCs (P Fluorescence microscopy identified the presence of ICG in the canalicular side of the cancer cell cytoplasm, and pseudoglands of the HCCs showed a cancerous-type fluorescence pattern. The ratio of the gene and protein expression levels in the cancerous to non-cancerous tissues for Na(+)/taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide 8 (OATP8), which are associated with portal uptake of ICG by hepatocytes that tended to be higher in the HCCs that showed cancerous-type fluorescence than in those that showed rim-type fluorescence. Preserved portal uptake of ICG in differentiated HCC cells by NTCP and OATP8 with concomitant biliary excretion disorders causes accumulation of ICG in the cancerous tissues after preoperative intravenous administration. This enables highly sensitive identification of HCC by intraoperative ICG fluorescence imaging.

  1. An image fiber based fluorescent probe with associated signal processing scheme for biomedical diagnostics

    International Nuclear Information System (INIS)

    Vaishakh, M; Murukeshan, V M; Seah, L K


    A dual-modality image fiber based fluorescent probe that can be used for depth sensitive imaging and suppression of fluorescent emissions with nanosecond lifetime difference is proposed and illustrated in this paper. The system can give high optical sectioning and employs an algorithm for obtaining phase sensitive images. The system can find main application in in vivo biomedical diagnostics for detecting biochemical changes for distinguishing malignant tissue from healthy tissue

  2. 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


    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.

  3. Multiplexed phase-space imaging for 3D fluorescence microscopy. (United States)

    Liu, Hsiou-Yuan; Zhong, Jingshan; Waller, Laura


    Optical phase-space functions describe spatial and angular information simultaneously; examples of optical phase-space functions include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order to sequentially pattern multiplexed coded apertures while capturing images in real space. Then, we reconstruct the 3D fluorescence distribution of our sample by solving an inverse problem via regularized least squares with a proximal accelerated gradient descent solver. We experimentally reconstruct a 101 Megavoxel 3D volume (1010×510×500µm with NA 0.4), demonstrating improved acquisition time, light throughput and resolution compared to scanning aperture methods. Our flexible patterning scheme further allows sparsity in the sample to be exploited for reduced data capture.

  4. Conditional-sampling spectrograph detection system for fluorescence measurements of individual airborne biological particles (United States)

    Nachman, Paul; Pinnick, R. G.; Hill, Steven C.; Chen, Gang; Chang, Richard K.; Mayo, Michael W.; Fernandez, Gilbert L.


    We report the design and operation of a prototype conditional-sampling spectrograph detection system that can record the fluorescence spectra of individual, micrometer-sized aerosols as they traverse an intense 488-nm intracavity laser beam. The instrument's image-intensified CCD detector is gated by elastic scattering or by undispersed fluorescence from particles that enter the spectrograph's field of view. It records spectra only from particles with preselected scattering-fluorescence levels (a fiber-optic-photomultiplier subsystem provides the gating signal). This conditional-sampling procedure reduces data-handling rates and increases the signal-to-noise ratio by restricting the system's exposures to brief periods when aerosols traverse the beam. We demonstrate these advantages by reliably capturing spectra from individual fluorescent microspheres dispersed in an airstream. The conditional-sampling procedure also permits some discrimination among different types of particles, so that spectra may be recorded from the few interesting particles present in a cloud of background aerosol. We demonstrate such discrimination by measuring spectra from selected fluorescent microspheres in a mixture of two types of microspheres, and from bacterial spores in a mixture of spores and nonfluorescent kaolin particles.

  5. X-ray fluorescence method for trace analysis and imaging

    International Nuclear Information System (INIS)

    Hayakawa, Shinjiro


    X-ray fluorescence analysis has a long history as conventional bulk elemental analysis with medium sensitivity. However, with the use of synchrotron radiation x-ray fluorescence method has become a unique analytical technique which can provide tace elemental information with the spatial resolution. To obtain quantitative information of trace elemental distribution by using the x-ray fluorescence method, theoretical description of x-ray fluorescence yield is described. Moreover, methods and instruments for trace characterization with a scanning x-ray microprobe are described. (author)

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

    Directory of Open Access Journals (Sweden)

    Alain Berinstain


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

  7. Deployment of a Fully-Automated Green Fluorescent Protein Imaging System in a High Arctic Autonomous Greenhouse (United States)

    Abboud, Talal; Bamsey, Matthew; Paul, Anna-Lisa; Graham, Thomas; Braham, Stephen; Noumeir, Rita; Berinstain, Alain; Ferl, Robert


    Higher plants are an integral part of strategies for sustained human presence in space. Space-based greenhouses have the potential to provide closed-loop recycling of oxygen, water and food. Plant monitoring systems with the capacity to remotely observe the condition of crops in real-time within these systems would permit operators to take immediate action to ensure optimum system yield and reliability. One such plant health monitoring technique involves the use of reporter genes driving fluorescent proteins as biological sensors of plant stress. In 2006 an initial prototype green fluorescent protein imager system was deployed at the Arthur Clarke Mars Greenhouse located in the Canadian High Arctic. This prototype demonstrated the advantageous of this biosensor technology and underscored the challenges in collecting and managing telemetric data from exigent environments. We present here the design and deployment of a second prototype imaging system deployed within and connected to the infrastructure of the Arthur Clarke Mars Greenhouse. This is the first imager to run autonomously for one year in the un-crewed greenhouse with command and control conducted through the greenhouse satellite control system. Images were saved locally in high resolution and sent telemetrically in low resolution. Imager hardware is described, including the custom designed LED growth light and fluorescent excitation light boards, filters, data acquisition and control system, and basic sensing and environmental control. Several critical lessons learned related to the hardware of small plant growth payloads are also elaborated. PMID:23486220

  8. Comparison of iodine K-edge subtraction and fluorescence subtraction imaging in an animal system

    International Nuclear Information System (INIS)

    Zhang, H.; Zhu, Y.; Bewer, B.; Zhang, L.; Korbas, M.; Pickering, I.J.; George, G.N.; Gupta, M.; Chapman, D.


    K-Edge Subtraction (KES) utilizes the discontinuity in the X-ray absorption across the absorption edge of the selected contrast element and creates an image of the projected density of the contrast element from two images acquired just above and below the K-edge of the contrast element. KES has proved to be powerful in coronary angiography, micro-angiography, bronchography, and lymphatic imaging. X-ray fluorescence imaging is a successful technique for the detection of dilute quantities of elements in specimens. However, its application at high X-ray energies (e.g. at the iodine K-edge) is complicated by significant Compton background, which may enter the energy window set for the contrast material's fluorescent X-rays. Inspired by KES, Fluorescence Subtraction Imaging (FSI) is a technique for high-energy (>20 keV) fluorescence imaging using two different incident beam energies just above and below the absorption edge of a contrast element (e.g. iodine). The below-edge image can be assumed as a 'background' image, which includes Compton scatter and fluorescence from other elements. The above-edge image will contain nearly identical spectral content as the below-edge image but will contain the additional fluorescence of the contrast element. This imaging method is especially promising with thick objects with dilute contrast materials, significant Compton background, and/or competing fluorescence lines from other materials. A quality factor is developed to facilitate the comparison. The theoretical value of the quality factor sets the upper limit that an imaging method can achieve when the noise is Poisson limited. The measured value of this factor makes two or more imaging methods comparable. Using the Hard X-ray Micro-Analysis (HXMA) beamline at the Canadian Light Source (CLS), the techniques of FSI and KES were critically compared, with reference to radiation dose, image acquisition time, resolution, signal-to-noise ratios, and quality factor

  9. A Biologically Inspired CMOS Image Sensor

    CERN Document Server

    Sarkar, Mukul


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

  10. Quantitative generalized ratiometric fluorescence spectroscopy for turbid media based on probe encapsulated by biologically localized embedding

    International Nuclear Information System (INIS)

    Yan, Xiu-Fang; Chen, Zeng-Ping; Cui, Yin-Yin; Hu, Yuan-Liang; Yu, Ru-Qin


    PEBBLE (probe encapsulated by biologically localized embedding) nanosensor encapsulating an intensity-based fluorescence indicator and an inert reference fluorescence dye inside the pores of stable matrix can be used as a generalized wavelength-ratiometric probe. However, the lack of an efficient quantitative model render the choices of inert reference dyes and intensity-based fluorescence indicators used in PEBBLEs based generalized wavelength-ratiometric probes rather limited. In this contribution, an extended quantitative fluorescence model was derived specifically for generalized wavelength-ratiometric probes based on PEBBLE technique (QFM GRP ) with a view to simplify the design of PEBBLEs and hence further extend their application potentials. The effectiveness of QFM GRP has been tested on the quantitative determination of free Ca 2+ in both simulated and real turbid media using a Ca 2+ sensitive PEBBLE nanosensor encapsulating Rhod-2 and eosin B inside the micropores of stable polyacrylamide matrix. Experimental results demonstrated that QFM GRP could realize precise and accurate quantification of free Ca 2+ in turbid samples, even though there is serious overlapping between the fluorescence excitation peaks of eosin B and Ca 2+ bound Rhod-2. The average relative predictive error value of QFM GRP for the test simulated turbid samples was 5.9%, about 2–4 times lower than the corresponding values of partial least squares calibration model and the empirical ratiometric model based on the ratio of fluorescence intensities at the excitation peaks of Ca 2+ bound Rhod-2 and eosin B. The recovery rates of QFM GRP for the real and spiked turbid samples varied from 93.1% to 101%, comparable to the corresponding results of atomic absorption spectrometry. - Highlights: • An advanced model was derived for generalized wavelength-ratiometric PEBBLEs. • The model can simplify the design of generalized wavelength-ratiometric PEBBLEs. • The model realized accurate

  11. Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Michael G Giacomelli

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

  13. Bladder cancer diagnosis with fluorescence-image-guided optical coherence tomography (United States)

    Wang, Z. G.; Durand, D. B.; Adler, H.; Pan, Y. T.


    A fluorescence-image-guided OCT (FIG-OCT) system is described, and its ability to enhance the sensitivity and specificity is examined in an animal bladder cancer model. Total 97 specimens were examined by fluorescence imaging, OCT and histological microscopy. The sensitivity and specificity of FIG-OCT is 100% and 93% respectively, compared to 79% and 53% for fluorescence imaging, while the OCT examination time has been dramatically decreased by 3~4 times. In combination of endoscopic OCT, FIG-OCT is a promising technique for effective early bladder cancer diagnosis.

  14. 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.

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

    International Nuclear Information System (INIS)

    Zang, Zhigang; Tang, Xiaosheng


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

  16. Image navigation as a means to expand the boundaries of fluorescence-guided surgery. (United States)

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


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

  17. Current limitations in super-resolution fluorescence microscopy for biological specimens: How deep can we go from the cover glass? (United States)

    Okada, Yasushi


    Diffraction limit of resolution has been one of the biggest limitations in the optical microscopy. Super-resolution fluorescence microscopy has enabled us to break this limit. However, for the observations of real biological specimens, especially for the imaging of tissues or whole body, the target structures of interest are often embedded deep inside the specimen. Here, we would present our results to extend the target of the super-resolution microscopy deeper into the cells. Confocal microscope optics work effectively to minimize the effect by the aberrations by the cellular components, but at the expense of the signal intensities. Spherical aberrations by the refractive index mismatch between the cellular environment and the immersion liquid can be much larger, but can be reduced by adjusting the correction collar at the objective lens.

  18. 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


    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.

  19. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine. (United States)

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


    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.

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

    International Nuclear Information System (INIS)

    Sung, Chang Kyu; Hong, Kyung Ah; Lin, Shun Mei


    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

  1. Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging. (United States)

    Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya


    We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

  2. Application of fluorescence spectroscopy and imaging in the detection of a photosensitizer in photodynamic therapy (United States)

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


    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.

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

  4. Multispectral fluorescence image algorithms for detection of frass on mature tomatoes (United States)

    A multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet LED excitation was developed for the detection of frass contamination on mature tomatoes. The algorithm utilized the fluorescence intensities at five wavebands, 515 nm, 640 nm, 664 nm, 690 nm, and 724 nm...

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

    Directory of Open Access Journals (Sweden)

    Davidson Michael W


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

  6. In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver (United States)

    Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.


    A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

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

    International Nuclear Information System (INIS)

    Al-Gubory, Kais H.


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

  8. Facilitating in vivo tumor localization by principal component analysis based on dynamic fluorescence molecular imaging (United States)

    Gao, Yang; Chen, Maomao; Wu, Junyu; Zhou, Yuan; Cai, Chuangjian; Wang, Daliang; Luo, Jianwen


    Fluorescence molecular imaging has been used to target tumors in mice with xenograft tumors. However, tumor imaging is largely distorted by the aggregation of fluorescent probes in the liver. A principal component analysis (PCA)-based strategy was applied on the in vivo dynamic fluorescence imaging results of three mice with xenograft tumors to facilitate tumor imaging, with the help of a tumor-specific fluorescent probe. Tumor-relevant features were extracted from the original images by PCA and represented by the principal component (PC) maps. The second principal component (PC2) map represented the tumor-related features, and the first principal component (PC1) map retained the original pharmacokinetic profiles, especially of the liver. The distribution patterns of the PC2 map of the tumor-bearing mice were in good agreement with the actual tumor location. The tumor-to-liver ratio and contrast-to-noise ratio were significantly higher on the PC2 map than on the original images, thus distinguishing the tumor from its nearby fluorescence noise of liver. The results suggest that the PC2 map could serve as a bioimaging marker to facilitate in vivo tumor localization, and dynamic fluorescence molecular imaging with PCA could be a valuable tool for future studies of in vivo tumor metabolism and progression.

  9. Intracellular distribution and stability of a luminescent rhenium(I) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging

    International Nuclear Information System (INIS)

    Wedding, Jason L.; Harris, Hugh H.; Bader, Christie A.; Plush, Sally E.; Mak, Rachel


    Optical fluorescence microscopy was used in conjunction with X-ray fluorescence microscopy to monitor the stability and intracellular distribution of the luminescent rhenium(I) complex fac-[Re(CO) 3 (phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex, in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence techniques. Furthermore, X-ray fluorescence also showed that the Re-I complex disrupted the homeostasis of some biologically relevant elements, such as chlorine, potassium and zinc.

  10. Imaging and the new biology: What's wrong with this picture? (United States)

    Vannier, Michael W.


    The Human Genome has been defined, giving us one part of the equation that stems from the central dogma of molecular biology. Despite this awesome scientific achievement, the correspondence between genomics and imaging is weak, since we cannot predict an organism's phenotype from even perfect knowledge of its genetic complement. Biological knowledge comes in several forms, and the genome is perhaps the best known and most completely understood type. Imaging creates another form of biological information, providing the ability to study morphology, growth and development, metabolic processes, and diseases in vitro and in vivo at many levels of scale. The principal challenge in biomedical imaging for the future lies in the need to reconcile the data provided by one or multiple modalities with other forms of biological knowledge, most importantly the genome, proteome, physiome, and other "-ome's." To date, the imaging science community has not set a high priority on the unification of their results with genomics, proteomics, and physiological functions in most published work. Images are relatively isolated from other forms of biological data, impairing our ability to conceive and address many fundamental questions in research and clinical practice. This presentation will explain the challenge of biological knowledge integration in basic research and clinical applications from the standpoint of imaging and image processing. The impediments to progress, isolation of the imaging community, and mainstream of new and future biological science will be identified, so the critical and immediate need for change can be highlighted.

  11. Improved image alignment method in application to X-ray images and biological images. (United States)

    Wang, Ching-Wei; Chen, Hsiang-Chou


    Alignment of medical images is a vital component of a large number of applications throughout the clinical track of events; not only within clinical diagnostic settings, but prominently so in the area of planning, consummation and evaluation of surgical and radiotherapeutical procedures. However, image registration of medical images is challenging because of variations on data appearance, imaging artifacts and complex data deformation problems. Hence, the aim of this study is to develop a robust image alignment method for medical images. An improved image registration method is proposed, and the method is evaluated with two types of medical data, including biological microscopic tissue images and dental X-ray images and compared with five state-of-the-art image registration techniques. The experimental results show that the presented method consistently performs well on both types of medical images, achieving 88.44 and 88.93% averaged registration accuracies for biological tissue images and X-ray images, respectively, and outperforms the benchmark methods. Based on the Tukey's honestly significant difference test and Fisher's least square difference test tests, the presented method performs significantly better than all existing methods (P ≤ 0.001) for tissue image alignment, and for the X-ray image registration, the proposed method performs significantly better than the two benchmark b-spline approaches (P < 0.001). The software implementation of the presented method and the data used in this study are made publicly available for scientific communities to use (∼cweiwang/ImprovedImageRegistration/).

  12. Liposomal encapsulation of a near-infrared fluorophore enhances fluorescence quenching and reliable whole body optical imaging upon activation in vivo. (United States)

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


    In the past decade, there has been significant progress in the development of water soluble near-infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650-900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto-fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY-676-COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY-676-COOH reveal strong fluorescence quenching. It is demonstrated that the non-targeted PEGylated fluorescence-activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan-induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY-676-COOH, prolonged stability and retention of liposomal-DY-676-COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY-676-COOH-loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. X-ray fluorescence imaging with polycapillary X-ray optics

    International Nuclear Information System (INIS)

    Yonehara, Tasuku; Yamaguchi, Makoto; Tsuji, Kouichi


    X-ray fluorescence spectrometry imaging is a powerful tool to provide information about the chemical composition and elemental distribution of a specimen. X-ray fluorescence spectrometry images were conventionally obtained by using a μ-X-ray fluorescence spectrometry spectrometer, which requires scanning a sample. Faster X-ray fluorescence spectrometry imaging would be achieved by eliminating the process of sample scanning. Thus, we developed an X-ray fluorescence spectrometry imaging instrument without sample scanning by using polycapillary X-ray optics, which had energy filter characteristics caused by the energy dependence of the total reflection phenomenon. In the present paper, we show that two independent straight polycapillary X-ray optics could be used as an energy filter of X-rays for X-ray fluorescence. Only low energy X-rays were detected when the angle between the two optical axes was increased slightly. Energy-selective X-ray fluorescence spectrometry images with projection mode were taken by using an X-ray CCD camera equipped with two polycapillary optics. It was shown that Fe Kα (6.40 keV) and Cu Kα (8.04 keV) could be discriminated for Fe and Cu foils.

  14. Single-atom-resolved fluorescence imaging of an atomic Mott insulator

    DEFF Research Database (Denmark)

    Sherson, Jacob; Weitenberg, Christof; Andres, Manuel


    in situ images of a quantum fluid in which each underlying quantum particle is detected. Here we report fluorescence imaging of strongly interacting bosonic Mott insulators in an optical lattice with single-atom and single-site resolution. From our images, we fully reconstruct the atom distribution...

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

    International Nuclear Information System (INIS)

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


    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

  16. An instrument for small-animal imaging using time-resolved diffuse and fluorescence optical methods

    International Nuclear Information System (INIS)

    Montcel, Bruno; Poulet, Patrick


    We describe time-resolved optical methods that use diffuse near-infrared photons to image the optical properties of tissues and their inner fluorescent probe distribution. The assembled scanner uses picosecond laser diodes at 4 wavelengths, an 8-anode photo-multiplier tube and time-correlated single photon counting. Optical absorption and reduced scattering images as well as fluorescence emission images are computed from temporal profiles of diffuse photons. This method should improve the spatial resolution and the quantification of fluorescence signals. We used the diffusion approximation of the radiation transport equation and the finite element method to solve the forward problem. The inverse problem is solved with an optimization algorithm such as ART or conjugate gradient. The scanner and its performances are presented, together with absorption, scattering and fluorescent images obtained with it


    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...

  18. In Vivo Deep Tissue Fluorescence and Magnetic Imaging Employing Hybrid Nanostructures. (United States)

    Ortgies, Dirk H; de la Cueva, Leonor; Del Rosal, Blanca; Sanz-Rodríguez, Francisco; Fernández, Nuria; Iglesias-de la Cruz, M Carmen; Salas, Gorka; Cabrera, David; Teran, Francisco J; Jaque, Daniel; Martín Rodríguez, Emma


    Breakthroughs in nanotechnology have made it possible to integrate different nanoparticles in one single hybrid nanostructure (HNS), constituting multifunctional nanosized sensors, carriers, and probes with great potential in the life sciences. In addition, such nanostructures could also offer therapeutic capabilities to achieve a wider variety of multifunctionalities. In this work, the encapsulation of both magnetic and infrared emitting nanoparticles into a polymeric matrix leads to a magnetic-fluorescent HNS with multimodal magnetic-fluorescent imaging abilities. The magnetic-fluorescent HNS are capable of simultaneous magnetic resonance imaging and deep tissue infrared fluorescence imaging, overcoming the tissue penetration limits of classical visible-light based optical imaging as reported here in living mice. Additionally, their applicability for magnetic heating in potential hyperthermia treatments is assessed.

  19. Application of indocyanine green-fluorescence imaging to full-thickness cholecystectomy. (United States)

    Morita, Kiyomi; Ishizawa, Takeaki; Tani, Keigo; Harada, Nobuhiro; Shimizu, Atsushi; Yamamoto, Satoshi; Takemura, Nobuyuki; Kaneko, Junichi; Aoki, Taku; Sakamoto, Yoshihiro; Sugawara, Yasuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro


    Fluorescence imaging using indocyanine green (ICG) has recently been applied to laparoscopic surgery to identify cancerous tissues, lymph nodes, and vascular anatomy. Here we report the application of ICG-fluorescence imaging to visualize the boundary between the liver and subserosal tissues of the gallbladder during laparoscopic full-thickness cholecystectomy. A patient with a potentially malignant gallbladder lesion was administered 2.5-mg intravenous ICG just before laparoscopic full-thickness cholecystectomy. Intraoperative fluorescence imaging enabled the real-time delineation of both extrahepatic bile duct anatomy and hepatic parenchyma throughout the procedure, which resulted in complete removal of subserosal tissues between liver and gallbladder. Safe and feasible ICG-fluorescence imaging can be widely applied to laparoscopic hepatobiliary surgery by utilizing a biliary excretion property of ICG. © 2014 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and Wiley Publishing Asia Pty Ltd.


    In this study, hyperspectral fluorescence imaging techniques were investigated for detection of microbial biofilm on stainless steel plates typically used to manufacture food processing equipment. Stainless steel coupons were immersed in bacterium cultures consisting of nonpathogenic E. coli, Pseudo...

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

    Directory of Open Access Journals (Sweden)

    Stefania Seidenari


    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.

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

    Directory of Open Access Journals (Sweden)

    Shilpa Dilipkumar


    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.

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

    International Nuclear Information System (INIS)

    Lue M, Marco P.; Hernandez-Caraballo, Edwin A.


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

  4. Fabrication of fluorescent silica nanoparticles with aggregation-induced emission luminogens for cell imaging. (United States)

    Chen, Sijie; Lam, Jacky W Y; Tang, Ben Zhong


    Fluorescence-based techniques have found wide applications in life science. Among various luminogenic materials, fluorescent nanoparticles have attracted much attention due to their fabulous emission properties and potential applications as sensors. Here, we describe the fabrication of fluorescent silica nanoparticles (FSNPs) containing aggregation-induced emission (AIE) luminogens. By employing surfactant-free sol-gel reaction, FSNPs with uniform size and high surface charge and colloidal stability are generated. The FSNPs emit strong light upon photoexcitation, due to the AIE characteristic of the silole -aggregates in the hybrid nanoparticles. The FSNPs are cytocompatible and can be utilized as fluorescent visualizer for intracellular imaging for HeLa cells.

  5. Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes. (United States)

    Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus


    Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  6. 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.

  7. Automatic extraction of nuclei centroids of mouse embryonic cells from fluorescence microscopy images.

    Directory of Open Access Journals (Sweden)

    Md Khayrul Bashar

    Full Text Available Accurate identification of cell nuclei and their tracking using three dimensional (3D microscopic images is a demanding task in many biological studies. Manual identification of nuclei centroids from images is an error-prone task, sometimes impossible to accomplish due to low contrast and the presence of noise. Nonetheless, only a few methods are available for 3D bioimaging applications, which sharply contrast with 2D analysis, where many methods already exist. In addition, most methods essentially adopt segmentation for which a reliable solution is still unknown, especially for 3D bio-images having juxtaposed cells. In this work, we propose a new method that can directly extract nuclei centroids from fluorescence microscopy images. This method involves three steps: (i Pre-processing, (ii Local enhancement, and (iii Centroid extraction. The first step includes two variations: first variation (Variant-1 uses the whole 3D pre-processed image, whereas the second one (Variant-2 modifies the preprocessed image to the candidate regions or the candidate hybrid image for further processing. At the second step, a multiscale cube filtering is employed in order to locally enhance the pre-processed image. Centroid extraction in the third step consists of three stages. In Stage-1, we compute a local characteristic ratio at every voxel and extract local maxima regions as candidate centroids using a ratio threshold. Stage-2 processing removes spurious centroids from Stage-1 results by analyzing shapes of intensity profiles from the enhanced image. An iterative procedure based on the nearest neighborhood principle is then proposed to combine if there are fragmented nuclei. Both qualitative and quantitative analyses on a set of 100 images of 3D mouse embryo are performed. Investigations reveal a promising achievement of the technique presented in terms of average sensitivity and precision (i.e., 88.04% and 91.30% for Variant-1; 86.19% and 95.00% for Variant-2

  8. The use of near-infrared fluorescence imaging in endocrine surgical procedures. (United States)

    Kahramangil, Bora; Berber, Eren


    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.

  9. Dual purpose scanner for thyroid imaging in the fluorescence and emission modes

    International Nuclear Information System (INIS)

    Charleston, D.; Beck, R.; Yasillo, N.; Atkins, F.; Cooper, M.; Kirchner, P.


    Quantitative elemental analysis by the use of stimulated fluorescence x-rays has been applied in an imaging modality whereby the relative concentration of iodine-127 in the thyroid can be mapped, and total iodine in the gland estimated for the diagnosis of malignant and benign nodules. To further the development of fluorescence imaging of the thyroid, three areas of work are described which include theoretical studies, empirical studies and hardware development, and clinical feasibility studies

  10. Lipid nanoparticle vectorization of indocyanine green improves fluorescence imaging for tumor diagnosis and lymph node resection. (United States)

    Navarro, Fabrice P; Berger, Michel; Guillermet, Stéphanie; Josserand, Véronique; Guyon, Laurent; Neumann, Emmanuelle; Vinet, Françoise; Texier, Isabelle


    Fluorescence imaging is opening a new era in image-guided surgery and other medical applications. The only FDA approved contrast agent in the near infrared is IndoCyanine Green (ICG), which despites its low toxicity, displays poor chemical and optical properties for long-term and sensitive imaging applications in human. Lipid nanoparticles are investigated for improving ICG optical properties and in vivo fluorescence imaging sensitivity. 30 nm diameter lipid nanoparticles (LNP) are loaded with ICG. Their characterization and use for tumor and lymph node imaging are described. Nano-formulation benefits dye optical properties (6 times improved brightness) and chemical stability (>6 months at 4 degrees C in aqueous buffer). More importantly, LNP vectorization allows never reported sensitive and prolonged (>1 day) labeling of tumors and lymph nodes. Composed of human-use approved ingredients, this novel ICG nanometric formulation is foreseen to expand rapidly the field of clinical fluorescence imaging applications.

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

    International Nuclear Information System (INIS)

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


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

  12. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo (United States)

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


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

  13. Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging (United States)

    Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei


    Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

  14. Biological imaging in radiation therapy: role of positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Nestle, Ursula; Hentschel, Michael; Grosu, Anca-Ligia [Departments of Radiation Oncology, University of Freiburg, Robert Koch Str. 3, 79106 Freiburg (Germany); Weber, Wolfgang [Nuclear Medicine, University of Freiburg, Robert Koch Str. 3, 79106 Freiburg (Germany)], E-mail:


    In radiation therapy (RT), staging, treatment planning, monitoring and evaluation of response are traditionally based on computed tomography (CT) and magnetic resonance imaging (MRI). These radiological investigations have the significant advantage to show the anatomy with a high resolution, being also called anatomical imaging. In recent years, so called biological imaging methods which visualize metabolic pathways have been developed. These methods offer complementary imaging of various aspects of tumour biology. To date, the most prominent biological imaging system in use is positron emission tomography (PET), whose diagnostic properties have clinically been evaluated for years. The aim of this review is to discuss the valences and implications of PET in RT. We will focus our evaluation on the following topics: the role of biological imaging for tumour tissue detection/delineation of the gross tumour volume (GTV) and for the visualization of heterogeneous tumour biology. We will discuss the role of fluorodeoxyglucose-PET in lung and head and neck cancer and the impact of amino acids (AA)-PET in target volume delineation of brain gliomas. Furthermore, we summarize the data of the literature about tumour hypoxia and proliferation visualized by PET. We conclude that, regarding treatment planning in radiotherapy, PET offers advantages in terms of tumour delineation and the description of biological processes. However, to define the real impact of biological imaging on clinical outcome after radiotherapy, further experimental, clinical and cost/benefit analyses are required. (topical review)

  15. Biological imaging in radiation therapy: role of positron emission tomography. (United States)

    Nestle, Ursula; Weber, Wolfgang; Hentschel, Michael; Grosu, Anca-Ligia


    In radiation therapy (RT), staging, treatment planning, monitoring and evaluation of response are traditionally based on computed tomography (CT) and magnetic resonance imaging (MRI). These radiological investigations have the significant advantage to show the anatomy with a high resolution, being also called anatomical imaging. In recent years, so called biological imaging methods which visualize metabolic pathways have been developed. These methods offer complementary imaging of various aspects of tumour biology. To date, the most prominent biological imaging system in use is positron emission tomography (PET), whose diagnostic properties have clinically been evaluated for years. The aim of this review is to discuss the valences and implications of PET in RT. We will focus our evaluation on the following topics: the role of biological imaging for tumour tissue detection/delineation of the gross tumour volume (GTV) and for the visualization of heterogeneous tumour biology. We will discuss the role of fluorodeoxyglucose-PET in lung and head and neck cancer and the impact of amino acids (AA)-PET in target volume delineation of brain gliomas. Furthermore, we summarize the data of the literature about tumour hypoxia and proliferation visualized by PET. We conclude that, regarding treatment planning in radiotherapy, PET offers advantages in terms of tumour delineation and the description of biological processes. However, to define the real impact of biological imaging on clinical outcome after radiotherapy, further experimental, clinical and cost/benefit analyses are required.

  16. Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking (United States)

    Ryu, Inkeon; Kim, Daekeun


    A typical selective plane illumination microscopy (SPIM) image size is basically limited by the field of view, which is a characteristic of the objective lens. If an image larger than the imaging area of the sample is to be obtained, image stitching, which combines step-scanned images into a single panoramic image, is required. However, accurately registering the step-scanned images is very difficult because the SPIM system uses a customized sample mount where uncertainties for the translational and the rotational motions exist. In this paper, an image registration technique based on multiple fluorescent microsphere tracking is proposed in the view of quantifying the constellations and measuring the distances between at least two fluorescent microspheres embedded in the sample. Image stitching results are demonstrated for optically cleared large tissue with various staining methods. Compensation for the effect of the sample rotation that occurs during the translational motion in the sample mount is also discussed.

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

    DEFF Research Database (Denmark)

    Hojman, Pernille; Eriksen, Jens; Gehl, Julie


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

  18. Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence. (United States)

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


    A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

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

    Directory of Open Access Journals (Sweden)

    Zhang Y


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

  20. Responses of sun-induced chlorophyll fluorescence to biological and environmental variations measured with a versatile Fluorescence Auto-Measurement Equipment (FAME) (United States)

    Gu, L.


    In this study, we examine responses of sun-induced chlorophyll fluorescence to biological and environmental variations measured with a versatile Fluorescence Auto-Measurement Equipment (FAME). FAME was developed to automatically and continuously measure chlorophyll fluorescence (F) of a leaf, plant or canopy in both laboratory and field environments, excited by either artificial light source or sunlight. FAME is controlled by a datalogger and allows simultaneous measurements of environmental variables complementary to the F signals. A built-in communication system allows FAME to be remotely monitored and data-downloaded. Radiance and irradiance calibrations can be done online. FAME has been applied in a variety of environments, allowing an investigation of biological and environmental controls on F emission.

  1. Cu2+-labeled dansyl compounds as fluorescent and PET probes for imaging apoptosis. (United States)

    Han, Junyan; Wang, Xukui; Yu, MeiXiang


    Compound DNSTT-Cu 2+ , a novel chelate of Cu 2+ with DOTA conjugated to a fluorescent dansyl fragment, is developed for imaging cell apoptosis. Apoptotic U-87MG cells could be selectively visualized by the fluorescence of DNSTT-Cu 2+ from cytoplasm of cells, confirmed by the fluorescence of apoptosis cells co-labeled with Alexa Fluor 568-labeled annexin V, a conventional probe for selectively labeling membranes of apoptosis cells. A radioactive 64 Cu 2 + analog, DNSTT- 64 Cu 2+ , was easily synthesized, providing a potential PET probe for imaging apoptosis in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. The MicroAnalysis Toolkit: X-ray Fluorescence Image Processing Software

    International Nuclear Information System (INIS)

    Webb, S. M.


    The MicroAnalysis Toolkit is an analysis suite designed for the processing of x-ray fluorescence microprobe data. The program contains a wide variety of analysis tools, including image maps, correlation plots, simple image math, image filtering, multiple energy image fitting, semi-quantitative elemental analysis, x-ray fluorescence spectrum analysis, principle component analysis, and tomographic reconstructions. To be as widely useful as possible, data formats from many synchrotron sources can be read by the program with more formats available by request. An overview of the most common features will be presented.

  3. Comparative study of protoporphyrin IX fluorescence image enhancement methods to improve an optical imaging system for oral cancer detection (United States)

    Jiang, Ching-Fen; Wang, Chih-Yu; Chiang, Chun-Ping


    Optoelectronics techniques to induce protoporphyrin IX fluorescence with topically applied 5-aminolevulinic acid on the oral mucosa have been developed to noninvasively detect oral cancer. Fluorescence imaging enables wide-area screening for oral premalignancy, but the lack of an adequate fluorescence enhancement method restricts the clinical imaging application of these techniques. This study aimed to develop a reliable fluorescence enhancement method to improve PpIX fluorescence imaging systems for oral cancer detection. Three contrast features, red-green-blue reflectance difference, R/B ratio, and R/G ratio, were developed first based on the optical properties of the fluorescence images. A comparative study was then carried out with one negative control and four biopsy confirmed clinical cases to validate the optimal image processing method for the detection of the distribution of malignancy. The results showed the superiority of the R/G ratio in terms of yielding a better contrast between normal and neoplastic tissue, and this method was less prone to errors in detection. Quantitative comparison with the clinical diagnoses in the four neoplastic cases showed that the regions of premalignancy obtained using the proposed method accorded with the expert's determination, suggesting the potential clinical application of this method for the detection of oral cancer.

  4. Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window (United States)

    Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.


    Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

  5. Recent trends in total reflection X-ray fluorescence spectrometry for biological applications

    International Nuclear Information System (INIS)

    Szoboszlai, Norbert; Polgari, Zsofia; Mihucz, Victor G.; Zaray, Gyula


    This review is focused on the application of total reflection X-ray fluorescence (TXRF) spectrometry in the field of biological research. In the last decade, most papers were published by authors who applied laboratory-scale TXRF equipments. The application of synchrotron radiation as excitation source (SR-TXRF) shows a slowly increasing tendency. In the cited papers the micro-, trace and multielement capability of these TXRF techniques was demonstrated in the clinical and medical laboratory practice, as well as in various plant physiological studies. For speciation of elements in biological matrices, the TXRF was used as element specific detector following an off-line separation step (e.g., thin layer chromatography, high performance liquid chromatography), however, these off-line methods are not competitive with the on-line coupled HPLC-inductively coupled plasma mass spectrometry

  6. Vascular thrombus imaging in vivo via near-infrared fluorescent nanodiamond particles bioengineered with the disintegrin bitistatin (Part II

    Directory of Open Access Journals (Sweden)

    Gerstenhaber JA


    Full Text Available Jonathan A Gerstenhaber,1,* Frank C Barone,2,* Cezary Marcinkiewicz,1,3 Jie Li,2 Aaron O Shiloh,4 Mark Sternberg,3 Peter I Lelkes,1,* Giora Feuerstein1,3,* 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA, 2Department of Neurology, State University of New York Downstate Medical Center, Brooklyn, NY, 3Debina Diagnostic Inc., Newtown Square, 4Diagnostic Imaging, Inc., Philadelphia, PA, USA *These authors contributed equally to this work Abstract: The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP covalently conjugated with bitistatin (F-NDP-Bit to detect vascular blood clots in vivo using extracorporeal near-infrared (NIR imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDPNV and N-V-N color centers and sizes (100–10,000 nm. Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin was obtained for F-NDPNV with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS] in vitro revealed that F-NDPNV-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm. In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl3 in the carotid artery bifurcation. Following systemic infusions of F-NDPNV-Bit (3 or 15 mg/kg via the external carotid artery or femoral vein (N=3, presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDPNV in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDPNV-Bit associate with vascular blood clots, presumably by binding

  7. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques. (United States)

    Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine


    We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. 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). 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. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

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


    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.

  9. Near infrared spatial frequency domain fluorescence imaging of tumor phantoms containing erythrocyte-derived optical nanoplatforms (United States)

    Burns, Joshua M.; Schaefer, Elise; Anvari, Bahman


    Light-activated theranostic constructs provide a multi-functional platform for optical imaging and phototherapeutic applications. Our group has engineered nano-sized vesicles derived from erythrocytes that encapsulate the FDAapproved near infrared (NIR) absorber indocyanine green (ICG). We refer to these constructs as NIR erythrocytemimicking transducers (NETs). Once photo-excited by NIR light these constructs can transduce the photons energy to emit fluorescence, generate heat, or induce chemical reactions. In this study, we investigated fluorescence imaging of NETs embedded within tumor phantoms using spatial frequency domain imaging (SFDI). Using SFDI, we were able to fluorescently image simulated tumors doped with different concentration of NETs. These preliminary results suggest that NETs can be used in conjunction with SFDI for potential tumor imaging applications.

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

    International Nuclear Information System (INIS)

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P; Zhu, R; Mayer, B; Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F; Salio, M; Shepherd, D; Polzella, P; Cerundolo, V; Dieudonne, M


    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 α-galactosylceramide (αGalCer)-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 ∼ 25 to ∼ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  11. A parallel adaptive finite element simplified spherical harmonics approximation solver for frequency domain fluorescence molecular imaging

    International Nuclear Information System (INIS)

    Lu Yujie; Zhu Banghe; Rasmussen, John C; Sevick-Muraca, Eva M; Shen Haiou; Wang Ge


    Fluorescence molecular imaging/tomography may play an important future role in preclinical research and clinical diagnostics. Time- and frequency-domain fluorescence imaging can acquire more measurement information than the continuous wave (CW) counterpart, improving the image quality of fluorescence molecular tomography. Although diffusion approximation (DA) theory has been extensively applied in optical molecular imaging, high-order photon migration models need to be further investigated to match quantitation provided by nuclear imaging. In this paper, a frequency-domain parallel adaptive finite element solver is developed with simplified spherical harmonics (SP N ) approximations. To fully evaluate the performance of the SP N approximations, a fast time-resolved tetrahedron-based Monte Carlo fluorescence simulator suitable for complex heterogeneous geometries is developed using a convolution strategy to realize the simulation of the fluorescence excitation and emission. The validation results show that high-order SP N can effectively correct the modeling errors of the diffusion equation, especially when the tissues have high absorption characteristics or when high modulation frequency measurements are used. Furthermore, the parallel adaptive mesh evolution strategy improves the modeling precision and the simulation speed significantly on a realistic digital mouse phantom. This solver is a promising platform for fluorescence molecular tomography using high-order approximations to the radiative transfer equation.

  12. 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: [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)


    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.

  13. Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement. (United States)

    Hong, Guosong; Lee, Jerry C; Jha, Arshi; Diao, Shuo; Nakayama, Karina H; Hou, Luqia; Doyle, Timothy C; Robinson, Joshua T; Antaris, Alexander L; Dai, Hongjie; Cooke, John P; Huang, Ngan F


    Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths. Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (Pimaging make it a useful imaging tool for murine models of vascular disease. © 2014 American Heart Association, Inc.

  14. Intraoperative Detection of Superficial Liver Tumors by Fluorescence Imaging Using Indocyanine Green and 5-aminolevulinic Acid. (United States)

    Kaibori, Masaki; Matsui, Kosuke; Ishizaki, Morihiko; Iida, Hiroya; Okumura, Tadayoshi; Sakaguchi, Tatsuma; Inoue, Kentaro; Ikeura, Tsukasa; Asano, Hiroaki; Kon, Masanori


    Indocyanine green (ICG) and the porphyrin precursor 5-aminolevulinic acid (5-ALA) have been approved as fluorescence imaging agents in the clinical setting. This study evaluated the usefulness of fluorescence imaging with both ICG and 5-ALA for intraoperative identification of latent small liver tumors. There were 48 patients who had main tumors within 5 mm of the liver surface. 5-ALA hydrochloride was orally administered to patients 3 h before surgery. ICG had been intravenously injected within 14 days prior to surgery. Intraoperatively, after visual inspection, manual palpation and ultrasonography fluorescence images of the liver surface were obtained with ICG and 5-ALA prior to resection. With ICG, the sensitivity, specificity and accuracy for detecting the preoperatively identified main tumors were 96%, 50% and 94%, respectively. Twelve latent small tumors were newly detected on the liver surface using ICG, five of which proved to be carcinomas. With 5-ALA, the sensitivity, specificity and accuracy for detecting the main tumors were 57%, 100% and 58%, respectively. Five latent small tumors were newly detected using 5-ALA; all were carcinomas. Overall, five new tumors were detected by both ICG and 5-ALA fluorescence imaging; two were hepatocellular carcinomas (HCCs) and three were metastases of colorectal cancer. The sensitivity and specificity of ICG fluorescence imaging for main tumor detection were relatively high and low, respectively, but the opposite was true of 5-ALA imaging. Fluorescence imaging using 5-ALA may provide greater specificity in the detection of surface-invisible malignant liver tumors than using ICG fluorescence imaging alone. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Matthew D., E-mail:; 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)


    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.

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

    International Nuclear Information System (INIS)

    Wilson, Matthew D.; Seller, Paul; Veale, Matthew C.; Connolley, Thomas; Dolbnya, Igor P.; Malandain, Andrew; Sawhney, Kawal; Grant, Patrick S.; Liotti, Enzo; Lui, Andrew


    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"2 with one of the 80×80 pixels imaging an area equivalent to 13µm"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.

  17. Clinical multi-colour fluorescence imaging of malignant tumours - initial experience

    International Nuclear Information System (INIS)

    Svanberg, K.; Wang, I.; Montan, S.; Andersson-Engels, S.; Svanberg, S.; Lund Inst. of Technology


    The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation, ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s was mixed with a normal colour video image of the tissue. A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in vitro investigations of resected breast cancer. (orig./MG)

  18. A high brightness probe of polymer nanoparticles for biological imaging (United States)

    Zhou, Sirong; Zhu, Jiarong; Li, Yaping; Feng, Liheng


    Conjugated polymer nanoparticles (CPNs) with high brightness in long wavelength region were prepared by the nano-precipitation method. Based on fluorescence resonance energy transfer (FRET) mechanism, the high brightness property of the CPNs was realized by four different emission polymers. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) displayed that the CPNs possessed a spherical structure and an average diameter of 75 nm. Analysis assays showed that the CPNs had excellent biocompatibility, good photostability and low cytotoxicity. The CPNs were bio-modified with a cell penetrating peptide (Tat, a targeted element) through covalent link. Based on the entire wave fluorescence emission, the functionalized CPNs1-4 can meet multichannel and high throughput assays in cell and organ imaging. The contribution of the work lies in not only providing a new way to obtain a high brightness imaging probe in long wavelength region, but also using targeted cell and organ imaging.

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

    KAUST Repository

    Yabiku, Y.; Kubo, S.; Nakagawa, M.; Vacha, M.; Habuchi, Satoshi


    We applied a super-resolution fluorescence imaging based on selective adsorption and redox switching of the fluorescent dye molecules for studying polymer nanostructures. We demonstrate that nano-scale structures of polymer thin films can

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

    International Nuclear Information System (INIS)

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


    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 <0.66, 0.66–1.06, 1.06–1.62, and >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. (paper)

  1. Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis (United States)

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


    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.

  2. 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: [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)


    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.

  3. Attributed relational graphs for cell nucleus segmentation in fluorescence microscopy images. (United States)

    Arslan, Salim; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem


    More rapid and accurate high-throughput screening in molecular cellular biology research has become possible with the development of automated microscopy imaging, for which cell nucleus segmentation commonly constitutes the core step. Although several promising methods exist for segmenting the nuclei of monolayer isolated and less-confluent cells, it still remains an open problem to segment the nuclei of more-confluent cells, which tend to grow in overlayers. To address this problem, we propose a new model-based nucleus segmentation algorithm. This algorithm models how a human locates a nucleus by identifying the nucleus boundaries and piecing them together. In this algorithm, we define four types of primitives to represent nucleus boundaries at different orientations and construct an attributed relational graph on the primitives to represent their spatial relations. Then, we reduce the nucleus identification problem to finding predefined structural patterns in the constructed graph and also use the primitives in region growing to delineate the nucleus borders. Working with fluorescence microscopy images, our experiments demonstrate that the proposed algorithm identifies nuclei better than previous nucleus segmentation algorithms.

  4. Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules. (United States)

    Elliott, Jonathan T; Dsouza, Alisha V; Marra, Kayla; Pogue, Brian W; Roberts, David W; Paulsen, Keith D


    Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials.

  5. Rapid Analysis and Exploration of Fluorescence Microscopy Images


    Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason; Steininger, Robert J; Wu, Lani; Altschuler, Steven


    Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.

  6. The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis. (United States)

    Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong


    Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.

  7. Multistage morphological segmentation of bright-field and fluorescent microscopy images (United States)

    Korzyńska, A.; Iwanowski, M.


    This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".

  8. A novel approach for phytotoxicity assessment by CCD fluorescence imaging

    Czech Academy of Sciences Publication Activity Database

    Gavel, Alan; Maršálek, Blahoslav


    Roč. 19, - (2004), s. 429-432 ISSN 1520-4081 R&D Projects: GA AV ČR KSK6005114 Institutional research plan: CEZ:AV0Z6005908 Keywords : phytotoxicity * chlorophyll fluorescence * algal bioassay Subject RIV: EF - Botanics Impact factor: 1.373, year: 2004

  9. Self-assembled fluorescent organic nanoparticles for live cell imaging

    NARCIS (Netherlands)

    Fischer, I.; Petkau, K.; Dorland, Y.L.; Schenning, A.P.H.J.; Brunsveld, L.


    Fluorescent, cell-permeable, organic nanoparticles based on self-assembled p-conjugated oligomers with high absorption cross-sections and high quantum yields have been developed. The nanoparticles are generated with a tuneable density of amino groups for charge-mediated cellular uptake by a

  10. Preliminary Results of Nuclear Fluorescence Imaging of Alpha and Beta Emitting Sources

    International Nuclear Information System (INIS)

    Feener, Jessica S.; Charlton, William S.


    The preliminary results from a series of nuclear fluorescence imaging experiments using a variety of radioactive sources and shielding are given. These experiments were done as part of a proof of concept to determine if nuclear fluorescence imaging could be used as a safeguards measurements tool or for nuclear warhead verification for nuclear arms control treaties such as the New Strategic Arms Reduction Treaty and the Fissile Material Cut-Off Treaty. An off-the-shelf Princeton Instruments charged coupled device camera system was used to image the emission of fluorescence photons from the de-excitation of nitrogen molecules in air that have been excited by ionizing radiation. The fluorescence emissions are primarily in the near ultraviolet range; between the wavelengths of 300 and 400 nm. Fluorescent imaging techniques are currently being investigated in a number of applications. A French research team has successfully demonstrated this concept for remote imaging of alpha contamination. It has also been shown that the phenomenon can be seen through translucent materials and that alpha radiation can be seen in the presence of large gamma backgrounds. Additionally, fluorescence telescopes and satellites utilize the de-excitation of nitrogen molecules to observe cosmic ray showers in the atmosphere. In cosmic ray shower detection, electrons are the main contributor to the excitation of the of nitrogen molecules in air. The experiments presented in this paper were designed to determine if the imaging system could observe beta emitting sources, differentiate between beta emitters and alpha emitting materials such as uranium oxide and uranium metal, and to further investigate the phenomenon through translucent and non-translucent materials. The initial results show that differentiation can be made between beta and alpha emitting sources and that the device can observe the phenomenon through very thin non-transparent material. Additionally, information is given on the

  11. Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging. (United States)

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


    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.

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

    International Nuclear Information System (INIS)

    Sovik, Aste; Malinen, Eirik; Olsen, Dag Rune


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

  13. Red fluorescence imaging for dental plaque detection and quantification: pilot study (United States)

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


    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 (phygiene assessment.

  14. Wide-field spectrally resolved quantitative fluorescence imaging system: toward neurosurgical guidance in glioma resection (United States)

    Xie, Yijing; Thom, Maria; Ebner, Michael; Wykes, Victoria; Desjardins, Adrien; Miserocchi, Anna; Ourselin, Sebastien; McEvoy, Andrew W.; Vercauteren, Tom


    In high-grade glioma surgery, tumor resection is often guided by intraoperative fluorescence imaging. 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) provides fluorescent contrast between normal brain tissue and glioma tissue, thus achieving improved tumor delineation and prolonged patient survival compared with conventional white-light-guided resection. However, commercially available fluorescence imaging systems rely solely on visual assessment of fluorescence patterns by the surgeon, which makes the resection more subjective than necessary. We developed a wide-field spectrally resolved fluorescence imaging system utilizing a Generation II scientific CMOS camera and an improved computational model for the precise reconstruction of the PpIX concentration map. In our model, the tissue's optical properties and illumination geometry, which distort the fluorescent emission spectra, are considered. We demonstrate that the CMOS-based system can detect low PpIX concentration at short camera exposure times, while providing high-pixel resolution wide-field images. We show that total variation regularization improves the contrast-to-noise ratio of the reconstructed quantitative concentration map by approximately twofold. Quantitative comparison between the estimated PpIX concentration and tumor histopathology was also investigated to further evaluate the system.

  15. A portable UV-fluorescence multispectral imaging system for the analysis of painted surfaces (United States)

    Comelli, Daniela; Valentini, Gianluca; Nevin, Austin; Farina, Andrea; Toniolo, Lucia; Cubeddu, Rinaldo


    A portable fluorescence multispectral imaging system was developed and has been used for the analysis of artistic surfaces. The imaging apparatus exploits two UV lamps for fluorescence excitation and a liquid crystal tunable filter coupled to a low-noise charge coupled device as the image detector. The main features of the system are critically presented, outlining the assets, drawbacks, and practical considerations of portability. A multivariate statistical treatment of spectral data is further considered. Finally, the in situ analysis with the new apparatus of recently restored Renaissance wall paintings is presented.

  16. Current Concepts and Future Perspectives on Intraoperative Fluorescence Imaging in Cancer : Clinical Need

    NARCIS (Netherlands)

    van Dam, Gooitzen M.; Ntziachristos, Vasilis

    Progress with technology and regulatory approvals has recently allowed the successful clinical translation of fluorescence molecular imaging to intra-operative applications. Initial studies have demonstrated a promising outlook for imaging cancer micro-foci, margins and lymph-nodes. However, not all

  17. Red to far-red multispectral fluorescence image fusion for detection of fecal contamination on apples (United States)

    This research developed a multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet/blue LED excitation for detection of fecal contamination on Golden Delicious apples. Using a hyperspectral line-scan imaging system consisting of an EMCCD camera, spectrograph, an...

  18. Fluorescence hyperspectral imaging technique for foreign substance detection on fresh-cut lettuce. (United States)

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


    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.

  19. Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer. (United States)

    Ale, Angelique; Ermolayev, Vladimir; Deliolanis, Nikolaos C; Ntziachristos, Vasilis


    The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.

  20. Very High Spectral Resolution Imaging Spectroscopy: the Fluorescence Explorer (FLEX) Mission (United States)

    Moreno, Jose F.; Goulas, Yves; Huth, Andreas; Middleton, Elizabeth; Miglietta, Franco; Mohammed, Gina; Nedbal, Ladislav; Rascher, Uwe; Verhoef, Wouter; Drusch, Matthias


    The Fluorescence Explorer (FLEX) mission has been recently selected as the 8th Earth Explorer by the European Space Agency (ESA). It will be the first mission specifically designed to measure from space vegetation fluorescence emission, by making use of very high spectral resolution imaging spectroscopy techniques. Vegetation fluorescence is the best proxy to actual vegetation photosynthesis which can be measurable from space, allowing an improved quantification of vegetation carbon assimilation and vegetation stress conditions, thus having key relevance for global mapping of ecosystems dynamics and aspects related with agricultural production and food security. The FLEX mission carries the FLORIS spectrometer, with a spectral resolution in the range of 0.3 nm, and is designed to fly in tandem with Copernicus Sentinel-3, in order to provide all the necessary spectral / angular information to disentangle emitted fluorescence from reflected radiance, and to allow proper interpretation of the observed fluorescence spatial and temporal dynamics.

  1. Simultaneous AFM and fluorescence imaging: A method for aligning an AFM-tip with an excitation beam using a 2D galvanometer (United States)

    Moores, A. N.; Cadby, A. J.


    Correlative fluorescence and atomic force microscopy (AFM) imaging is a highly attractive technique for use in biological imaging, enabling force and mechanical measurements of particular structures whose locations are known due to the specificity of fluorescence imaging. The ability to perform these two measurements simultaneously (rather than consecutively with post-processing correlation) is highly valuable because it would allow the mechanical properties of a structure to be tracked over time as changes in the sample occur. We present an instrument which allows simultaneous AFM and fluorescence imaging by aligning an incident fluorescence excitation beam with an AFM-tip. Alignment was performed by calibrating a 2D galvanometer present in the excitation beam path and using it to reposition the incident beam. Two programs were developed (one manual and one automated) which correlate sample features between the AFM and fluorescence images, calculating the distance required to translate the incident beam towards the AFM-tip. Using this method, we were able to obtain beam-tip alignment (and therefore field-of-view alignment) from an offset of >15 μm to within one micron in two iterations of the program. With the program running alongside data acquisition for real-time feedback between AFM and optical images, this offset was maintained over a time period of several hours. Not only does this eliminate the need to image large areas with both techniques to ensure that fields-of-view overlap, but it also raises the possibility of using this instrument for tip-enhanced fluorescence applications, a technique in which super-resolution images have previously been achieved.

  2. Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation) (United States)

    Yilmaz, Hasan


    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).

  3. Synthesis and characterization of bioresorbable calcium phosphosilicate nanocomposite particles for fluorescence imaging and biomedical applications (United States)

    Morgan, Thomas T.

    Organically doped calcium phosphosilicate nanoparticles (CPSNPs) were developed and characterized, driven by the need for non-toxic vectors for drug delivery and fluorescence biological imaging applications. In particular, advancement in drug delivery for the chemotherapeutic treatment of cancers is required to increase drug efficacy and improve patient quality of life. Additionally, brighter and more photostable fluorophores are needed to meet demands for improved sensitivity and experimental diversity, which may lead to improvements in early detection of solid tumors and advancement in understanding of biological processes. A literature survey on the state of the field for nanoparticle based biological fluorescence imaging and drug delivery is presented in Chapter 1. Chapter 2 focuses on the characterization techniques used in this work. The development and optical characterization of 20-40 nm diameter, citrate functionalized Cy3 amidite doped calcium phosphosilicate nanoparticles (Cy3 CPSNPs) for in vitro fluorescence imaging is outlined in Chapters 3 and 4, respectively. In particular, sodium citrate was used to functionalize the surface and provide electrosteric dispersion of these particles. CPSNPs stabilized with sodium citrate routinely exhibited highly negative zeta potentials greater than -25 mV in magnitude. Furthermore, the fluorescence quantum yield of the encapsulated fluorophore was improved by more than 4.5-fold when compared to the unencapsulated dye. The bioimaging and drug delivery capability of CPSNPs was explored. Cy3 CPSNPs dissolved quickly in the acidic environment experienced during endocytosis, releasing the encapsulated fluorophore. This is consistent with solution phase experiments that show the particles are dissolved at pH 5. CPSNPs loaded with fluorescein and a hydrophobic growth inhibitor, ceramide C6, proved the ability to simultaneously image and delivery of the hydrophobic drug to cells in vitro. Chapter 5 examined the colloidal

  4. Catheter-based time-gated near-infrared fluorescence/OCT imaging system (United States)

    Lu, Yuankang; Abran, Maxime; Cloutier, Guy; Lesage, Frédéric


    We developed a new dual-modality intravascular imaging system based on fast time-gated fluorescence intensity imaging and spectral domain optical coherence tomography (SD-OCT) for the purpose of interventional detection of atherosclerosis. A pulsed supercontinuum laser was used for fluorescence and OCT imaging. A double-clad fiber (DCF)- based side-firing catheter was designed and fabricated to have a 23 μm spot size at a 2.2 mm working distance for OCT imaging. Its single-mode core is used for OCT, while its inner cladding transports fluorescence excitation light and collects fluorescent photons. The combination of OCT and fluorescence imaging was achieved by using a DCF coupler. For fluorescence detection, we used a time-gated technique with a novel single-photon avalanche diode (SPAD) working in an ultra-fast gating mode. A custom-made delay chip was integrated in the system to adjust the delay between the excitation laser pulse and the SPAD gate-ON window. This technique allowed to detect fluorescent photons of interest while rejecting most of the background photons, thus leading to a significantly improved signal to noise ratio (SNR). Experiments were carried out in turbid media mimicking tissue with an indocyanine green (ICG) inclusion (1 mM and 100 μM) to compare the time-gated technique and the conventional continuous detection technique. The gating technique increased twofold depth sensitivity, and tenfold SNR at large distances. The dual-modality imaging capacity of our system was also validated with a silicone-based tissue-mimicking phantom.

  5. A Geometric Dictionary Learning Based Approach for Fluorescence Spectroscopy Image Fusion

    Directory of Open Access Journals (Sweden)

    Zhiqin Zhu


    Full Text Available In recent years, sparse representation approaches have been integrated into multi-focus image fusion methods. The fused images of sparse-representation-based image fusion methods show great performance. Constructing an informative dictionary is a key step for sparsity-based image fusion method. In order to ensure sufficient number of useful bases for sparse representation in the process of informative dictionary construction, image patches from all source images are classified into different groups based on geometric similarities. The key information of each image-patch group is extracted by principle component analysis (PCA to build dictionary. According to the constructed dictionary, image patches are converted to sparse coefficients by simultaneous orthogonal matching pursuit (SOMP algorithm for representing the source multi-focus images. At last the sparse coefficients are fused by Max-L1 fusion rule and inverted to fused image. Due to the limitation of microscope, the fluorescence image cannot be fully focused. The proposed multi-focus image fusion solution is applied to fluorescence imaging area for generating all-in-focus images. The comparison experimentation results confirm the feasibility and effectiveness of the proposed multi-focus image fusion solution.

  6. Near-infrared-fluorescence imaging of lymph nodes by using liposomally formulated indocyanine green derivatives. (United States)

    Toyota, Taro; Fujito, Hiromichi; Suganami, Akiko; Ouchi, Tomoki; Ooishi, Aki; Aoki, Akira; Onoue, Kazutaka; Muraki, Yutaka; Madono, Tomoyuki; Fujinami, Masanori; Tamura, Yutaka; Hayashi, Hideki


    Liposomally formulated indocyanine green (LP-ICG) has drawn much attention as a highly sensitive near-infrared (NIR)-fluorescence probe for tumors or lymph nodes in vivo. We synthesized ICG derivatives tagged with alkyl chains (ICG-Cn), and we examined NIR-fluorescence imaging for lymph nodes in the lower extremities of mice by using liposomally formulated ICG-Cn (LP-ICG-Cn) as well as conventional liposomally formulated ICG (LP-ICG) and ICG. Analysis with a noninvasive preclinical NIR-fluorescence imaging system revealed that LP-ICG-Cn accumulates in only the popliteal lymph node 1h after injection into the footpad, whereas LP-ICG and ICG accumulate in the popliteal lymph node and other organs like the liver. This result indicates that LP-ICG-Cn is a useful NIR-fluorescence probe for noninvasive in vivo bioimaging, especially for the sentinel lymph node. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    International Nuclear Information System (INIS)

    Daldrup-Link, Heike E.; Rudelius, Martina; Piontek, Guido; Schlegel, Juergen; Metz, Stephan; Settles, Marcus; Rummeny, Ernst J.; Pichler, Bernd; Heinzmann, Ulrich; Oostendorp, Robert A.J.


    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10 6 -3 x 10 8 labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10 6 cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells. (orig.)

  8. Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. (United States)

    Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per


    Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

  9. Introduction to basic molecular biologic techniques for molecular imaging researches

    International Nuclear Information System (INIS)

    Kang, Joo Hyun


    Molecular imaging is a rapidly growing field due to the advances in molecular biology and imaging technologies. With the introduction of imaging reporter genes into the cell, diverse cellular processes can be monitored, quantified and imaged non-invasively in vivo. These processes include the gene expression, protein-protein interactions, signal transduction pathways, and monitoring of cells such as cancer cells, immune cells, and stem cells. In the near future, molecular imaging analysis will allow us to observe the incipience and progression of the disease. These will make us easier to give a diagnosis in the early stage of intractable diseases such as cancer, neuro-degenerative disease, and immunological disorders. Additionally, molecular imaging method will be a valuable tool for the real-time evaluation of cells in molecular biology and the basic biological studies. As newer and more powerful molecular imaging tools become available, it will be necessary to corporate clinicians, molecular biologists and biochemists for the planning, interpretation, and application of these techniques to their fullest potential. In order for such a multidisciplinary team to be effective, it is essential that a common understanding of basic biochemical and molecular biologic techniques is achieved. Basic molecular techniques for molecular imaging methods are presented in this paper

  10. 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)


    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.

  11. Automated parasite faecal egg counting using fluorescence labelling, smartphone image capture and computational image analysis. (United States)

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


    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 (P<0.0001). This demonstrates the feasibility of a simple, automated on-site test to detect and/or enumerate parasite eggs in mammalian faeces without the need for a laboratory microscope, and highlights the potential of smartphones as relatively sophisticated, inexpensive and portable medical diagnostic devices. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  12. Diagnostic imaging of cervical intraepithelial neoplasia based on hematoxylin and eosin fluorescence. (United States)

    Castellanos, Mario R; Szerszen, Anita; Gundry, Stephen; Pirog, Edyta C; Maiman, Mitchell; Rajupet, Sritha; Gomez, John Paul; Davidov, Adi; Debata, Priya Ranjan; Banerjee, Probal; Fata, Jimmie E


    Pathological classification of cervical intraepithelial neoplasia (CIN) is problematic as it relies on subjective criteria. We developed an imaging method that uses spectroscopy to assess the fluorescent intensity of cervical biopsies derived directly from hematoxylin and eosin (H&E) stained tissues. Archived H&E slides were identified containing normal cervical tissue, CIN I, and CIN III cases, from a Community Hospital and an Academic Medical Center. Cases were obtained by consensus review of at least 2 senior pathologists. Images from H&E slides were captured first with bright field illumination and then with fluorescent illumination. We used a Zeiss Axio Observer Z1 microscope and an AxioVision 4.6.3-AP1 camera at excitation wavelength of 450-490 nm with emission captured at 515-565 nm. The 32-bit grayscale fluorescence images were used for image analysis. We reviewed 108 slides: 46 normal, 33 CIN I and 29 CIN III. Fluorescent intensity increased progressively in normal epithelial tissue as cells matured and advanced from the basal to superficial regions of the epithelium. In CIN I cases this change was less prominent as compared to normal. In high grade CIN lesions, there was a slight or no increase in fluorescent intensity. All groups examined were statistically different. Presently, there are no markers to help in classification of CIN I-III lesions. Our imaging method may complement standard H&E pathological review and provide objective criteria to support the CIN diagnosis.

  13. Parallel scan hyperspectral fluorescence imaging system and biomedical application for microarrays

    International Nuclear Information System (INIS)

    Liu Zhiyi; Ma Suihua; Liu Le; Guo Jihua; He Yonghong; Ji Yanhong


    Microarray research offers great potential for analysis of gene expression profile and leads to greatly improved experimental throughput. A number of instruments have been reported for microarray detection, such as chemiluminescence, surface plasmon resonance, and fluorescence markers. Fluorescence imaging is popular for the readout of microarrays. In this paper we develop a quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging system for microarray research. Hyperspectral imaging records the entire emission spectrum for every voxel within the imaged area in contrast to recording only fluorescence intensities of filter-based scanners. Coupled with data analysis, the recorded spectral information allows for quantitative identification of the contributions of multiple, spectrally overlapping fluorescent dyes and elimination of unwanted artifacts. The mechanism of quasi-confocal imaging provides a high signal-to-noise ratio, and parallel scan makes this approach a high throughput technique for microarray analysis. This system is improved with a specifically designed spectrometer which can offer a spectral resolution of 0.2 nm, and operates with spatial resolutions ranging from 2 to 30 μm . Finally, the application of the system is demonstrated by reading out microarrays for identification of bacteria.

  14. Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging (United States)

    Gravier, Julien; Navarro, Fabrice P.; Delmas, Thomas; Mittler, Frédérique; Couffin, Anne-Claude; Vinet, Françoise; Texier, Isabelle


    The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC50 > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.

  15. Real-time Fluorescence Image-Guided Oncologic Surgery (United States)

    Mondal, Suman B.; Gao, Shengkui; Zhu, Nan; Liang, Rongguang; Gruev, Viktor; Achilefu, Samuel


    Medical imaging plays a critical role in cancer diagnosis and planning. Many of these patients rely on surgical intervention for curative outcomes. This requires a careful identification of the primary and microscopic tumors, and the complete removal of cancer. Although there have been efforts to adapt traditional imaging modalities for intraoperative image guidance, they suffer from several constraints such as large hardware footprint, high operation cost, and disruption of the surgical workflow. Because of the ease of image acquisition, relatively low cost devices and intuitive operation, optical imaging methods have received tremendous interests for use in real-time image-guided surgery. To improve imaging depth under low interference by tissue autofluorescence, many of these applications utilize light in the near-infra red (NIR) wavelengths, which is invisible to human eyes. With the availability of a wide selection of tumor-avid contrast agents, advancements in imaging sensors, electronic and optical designs, surgeons are able to combine different attributes of NIR optical imaging techniques to improve treatment outcomes. The emergence of diverse commercial and experimental image guidance systems, which are in various stages of clinical translation, attests to the potential high impact of intraoperative optical imaging methods to improve speed of oncologic surgery with high accuracy and minimal margin positivity. PMID:25287689

  16. Facile method for CLSM imaging unfunctionalized Au nanoparticles through fluorescent channels

    International Nuclear Information System (INIS)

    Yuan Lan; Wei Wei; Li Juan; Sun, Zhiwei; Wang Hongfang; Zhang Xiuzhi; Chen Yueyue


    The microscopic visualization of metal nanoparticles has become a useful tool for the investigation of their applications in cell labeling and the study of their bio-effects. In the current study, we have developed a facile method with confocal laser scanning microscope (CLSM) to observe unfunctionalized Au nanoparticles through fluorescent channels. The sharp reflected signal and photostable property of the metal nanoparticles makes the present method very ideal for fluorescent co-localization, real-time imaging, and further quantitative analysis.

  17. A turn-on supramolecular fluorescent probe for sensing benzimidazole fungicides and its application in living cell imaging (United States)

    Tang, Qing; Zhang, Jing; Sun, Tao; Wang, Cheng-Hui; Huang, Ying; Zhou, Qingdi; Wei, Gang


    A cucurbit[8]uril-based turn-on supramolecular fluorescent probe between cucurbit[8]uril (Q[8]) and pyronine Y (PyY) (designated 2PyY@Q[8]) in acidic aqueous solution showed a remarkable fluorescence 'turn-on' response to benzimidazole fungicides such as thiabendazole, fuberidazole and carbendazim. The 2PyY@Q[8] fluorescent probe can be used to detect benzimidazole fungicides with high sensitivity and selectivity with a detection limit of 10- 8 mol/L. A good linear relationship of emission intensity at 580 nm for benzimidazole fungicides at concentrations of 0.4-5.0 μmol/L was observed. The proposed sensing mechanism was investigated using 1H NMR spectroscopy combined with density functional theory calculations at the B3LYP/6-31G(d) level. The cell imaging study showed that the 2PyY@Q[8] complex could be used to image benzimidazole fungicide in prostate cancer (PC3) cells, which may help to elucidate relevant biological processes at the molecular level.

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

    Directory of Open Access Journals (Sweden)

    Nan Guo


    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.

  19. Deep brain two-photon NIR fluorescence imaging for study of Alzheimer's disease (United States)

    Chen, Congping; Liang, Zhuoyi; Zhou, Biao; Ip, Nancy Y.; Qu, Jianan Y.


    Amyloid depositions in the brain represent the characteristic hallmarks of Alzheimer's disease (AD) pathology. The abnormal accumulation of extracellular amyloid-beta (Aβ) and resulting toxic amyloid plaques are considered to be responsible for the clinical deficits including cognitive decline and memory loss. In vivo two-photon fluorescence imaging of amyloid plaques in live AD mouse model through a chronic imaging window (thinned skull or craniotomy) provides a mean to greatly facilitate the study of the pathological mechanism of AD owing to its high spatial resolution and long-term continuous monitoring. However, the imaging depth for amyloid plaques is largely limited to upper cortical layers due to the short-wavelength fluorescence emission of commonly used amyloid probes. In this work, we reported that CRANAD-3, a near-infrared (NIR) probe for amyloid species with excitation wavelength at 900 nm and emission wavelength around 650 nm, has great advantages over conventionally used probes and is well suited for twophoton deep imaging of amyloid plaques in AD mouse brain. Compared with a commonly used MeO-X04 probe, the imaging depth of CRANAD-3 is largely extended for open skull cranial window. Furthermore, by using two-photon excited fluorescence spectroscopic imaging, we characterized the intrinsic fluorescence of the "aging pigment" lipofuscin in vivo, which has distinct spectra from CRANAD-3 labeled plaques. This study reveals the unique potential of NIR probes for in vivo, high-resolution and deep imaging of brain amyloid in Alzheimer's disease.

  20. Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis (United States)

    Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.


    Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

  1. Improved detection of soma location and morphology in fluorescence microscopy images of neurons. (United States)

    Kayasandik, Cihan Bilge; Labate, Demetrio


    Automated detection and segmentation of somas in fluorescent images of neurons is a major goal in quantitative studies of neuronal networks, including applications of high-content-screenings where it is required to quantify multiple morphological properties of neurons. Despite recent advances in image processing targeted to neurobiological applications, existing algorithms of soma detection are often unreliable, especially when processing fluorescence image stacks of neuronal cultures. In this paper, we introduce an innovative algorithm for the detection and extraction of somas in fluorescent images of networks of cultured neurons where somas and other structures exist in the same fluorescent channel. Our method relies on a new geometrical descriptor called Directional Ratio and a collection of multiscale orientable filters to quantify the level of local isotropy in an image. To optimize the application of this approach, we introduce a new construction of multiscale anisotropic filters that is implemented by separable convolution. Extensive numerical experiments using 2D and 3D confocal images show that our automated algorithm reliably detects somas, accurately segments them, and separates contiguous ones. We include a detailed comparison with state-of-the-art existing methods to demonstrate that our algorithm is extremely competitive in terms of accuracy, reliability and computational efficiency. Our algorithm will facilitate the development of automated platforms for high content neuron image processing. A Matlab code is released open-source and freely available to the scientific community. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles. (United States)

    Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J


    Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

  3. Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer

    Directory of Open Access Journals (Sweden)

    Nakase H


    Full Text Available Hiroshi Nakase,1,2 Shinji Sakuma,3 Takumi Fukuchi,4 Takuya Yoshino,1,5 Kohta Mohri,3 Kohei Miyata,3,6 Hironori Kumagai,6 Ken-Ichiro Hiwatari,6 Kazufumi Tsubaki,6 Tetsuya Ikejima,6 Etsuo Tobita,6 Meiying Zhu,7 Kevin J Wilson,7 Kay Washington,8 John C Gore,7,9–13 Wellington Pham7,9–12,14 1Division of Endoscopy, Kyoto University Hospital, Kyoto, Japan; 2Department of Gastroenterology and Hepatology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 3Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, Japan; 4Gastroenterology and Hepatology, Osakafu Saiseikai Nakatsu Hospital, Osaka, Japan; 5Division of Inflammatory Bowel Disease, Digestive Disease Center, Kitano Hospital, Osaka, Japan; 6Advanced Materials R&D Laboratory, ADEKA Corp., Tokyo, Japan; 7Department of Radiology, Vanderbilt University Institute of Imaging Science, 8Division of Gastrointestinal and Hepatic Pathology, Vanderbilt School of Medicine, 9Department of Radiology and Radiological Sciences, 10Department of Biomedical Engineering, 11Vanderbilt Ingram Cancer Center, Vanderbilt School of Medicine, 12Vanderbilt Brain Institute, 13Molecular Physiology and Biophysics, 14Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA Abstract: The Thomsen–Friedenreich (TF antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident. Keywords: TF antigen

  4. Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe. (United States)

    Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie


    Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions (United States)

    Sharifzadeh, Mohsen; Bernstein, Paul S.; Gellermann, Werner


    We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the blue-green wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.

  6. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo (United States)

    Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.


    There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

  7. Static Hyperspectral Fluorescence Imaging of Viscous Materials Based on a Linear Variable Filter Spectrometer

    Directory of Open Access Journals (Sweden)

    Alexander W. Koch


    Full Text Available This paper presents a low-cost hyperspectral measurement setup in a new application based on fluorescence detection in the visible (Vis wavelength range. The aim of the setup is to take hyperspectral fluorescence images of viscous materials. Based on these images, fluorescent and non-fluorescent impurities in the viscous materials can be detected. For the illumination of the measurement object, a narrow-band high-power light-emitting diode (LED with a center wavelength of 370 nm was used. The low-cost acquisition unit for the imaging consists of a linear variable filter (LVF and a complementary metal oxide semiconductor (CMOS 2D sensor array. The translucent wavelength range of the LVF is from 400 nm to 700 nm. For the confirmation of the concept, static measurements of fluorescent viscous materials with a non-fluorescent impurity have been performed and analyzed. With the presented setup, measurement surfaces in the micrometer range can be provided. The measureable minimum particle size of the impurities is in the nanometer range. The recording rate for the measurements depends on the exposure time of the used CMOS 2D sensor array and has been found to be in the microsecond range.

  8. Fluorescent metal nanoshell and CK19 detection on single cell image

    International Nuclear Information System (INIS)

    Zhang, Jian; Fu, Yi; Li, Ge; Lakowicz, Joseph R.; Zhao, Richard Y.


    Highlights: → Novel metal nanoshell as fluorescence imaging agent. → Fluorescent mAb-metal complex with enhanced intensity and shortened lifetime. → Immuno-interactions of mAb-metal complexes with CK19 molecules on CNCAP and HeLa cell surfaces. → Isolation of conjugated mAb-metal complexes from cellular autofluorescence on cell image. -- Abstract: In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.

  9. Single-Shot, Volumetrically Illuminated, Three-Dimensional, Tomographic Laser-Induced-Fluorescence Imaging in a Gaseous Free Jet (United States)


    Single-shot, volumetrically illuminated, three- dimensional, tomographic laser-induced- fluorescence imaging in a gaseous free jet Benjamin R. Halls...acquisition; (110.6955) Tomographic imaging ; (110.6960) Tomography; (280.2490) Flow diagnostics; (300.2530) Fluorescence , laser-induced...84 (1983). 2. I. van Cruyningen, A. Lozano, and R. K. Hanson, “Quantitative imaging of concentration by planar laser-induced fluorescence ,” Exp


    A video imaging system and the associated quantification methods have been developed for measurement of the transfers of a fluorescent tracer from surfaces to hands. The highly fluorescent compound riboflavin (Vitamin B2), which is also water soluble and non-toxic, was chosen as...

  11. Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research. (United States)

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


    Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

  12. A Conjugate of Pentamethine Cyanine and 18F as a Positron Emission Tomography/Near-Infrared Fluorescence Probe for Multimodality Tumor Imaging

    Directory of Open Access Journals (Sweden)

    Fei-Fei An


    Full Text Available The novel synthesis of a dual-modality, pentamethine cyanine (Cy5 fluorescent, 18F positron emission tomography (PET imaging probe is reported. The probe shows a large extinction coefficient and large quantum yield in the biologically transparent, near-infrared window (650–900 nm for in vivo fluorescent imaging. This fluorophore bears the isotope, 18F, giving a 18F-PET/near-infrared fluorescent (NIRF, bi-modal imaging probe, that combines the long-term stability of NIRF and the unlimited penetration depth of PET imaging. The bi-modal probe is labeled with 18F in a quick, one-step reaction, which is important in working with the rapid decay of 18F. The bi-modal probe bears a free carboxyl group, highlighting a PET/NIRF synthon that can be conjugated onto many advanced biomolecules for biomarker-specific in vivo dual-modal PET/NIR tumor imaging, confocal histology, and utility in multi-fluorophore, fluorescence-guided surgery. Its potential in vivo biocompatibility is explored in a quick proof-of-principal in vivo study. The dye is delivered to A549 xenograft flank-tumors to generate PET and NIRF signals at the tumor site. The tumor distribution is confirmed in ex vivo gamma counting and imaging. Pentamethine cyanine (Cy5 has the ability to preferentially accumulate in tumor xenografts. We substitute the PET/NIRF probe for Cy5, and explore this phenomenon.

  13. Fluorescent ratiometric pH indicator SypHer2: applications in neuroscience and regenerative biology (United States)

    Matlashov, Mikhail E.; Bogdanova, Yulia A.; Ermakova, Galina V.; Mishina, Natalia M.; Ermakova, Yulia G.; Nikitin, Evgeny S.; Balaban, Pavel M.; Okabe, Shigeo; Lukyanov, Sergey; Enikolopov, Grigori; Zaraisky, Andrey G.; Belousov, Vsevolod V.


    Background SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use. Methods Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer. Results Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. Conclusions SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. General significance The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies. PMID:26259819

  14. Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology. (United States)

    Matlashov, Mikhail E; Bogdanova, Yulia A; Ermakova, Galina V; Mishina, Natalia M; Ermakova, Yulia G; Nikitin, Evgeny S; Balaban, Pavel M; Okabe, Shigeo; Lukyanov, Sergey; Enikolopov, Grigori; Zaraisky, Andrey G; Belousov, Vsevolod V


    SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells. (United States)

    Montoya, Leticia A; Pluth, Michael D


    Hydrogen sulfide (H(2)S) is an important biological messenger but few biologically-compatible methods are available for its detection. Here we report two bright fluorescent probes that are selective for H(2)S over cysteine, glutathione and other reactive sulfur, nitrogen, and oxygen species. Both probes are demonstrated to detect H(2)S in live cells. This journal is © The Royal Society of Chemistry 2012

  16. A compact gamma camera for biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, E L; Cella, J; Majewski, S; Popov, V; Qian, Jianguo; Saha, M S; Smith, M F; Weisenberger, A G; Welsh, R E


    A compact detector, sized particularly for imaging a mouse, is described. The active area of the detector is approximately 46 mm; spl times/ 96 mm. Two flat-panel Hamamatsu H8500 position-sensitive photomultiplier tubes (PSPMTs) are coupled to a pixellated NaI(Tl) scintillator which views the animal through a copper-beryllium (CuBe) parallel-hole collimator specially designed for {sup 125}I. Although the PSPMTs have insensitive areas at their edges and there is a physical gap, corrections for scintillation light collection at the junction between the two tubes results in a uniform response across the entire rectangular area of the detector. The system described has been developed to optimize both sensitivity and resolution for in-vivo imaging of small animals injected with iodinated compounds. We demonstrate an in-vivo application of this detector, particularly to SPECT, by imaging mice injected with approximately 10-15; spl mu/Ci of {sup 125}I.

  17. Adaptive Spot Detection With Optimal Scale Selection in Fluorescence Microscopy Images. (United States)

    Basset, Antoine; Boulanger, Jérôme; Salamero, Jean; Bouthemy, Patrick; Kervrann, Charles


    Accurately detecting subcellular particles in fluorescence microscopy is of primary interest for further quantitative analysis such as counting, tracking, or classification. Our primary goal is to segment vesicles likely to share nearly the same size in fluorescence microscopy images. Our method termed adaptive thresholding of Laplacian of Gaussian (LoG) images with autoselected scale (ATLAS) automatically selects the optimal scale corresponding to the most frequent spot size in the image. Four criteria are proposed and compared to determine the optimal scale in a scale-space framework. Then, the segmentation stage amounts to thresholding the LoG of the intensity image. In contrast to other methods, the threshold is locally adapted given a probability of false alarm (PFA) specified by the user for the whole set of images to be processed. The local threshold is automatically derived from the PFA value and local image statistics estimated in a window whose size is not a critical parameter. We also propose a new data set for benchmarking, consisting of six collections of one hundred i