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Sample records for quantitative fluorescence microscopy

  1. Quantitative fluorescence microscopy and image deconvolution.

    Science.gov (United States)

    Swedlow, Jason R

    2013-01-01

    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

  2. Quantitative high dynamic range beam profiling for fluorescence microscopy

    International Nuclear Information System (INIS)

    Mitchell, T. J.; Saunter, C. D.; O’Nions, W.; Girkin, J. M.; Love, G. D.

    2014-01-01

    Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences

  3. Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

    Science.gov (United States)

    Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

    2004-07-01

    Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

  4. DNA origami-based standards for quantitative fluorescence microscopy.

    Science.gov (United States)

    Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

    2014-01-01

    Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

  5. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  6. Toward quantitative fluorescence microscopy with DNA origami nanorulers.

    Science.gov (United States)

    Beater, Susanne; Raab, Mario; Tinnefeld, Philip

    2014-01-01

    The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution. © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2013-02-01

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

  8. Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

    Science.gov (United States)

    Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

    2009-10-01

    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.

  9. Quantitative Fluorescence Sensing Through Highly Autofluorescent, Scattering, and Absorbing Media Using Mobile Microscopy

    KAUST Repository

    Göröcs, Zoltán

    2016-09-13

    Compact and cost-effective systems for in vivo fluorescence and near-infrared imaging in combination with activatable reporters embedded inside the skin to sample interstitial fluid or blood can enable a variety of biomedical applications. However, the strong autofluorescence of human skin creates an obstacle for fluorescence-based sensing. Here we introduce a method for quantitative fluorescence sensing through highly autofluorescent, scattering, and absorbing media. For this, we created a compact and cost-effective fluorescence microscope weighing <40 g and used it to measure various concentrations of a fluorescent dye embedded inside a tissue phantom, which was designed to mimic the optical characteristics of human skin. We used an elliptical Gaussian beam excitation to digitally separate tissue autofluorescence from target fluorescence, although they severely overlap in both space and optical spectrum. Using ∼10-fold less excitation intensity than the safety limit for skin radiation exposure, we successfully quantified the density of the embedded fluorophores by imaging the skin phantom surface and achieved a detection limit of ∼5 × 105 and ∼2.5 × 107 fluorophores within ∼0.01 μL sample volume that is positioned 0.5 and 2 mm below the phantom surface, corresponding to a concentration of 105.9 pg/mL and 5.3 ng/mL, respectively. We also confirmed that this approach can track the spatial misalignments of the mobile microscope with respect to the embedded target fluorescent volume. This wearable microscopy platform might be useful for designing implantable biochemical sensors with the capability of spatial multiplexing to continuously monitor a panel of biomarkers and chronic conditions even at patients’ home.

  10. Quantitative Fluorescence Sensing Through Highly Autofluorescent, Scattering, and Absorbing Media Using Mobile Microscopy

    KAUST Repository

    Gö rö cs, Zoltá n; Rivenson, Yair; Ceylan Koydemir, Hatice; Tseng, Derek; Troy, Tamara L.; Demas, Vasiliki; Ozcan, Aydogan

    2016-01-01

    Compact and cost-effective systems for in vivo fluorescence and near-infrared imaging in combination with activatable reporters embedded inside the skin to sample interstitial fluid or blood can enable a variety of biomedical applications. However, the strong autofluorescence of human skin creates an obstacle for fluorescence-based sensing. Here we introduce a method for quantitative fluorescence sensing through highly autofluorescent, scattering, and absorbing media. For this, we created a compact and cost-effective fluorescence microscope weighing <40 g and used it to measure various concentrations of a fluorescent dye embedded inside a tissue phantom, which was designed to mimic the optical characteristics of human skin. We used an elliptical Gaussian beam excitation to digitally separate tissue autofluorescence from target fluorescence, although they severely overlap in both space and optical spectrum. Using ∼10-fold less excitation intensity than the safety limit for skin radiation exposure, we successfully quantified the density of the embedded fluorophores by imaging the skin phantom surface and achieved a detection limit of ∼5 × 105 and ∼2.5 × 107 fluorophores within ∼0.01 μL sample volume that is positioned 0.5 and 2 mm below the phantom surface, corresponding to a concentration of 105.9 pg/mL and 5.3 ng/mL, respectively. We also confirmed that this approach can track the spatial misalignments of the mobile microscope with respect to the embedded target fluorescent volume. This wearable microscopy platform might be useful for designing implantable biochemical sensors with the capability of spatial multiplexing to continuously monitor a panel of biomarkers and chronic conditions even at patients’ home.

  11. In-focal-plane characterization of excitation distribution for quantitative fluorescence microscopy applications

    Science.gov (United States)

    Dietrich, Klaus; Brülisauer, Martina; ćaǧin, Emine; Bertsch, Dietmar; Lüthi, Stefan; Heeb, Peter; Stärker, Ulrich; Bernard, André

    2017-06-01

    The applications of fluorescence microscopy span medical diagnostics, bioengineering and biomaterial analytics. Full exploitation of fluorescent microscopy is hampered by imperfections in illumination, detection and filtering. Mainly, errors stem from deviations induced by real-world components inducing spatial or angular variations of propagation properties along the optical path, and they can be addressed through consistent and accurate calibration. For many applications, uniform signal to noise ratio (SNR) over the imaging area is required. Homogeneous SNR can be achieved by quantifying and compensating for the signal bias. We present a method to quantitatively characterize novel reference materials as a calibration reference for biomaterials analytics. The reference materials under investigation comprise thin layers of fluorophores embedded in polymer matrices. These layers are highly homogeneous in their fluorescence response, where cumulative variations do not exceed 1% over the field of view (1.5 x 1.1 mm). An automated and reproducible measurement methodology, enabling sufficient correction for measurement artefacts, is reported. The measurement setup is equipped with an autofocus system, ensuring that the measured film quality is not artificially increased by out-of-focus reduction of the system modulation transfer function. The quantitative characterization method is suitable for analysis of modified bio-materials, especially through patterned protein decoration. The imaging method presented here can be used to statistically analyze protein patterns, thereby increasing both precision and throughput. Further, the method can be developed to include a reference emitter and detector pair on the image surface of the reference object, in order to provide traceable measurements.

  12. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical

  13. Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

    Science.gov (United States)

    Schmid, Volker J; Cremer, Marion; Cremer, Thomas

    2017-07-01

    Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    OpenAIRE

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultipl...

  15. Quantitative comparison of two particle tracking methods in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2013-09-01

    Full Text Available that cannot be analysed efficiently by means of manual analysis. In this study we compare the performance of two computer-based tracking methods for tracking of bright particles in fluorescence microscopy image sequences. The methods under comparison are...

  16. Fluorescence (Multiwave) Confocal Microscopy.

    Science.gov (United States)

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

    Science.gov (United States)

    Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

    2015-12-01

    Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

  18. QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Merete Krog Raarup

    2011-05-01

    Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

  19. Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe

    Directory of Open Access Journals (Sweden)

    Dominika Żurek-Biesiada

    2016-06-01

    Full Text Available Single Molecule Localization Microscopy (SMLM is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015 [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.

  20. Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

    DEFF Research Database (Denmark)

    Fidorra, Matthias; Garcia, Alejandra; Ipsen, John Hjort

    2009-01-01

    We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks...

  1. Bridging fluorescence microscopy and electron microscopy

    NARCIS (Netherlands)

    Giepmans, Ben N. G.

    Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major

  2. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-12-14

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

  3. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-11-01

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

  4. Membranes and Fluorescence microscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2009-01-01

    Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s to study different aspects of membrane-related phenomena, i.e., mainly relating to lipid-lipid and lipid-protein (peptide) interactions. Even though fluorescence...

  5. Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

    Science.gov (United States)

    Johnson, Heath E; Haugh, Jason M

    2013-12-02

    This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

  6. Fluorescence confocal microscopy for pathologists.

    Science.gov (United States)

    Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

    2014-03-01

    Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

  7. Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

    Science.gov (United States)

    Wei, Linpeng; Chen, Ye; Yin, Chengbo; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

    2017-04-01

    Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

  8. Fluorescence confocal polarizing microscopy

    Indian Academy of Sciences (India)

    Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

  9. A method to validate quantitative high-frequency power doppler ultrasound with fluorescence in vivo video microscopy.

    Science.gov (United States)

    Pinter, Stephen Z; Kim, Dae-Ro; Hague, M Nicole; Chambers, Ann F; MacDonald, Ian C; Lacefield, James C

    2014-08-01

    Flow quantification with high-frequency (>20 MHz) power Doppler ultrasound can be performed objectively using the wall-filter selection curve (WFSC) method to select the cutoff velocity that yields a best-estimate color pixel density (CPD). An in vivo video microscopy system (IVVM) is combined with high-frequency power Doppler ultrasound to provide a method for validation of CPD measurements based on WFSCs in mouse testicular vessels. The ultrasound and IVVM systems are instrumented so that the mouse remains on the same imaging platform when switching between the two modalities. In vivo video microscopy provides gold-standard measurements of vascular diameter to validate power Doppler CPD estimates. Measurements in four image planes from three mice exhibit wide variation in the optimal cutoff velocity and indicate that a predetermined cutoff velocity setting can introduce significant errors in studies intended to quantify vascularity. Consistent with previously published flow-phantom data, in vivo WFSCs exhibited three characteristic regions and detectable plateaus. Selection of a cutoff velocity at the right end of the plateau yielded a CPD close to the gold-standard vascular volume fraction estimated using IVVM. An investigator can implement the WFSC method to help adapt cutoff velocity to current blood flow conditions and thereby improve the accuracy of power Doppler for quantitative microvascular imaging. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  10. Plasmonics Enhanced Smartphone Fluorescence Microscopy

    KAUST Repository

    Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

    2017-01-01

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  11. Plasmonics Enhanced Smartphone Fluorescence Microscopy

    KAUST Repository

    Wei, Qingshan

    2017-05-12

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  12. Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

    Science.gov (United States)

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

    2013-01-01

    To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

  13. Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

    Directory of Open Access Journals (Sweden)

    Jenna L Mueller

    Full Text Available To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA and the circle transform (CT was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity. For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

  14. Integrated Photoacoustic and Fluorescence Confocal Microscopy

    OpenAIRE

    Wang, Yu; Maslov, Konstantin; Kim, Chulhong; Hu, Song; Wang, Lihong V.

    2010-01-01

    We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous photoacoustic...

  15. Tracking Lithium Ions via Widefield Fluorescence Microscopy for Battery Diagnostics.

    Science.gov (United States)

    Padilla, Nicolas A; Rea, Morgan T; Foy, Michael; Upadhyay, Sunil P; Desrochers, Kyle A; Derus, Tyler; Knapper, Kassandra A; Hunter, Nathanael H; Wood, Sharla; Hinton, Daniel A; Cavell, Andrew C; Masias, Alvaro G; Goldsmith, Randall H

    2017-07-28

    Direct tracking of lithium ions with time and spatial resolution can provide an important diagnostic tool for understanding mechanisms in lithium ion batteries. A fluorescent indicator of lithium ions, 2-(2-hydroxyphenyl)naphthoxazole, was synthesized and used for real-time tracking of lithium ions via widefield fluorescence microscopy. The fluorophore can be excited with visible light and was shown to enable quantitative determination of the lithium ion diffusion constant in a microfluidic model system for a plasticized polymer electrolyte lithium battery. The use of widefield fluorescence microscopy for in situ tracking of lithium ions in batteries is discussed.

  16. Examining Thermally Sprayed Coats By Fluorescence Microscopy

    Science.gov (United States)

    Street, Kenneth W., Jr.; Leonhardt, Todd A.

    1994-01-01

    True flaws distinquished from those induced by preparation of specimens. Fluorescence microscopy reveals debonding, porosity, cracks, and other flaws in specimens of thermally sprayed coating materials. Specimen illuminated, and dye it contains fluoresces, emitting light at different wavelength. Filters emphasize contrast between excitation light and emission light. Specimen viewed directly or photographed on color film.

  17. Handheld Fluorescence Microscopy based Flow Analyzer.

    Science.gov (United States)

    Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

    2016-03-01

    Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

  18. Fast globally optimal segmentation of cells in fluorescence microscopy images.

    Science.gov (United States)

    Bergeest, Jan-Philip; Rohr, Karl

    2011-01-01

    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.

  19. Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

    Science.gov (United States)

    Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George S

    2015-06-01

    Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry.

  20. New tools for comparing microscopy images : Quantitative analysis of cell types in Bacillus subtilis

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-01-01

    Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy

  1. Use of astronomy filters in fluorescence microscopy.

    Science.gov (United States)

    Piper, Jörg

    2012-02-01

    Monochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this "low energy excitation," photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.

  2. BlobFinder, a tool for fluorescence microscopy image cytometry

    OpenAIRE

    Allalou, Amin; Wählby, Carolina

    2009-01-01

    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. Signal and noise modeling in confocal laser scanning fluorescence microscopy.

    Science.gov (United States)

    Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

    2012-01-01

    Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

  4. Multimodal quantitative phase and fluorescence imaging of cell apoptosis

    Science.gov (United States)

    Fu, Xinye; Zuo, Chao; Yan, Hao

    2017-06-01

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

  5. 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

    2007-01-01

    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

  6. Simultaneous correlative scanning electron and high-NA fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Nalan Liv

    Full Text Available Correlative light and electron microscopy (CLEM is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

  7. Quantitative fluorescence nanoscopy for cancer biomedicine

    Science.gov (United States)

    Huang, Tao; Nickerson, Andrew; Peters, Alec; Nan, Xiaolin

    2015-08-01

    Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.

  8. Optofluidic time-stretch quantitative phase microscopy.

    Science.gov (United States)

    Guo, Baoshan; Lei, Cheng; Wu, Yi; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Lee, Sangwook; Isozaki, Akihiro; Li, Ming; Jiang, Yiyue; Yasumoto, Atsushi; Di Carlo, Dino; Tanaka, Yo; Yatomi, Yutaka; Ozeki, Yasuyuki; Goda, Keisuke

    2018-03-01

    Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

    Science.gov (United States)

    Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

    2017-12-01

    Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

  10. Quantitative phase imaging with scanning holographic microscopy: an experimental assesment

    Directory of Open Access Journals (Sweden)

    Tada Yoshitaka

    2006-11-01

    Full Text Available Abstract This paper demonstrates experimentally how quantitative phase information can be obtained in scanning holographic microscopy. Scanning holography can operate in both coherent and incoherent modes, simultaneously if desired, with different detector geometries. A spatially integrating detector provides an incoherent hologram of the object's intensity distribution (absorption and/or fluorescence, for example, while a point detector in a conjugate plane of the pupil provides a coherent hologram of the object's complex amplitude, from which a quantitative measure of its phase distribution can be extracted. The possibility of capturing simultaneously holograms of three-dimensional specimens, leading to three-dimensional reconstructions with absorption contrast, reflectance contrast, fluorescence contrast, as was previously demonstrated, and quantitative phase contrast, as shown here for the first time, opens up new avenues for multimodal imaging in biological studies.

  11. Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

    DEFF Research Database (Denmark)

    Jensen, Thomas; Holten-Rossing, Henrik; Svendsen, Ida M H

    2016-01-01

    to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including...... staining may benefit. METHODS: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm......BACKGROUND: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar...

  12. Quantitative transmission electron microscopy at atomic resolution

    International Nuclear Information System (INIS)

    Allen, L J; D'Alfonso, A J; Forbes, B D; Findlay, S D; LeBeau, J M; Stemmer, S

    2012-01-01

    In scanning transmission electron microscopy (STEM) it is possible to operate the microscope in bright-field mode under conditions which, by the quantum mechanical principle of reciprocity, are equivalent to those in conventional transmission electron microscopy (CTEM). The results of such an experiment will be presented which are in excellent quantitative agreement with theory for specimens up to 25 nm thick. This is at variance with the large contrast mismatch (typically between two and five) noted in equivalent CTEM experiments. The implications of this will be discussed.

  13. Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Thomas Jensen

    2016-01-01

    Full Text Available Background: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including staining may benefit. Methods: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm are presented. Results: It is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. The output of the method is absolute quantitation along with accurate outlines of above-mentioned components. The digital quantitations are verified by comparison to point grid quantitations performed on the microsections after Van Gieson staining. Conclusion: The presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. The main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework.

  14. Quantitative imaging of bilirubin by photoacoustic microscopy

    Science.gov (United States)

    Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

    2013-03-01

    Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

  15. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

    Science.gov (United States)

    Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

    2014-02-01

    The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the

  16. Experimental assessment of fluorescence microscopy signal enhancement by stimulated emission

    Science.gov (United States)

    Dake, Fumihiro; Yazawa, Hiroki

    2017-10-01

    The quantity of photons generated during fluorescence microscopy is principally determined by the quantum yield of the fluorescence dyes and the optical power of the excitation beam. However, even though low quantum yields can produce poor images, it is challenging to tune this parameter, while increasing the power of the excitation beam often results in photodamage. Here, we propose the use of stimulated emission (SE) as a means of enhancing both the signal intensity and signal-to-noise ratio during confocal fluorescence microscopy. This work experimentally confirmed that both these factors can be enhanced by SE radiation, through generating a greater number of photons than are associated with the standard fluorescence signal. We also propose the concept of stimulated emission enhancing fluorescence (SEEF) microscopy, which employs both the SE and fluorescence signals, and demonstrate that the intensity of an SEEF signal is greater than those of the individual SE and fluorescence signals.

  17. Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Macháň, Radek; Kapusta, Peter; Hof, Martin

    Roč. 406 , č. 20 (2014), s. 4797-4813 ISSN 1618-2642 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Filtered fluorescence correlation spectroscopy * Fluorescence lifetime correlation spectroscopy * Fluorescence spectral correlation spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.436, year: 2014

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

    OpenAIRE

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

    2013-01-01

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

  19. Aorta Fluorescence Imaging by Using Confocal Microscopy

    OpenAIRE

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

    2011-01-01

    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...

  20. Calibration of a DG–model for fluorescence microscopy

    DEFF Research Database (Denmark)

    Hansen, Christian Valdemar

    It is well known that diseases like Alzheimer, Parkinson, Corea Huntington and Arteriosclerosis are caused by a jam in intracellular membrane traffic [2]. Hence to improve treatment, a quantitative analysis of intracellular transport is essential. Fluorescence loss in photobleaching (FLIP......) is an impor- tant and widely used microscopy method for visualization of molecular transport processes in living cells. Thus, the motivation for making an automated reliable analysis of the image data is high. In this contribution, we present and comment on the calibration of a Discontinuous......–Galerkin simulator [3, 4] on segmented cell images. The cell geometry is extracted from FLIP images using the Chan– Vese active contours algorithm [1] while the DG simulator is implemented in FEniCS [5]. Simulated FLIP sequences based on optimal parameters from the PDE model are presented, with an overall goal...

  1. Hybrid confocal Raman fluorescence microscopy on single cells using semiconductor quantum dots

    NARCIS (Netherlands)

    van Manen, H.J.; Otto, Cornelis

    2007-01-01

    We have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). Here we present a hybrid Raman fluorescence spectral imaging approach for single-cell microscopy applications. We

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

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

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

  3. Quantitative Image Restoration in Bright Field Optical Microscopy.

    Science.gov (United States)

    Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

    2017-11-07

    Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    CSIR Research Space (South Africa)

    Mabaso, M

    2012-10-01

    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...

  5. Fluorescence microscopy for the characterization of structural integrity

    Science.gov (United States)

    Street, Kenneth W.; Leonhardt, Todd A.

    1991-01-01

    The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

  6. Saturated virtual fluorescence emission difference microscopy based on detector array

    Science.gov (United States)

    Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Ge, Baoliang; Wang, Wensheng; Liu, Xu

    2017-07-01

    Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

  7. Widefield fluorescence sectioning with HiLo microscopy.

    Science.gov (United States)

    Mertz, Jerome; Lim, Daryl; Chu, Kengyeh K; Bozinovic, Nenad; Ford, Timothy

    2009-01-01

    HiLo microscopy is a widefield fluorescence imaging technique that provides depth discrimination by combining two images, one with non-uniform illumination and one with uniform illumination. We discuss the theory of this technique and a variety of practical implementations in brain-tissue imaging and fluorescence endomicroscopy.

  8. Preparation of tissue samples for X-ray fluorescence microscopy

    International Nuclear Information System (INIS)

    Chwiej, Joanna; Szczerbowska-Boruchowska, Magdalena; Lankosz, Marek; Wojcik, Slawomir; Falkenberg, Gerald; Stegowski, Zdzislaw; Setkowicz, Zuzanna

    2005-01-01

    As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain

  9. Preparation of tissue samples for X-ray fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chwiej, Joanna [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland)]. E-mail: jchwiej@novell.ftj.agh.edu.pl; Szczerbowska-Boruchowska, Magdalena [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Lankosz, Marek [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Wojcik, Slawomir [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Falkenberg, Gerald [Hamburger Synchrotronstrahlungslabor at Deutsches Elektronen-Synchrotron, Notkestr. 85, Hamburg (Germany); Stegowski, Zdzislaw [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Setkowicz, Zuzanna [Department of Neuroanatomy, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow (Poland)

    2005-12-15

    As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.

  10. Performance of LED fluorescence microscopy for the detection of ...

    African Journals Online (AJOL)

    Introduction: Ziehl-Neelsen (ZN) bright-field microscopy is time-consuming, with poor sensitivity, even under optimal conditions. Introduction of Primo Star iLED fluorescent microscopy (FM) may improve TB case finding at referral hospitals in Rwanda. The study aimed to determine the acceptability and effectiveness of iLED ...

  11. Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

    Science.gov (United States)

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

    2013-07-29

    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.

  12. Segmentation of fluorescence microscopy cell images using unsupervised mining.

    Science.gov (United States)

    Du, Xian; Dua, Sumeet

    2010-05-28

    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.

  13. Fluorescence confocal polarizing microscopy: Three-dimensional ...

    Indian Academy of Sciences (India)

    journal of. August 2003 physics pp. 373–384. Fluorescence confocal polarizing ... and focal conic domains in flat samples of lamellar LCs are practically indistinguishable. ... or less) LC layer confined between two transparent plates. ... in studies of electro-optic effects such as the Frederiks effect, defects, surface anchoring,.

  14. Fluorescence confocal polarizing microscopy: Three-dimensional ...

    Indian Academy of Sciences (India)

    Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

  15. Super-resolution fluorescence microscopy by stepwise optical saturation

    Science.gov (United States)

    Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

    2018-01-01

    Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

  16. Quantitative fluorescence angiography for neurosurgical interventions.

    Science.gov (United States)

    Weichelt, Claudia; Duscha, Philipp; Steinmeier, Ralf; Meyer, Tobias; Kuß, Julia; Cimalla, Peter; Kirsch, Matthias; Sobottka, Stephan B; Koch, Edmund; Schackert, Gabriele; Morgenstern, Ute

    2013-06-01

    Present methods for quantitative measurement of cerebral perfusion during neurosurgical operations require additional technology for measurement, data acquisition, and processing. This study used conventional fluorescence video angiography--as an established method to visualize blood flow in brain vessels--enhanced by a quantifying perfusion software tool. For these purposes, the fluorescence dye indocyanine green is given intravenously, and after activation by a near-infrared light source the fluorescence signal is recorded. Video data are analyzed by software algorithms to allow quantification of the blood flow. Additionally, perfusion is measured intraoperatively by a reference system. Furthermore, comparing reference measurements using a flow phantom were performed to verify the quantitative blood flow results of the software and to validate the software algorithm. Analysis of intraoperative video data provides characteristic biological parameters. These parameters were implemented in the special flow phantom for experimental validation of the developed software algorithms. Furthermore, various factors that influence the determination of perfusion parameters were analyzed by means of mathematical simulation. Comparing patient measurement, phantom experiment, and computer simulation under certain conditions (variable frame rate, vessel diameter, etc.), the results of the software algorithms are within the range of parameter accuracy of the reference methods. Therefore, the software algorithm for calculating cortical perfusion parameters from video data presents a helpful intraoperative tool without complex additional measurement technology.

  17. Fundamentals of fluorescence microscopy exploring life with light

    CERN Document Server

    Mondal, Partha Pratim

    2014-01-01

    This book starts at an introductory level and leads reader to the most advanced developments in fluorescence imaging and super-resolution techniques that have enabled the emergence of new disciplines such as nanobioimaging, multiphoton microscopy, photodynamic therapy, nanometrology and nanosensors. The interdisciplinary subject of fluorescence microscopy and imaging requires complete knowledge of imaging optics and molecular physics. So, this book approaches the subject by introducing optical imaging concepts before going deep into the advanced imaging systems and their applications. Molecular orbital theory forms the basis for understanding fluorescent molecules and thereby facilitates complete explanation of light-matter interaction at the geometrical focus. The two disciplines have some overlap since light controls the states of molecules and conversely, molecular states control the emitted light. These two mechanisms together determine essential fluorescence  factors and phenomena such as, molecular cro...

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

    Science.gov (United States)

    Hassanzadeh, Abdollah

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

  19. Three Dimensional Fluorescence Microscopy Image Synthesis and Segmentation

    OpenAIRE

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

    2018-01-01

    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...

  20. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

    International Nuclear Information System (INIS)

    Miranda, Adelaide; De Beule, Pieter A. A.; Martins, Marco

    2015-01-01

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate

  1. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Miranda, Adelaide; De Beule, Pieter A. A., E-mail: pieter.de-beule@inl.int [Applied Nano-Optics Laboratory, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, s/n, 4715-330 Braga (Portugal); Martins, Marco [Nano-ICs Group, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, s/n, 4715-330 Braga (Portugal)

    2015-09-15

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

  2. 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

    2008-01-01

    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

  3. Robust tumor morphometry in multispectral fluorescence microscopy

    Science.gov (United States)

    Tabesh, Ali; Vengrenyuk, Yevgen; Teverovskiy, Mikhail; Khan, Faisal M.; Sapir, Marina; Powell, Douglas; Mesa-Tejada, Ricardo; Donovan, Michael J.; Fernandez, Gerardo

    2009-02-01

    Morphological and architectural characteristics of primary tissue compartments, such as epithelial nuclei (EN) and cytoplasm, provide important cues for cancer diagnosis, prognosis, and therapeutic response prediction. We propose two feature sets for the robust quantification of these characteristics in multiplex immunofluorescence (IF) microscopy images of prostate biopsy specimens. To enable feature extraction, EN and cytoplasm regions were first segmented from the IF images. Then, feature sets consisting of the characteristics of the minimum spanning tree (MST) connecting the EN and the fractal dimension (FD) of gland boundaries were obtained from the segmented compartments. We demonstrated the utility of the proposed features in prostate cancer recurrence prediction on a multi-institution cohort of 1027 patients. Univariate analysis revealed that both FD and one of the MST features were highly effective for predicting cancer recurrence (p <= 0.0001). In multivariate analysis, an MST feature was selected for a model incorporating clinical and image features. The model achieved a concordance index (CI) of 0.73 on the validation set, which was significantly higher than the CI of 0.69 for the standard multivariate model based solely on clinical features currently used in clinical practice (p < 0.0001). The contributions of this work are twofold. First, it is the first demonstration of the utility of the proposed features in morphometric analysis of IF images. Second, this is the largest scale study of the efficacy and robustness of the proposed features in prostate cancer prognosis.

  4. Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

    NARCIS (Netherlands)

    Tanner, Nathan A.; Loparo, Joseph J.; Oijen, Antoine M. van

    2009-01-01

    We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides. The

  5. Magnetic force microscopy : Quantitative issues in biomaterials

    NARCIS (Netherlands)

    Passeri, D.; Dong, C.; Reggente, M.; Angeloni, L.; Barteri, M.; Scaramuzzo, F.A.; De Angelis, F.; Marinelli, F.; Antonelli, F.; Rinaldi, F.; Marianecci, C.; Carafa, M.; Sorbo, A.; Sordi, D.; Arends, I.W.C.E.; Rossi, M.

    2014-01-01

    Magnetic force microscopy (MFM) is an atomic force microscopy (AFM) based technique in which an AFM tip with a magnetic coating is used to probe local magnetic fields with the typical AFM spatial resolution, thus allowing one to acquire images reflecting the local magnetic properties of the samples

  6. Boundary segmentation for fluorescence microscopy using steerable filters

    Science.gov (United States)

    Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

    2017-02-01

    Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

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

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

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

  8. Biostatistical analysis of quantitative immunofluorescence microscopy images.

    Science.gov (United States)

    Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

    2016-12-01

    Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

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

    Directory of Open Access Journals (Sweden)

    Gregor P. C. Drummen

    2012-11-01

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

  10. A Nanodiamond-peptide Bioconjugate for Fluorescence and ODMR Microscopy of a Single Actin Filament.

    Science.gov (United States)

    Genjo, Takuya; Sotoma, Shingo; Tanabe, Ryotaro; Igarashi, Ryuji; Shirakawa, Masahiro

    2016-01-01

    Recently, the importance of conformational changes in actin filaments induced by mechanical stimulation of a cell has been increasingly recognized, especially in terms of mechanobiology. Despite its fundamental importance, however, long-term observation of a single actin filament by fluorescent microscopy has been difficult because of the low photostability of traditional fluorescent molecules. This paper reports a novel molecular labeling system for actin filaments using fluorescent nanodiamond (ND) particles harboring nitrogen-vacancy centers; ND has flexible chemical modifiability, extremely high photostability and biocompatibility, and provides a variety of physical information quantitatively via optically detected magnetic resonance (ODMR) measurements. We performed the chemical surface modification of an ND with the actin filament-specific binding peptide Lifeact and observed colocalization of pure Lifeact-modified ND and actin filaments by the ODMR selective imaging protocol, suggesting the capability of long-term observation and quantitative analysis of a single molecule by using an ND particle.

  11. Detection of oxidative hair treatment using fluorescence microscopy.

    Science.gov (United States)

    Witt, Silvana; Wunder, Cora; Paulke, Alexander; Verhoff, Marcel A; Schubert-Zsilavecz, Manfred; Toennes, Stefan W

    2016-08-01

    In assessing abstinence from drug or alcohol abuse, hair analysis plays an important role. Cosmetic hair treatment influences the content of deposited drugs which is not always detectable during analysis. Since oxidation of melanin leads to an increase in fluorescence, a microscopic method was developed to distinguish natural from cosmetically treated hair. For validation, natural hair samples were treated with different types of cosmetics and inspected by fluorescence microscopy. Hair samples from 20 volunteers with documented cosmetic treatment and as a proof of concept 100 hair samples from forensic cases were analyzed by this method. Apart from autofluorescence with excitation at 365 nm, no obvious fluorescence was observed in untreated hair samples. Tinting and a natural plant product had no influence on fluorescence, but dyeing procedures including oxidation led to a marked increase in fluorescence. Proof of cosmetic treatment was achieved in hair samples from the 20 volunteers. In 100 forensic cases, 13 samples were characterized as oxidatively treated, which was in accordance with the respective disclosure except for one case where treatment was not admitted. This fluorescence microscopic procedure proved to be fast, easy, and reliable to identify oxidatively treated hair samples, which must be considered especially in evaluating cases of negative drug results. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  12. Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

    Science.gov (United States)

    Siegel, Nisan; Brooker, Gary

    2014-09-22

    FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

  13. MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

    Science.gov (United States)

    Prasath, V B S; Pelapur, R; Glinskii, O V; Glinsky, V V; Huxley, V H; Palaniappan, K

    2015-04-01

    Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

  14. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel

    2013-01-01

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

  15. Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

    Science.gov (United States)

    Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

    2004-01-01

    Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

  16. Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

    Science.gov (United States)

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

    2012-08-01

    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.

  17. Intradermal indocyanine green for in vivo fluorescence laser scanning microscopy of human skin: a pilot study.

    Directory of Open Access Journals (Sweden)

    Constanze Jonak

    Full Text Available BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of

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

    Science.gov (United States)

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

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

  19. Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

    Science.gov (United States)

    Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

    2015-10-01

    Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

  20. A portable microscopy system for fluorescence, polarized, and brightfield imaging

    Science.gov (United States)

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

    2018-02-01

    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.

  1. A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

    Science.gov (United States)

    Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

    2015-10-01

    Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. Copyright © 2015. Published by Elsevier Ltd.

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

    Directory of Open Access Journals (Sweden)

    Wang Ching-Wei

    2012-01-01

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

  3. Quantitative measurements of shear displacement using atomic force microscopy

    International Nuclear Information System (INIS)

    Wang, Wenbo; Wu, Weida; Sun, Ying; Zhao, Yonggang

    2016-01-01

    We report a method to quantitatively measure local shear deformation with high sensitivity using atomic force microscopy. The key point is to simultaneously detect both torsional and buckling motions of atomic force microscopy (AFM) cantilevers induced by the lateral piezoelectric response of the sample. This requires the quantitative calibration of torsional and buckling response of AFM. This method is validated by measuring the angular dependence of the in-plane piezoelectric response of a piece of piezoelectric α-quartz. The accurate determination of the amplitude and orientation of the in-plane piezoelectric response, without rotation, would greatly enhance the efficiency of lateral piezoelectric force microscopy.

  4. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

    Science.gov (United States)

    The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

  5. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics.

    Science.gov (United States)

    Jang, Changwon; Kim, Jonghyun; Clark, David C; Lee, Seungjae; Lee, Byoungho; Kim, Myung K

    2015-01-01

    Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: a wavefront sensor, wavefront corrector, and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, for example, lenslet arrays for sensing or multiactuator deformable mirrors for correcting. We have previously introduced an alternate approach based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile are possible not only with conventional coherent digital holography, but also with a new type of digital holography using incoherent light: selfinterference incoherent digital holography (SIDH). The SIDH generates a complex—i.e., amplitude plus phase—hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. Adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.

  6. Parallel detection experiment of fluorescence confocal microscopy using DMD.

    Science.gov (United States)

    Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

    2016-05-01

    Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  7. Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity

    International Nuclear Information System (INIS)

    Schorb, Martin; Briggs, John A.G.

    2014-01-01

    Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50 nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals. - Highlights: • Workflow for correlated cryo-fluorescence and cryo-electron microscopy. • Cryo-fluorescence microscopy setup incorporating a high numerical aperture objective. • Fluorescent signals located in cryo-electron micrographs with 50 nm spatial precision

  8. Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Schorb, Martin [Structural and Computational Biology Unit, European Molecular Biology Laboratory, D-69117 Heidelberg (Germany); Briggs, John A.G., E-mail: john.briggs@embl.de [Structural and Computational Biology Unit, European Molecular Biology Laboratory, D-69117 Heidelberg (Germany); Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, D-69117 Heidelberg (Germany)

    2014-08-01

    Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50 nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals. - Highlights: • Workflow for correlated cryo-fluorescence and cryo-electron microscopy. • Cryo-fluorescence microscopy setup incorporating a high numerical aperture objective. • Fluorescent signals located in cryo-electron micrographs with 50 nm spatial precision.

  9. Adaptive Spot Detection With Optimal Scale Selection in Fluorescence Microscopy Images.

    Science.gov (United States)

    Basset, Antoine; Boulanger, Jérôme; Salamero, Jean; Bouthemy, Patrick; Kervrann, Charles

    2015-11-01

    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 images each, which exploits backgrounds extracted from real microscopy images. We have carried out an extensive comparative evaluation on several data sets with ground-truth, which demonstrates that ATLAS outperforms existing methods. ATLAS does not need any fine parameter tuning and requires very low computation time. Convincing results are also reported on real total internal reflection fluorescence microscopy images.

  10. Scanless multitarget-matching multiphoton excitation fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Junpeng Qiu

    2018-03-01

    Full Text Available Using the combination of a reflective blazed grating and a reflective phase-only diffractive spatial light modulator (SLM, scanless multitarget-matching multiphoton excitation fluorescence microscopy (SMTM-MPM was achieved. The SLM shaped an incoming mode-locked, near-infrared Ti:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matched the samples of interest in the field of view. Temporal and spatial focusing were simultaneously realized by combining an objective lens and a blazed grating. The fluorescence signal from illuminated areas was recorded by a two-dimensional sCMOS camera. Compared with a conventional temporal focusing multiphoton microscope, our microscope achieved effective use of the laser power and decreased photodamage with higher axial resolution.

  11. Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

    Science.gov (United States)

    Cremer, Christoph; Birk, Udo

    2016-04-01

    The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

  12. Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

    Directory of Open Access Journals (Sweden)

    Christoph eCremer

    2016-04-01

    Full Text Available The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light, which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

  13. Rapid Analysis and Exploration of Fluorescence Microscopy Images

    OpenAIRE

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

    2014-01-01

    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.

  14. 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

    2004-01-01

    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

  15. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    Science.gov (United States)

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

  16. AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

    Science.gov (United States)

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

    2015-04-01

    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.

  17. Example-Based Super-Resolution Fluorescence Microscopy.

    Science.gov (United States)

    Jia, Shu; Han, Boran; Kutz, J Nathan

    2018-04-23

    Capturing biological dynamics with high spatiotemporal resolution demands the advancement in imaging technologies. Super-resolution fluorescence microscopy offers spatial resolution surpassing the diffraction limit to resolve near-molecular-level details. While various strategies have been reported to improve the temporal resolution of super-resolution imaging, all super-resolution techniques are still fundamentally limited by the trade-off associated with the longer image acquisition time that is needed to achieve higher spatial information. Here, we demonstrated an example-based, computational method that aims to obtain super-resolution images using conventional imaging without increasing the imaging time. With a low-resolution image input, the method provides an estimate of its super-resolution image based on an example database that contains super- and low-resolution image pairs of biological structures of interest. The computational imaging of cellular microtubules agrees approximately with the experimental super-resolution STORM results. This new approach may offer potential improvements in temporal resolution for experimental super-resolution fluorescence microscopy and provide a new path for large-data aided biomedical imaging.

  18. Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

    Science.gov (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.

  19. Community detection for fluorescent lifetime microscopy image segmentation

    Science.gov (United States)

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

    2014-03-01

    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.

  20. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    A. Beaudet

    1998-11-01

    Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

  1. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    Science.gov (United States)

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

    2008-01-01

    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

  2. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

    Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

  3. Reproducibility and Reliability of Repeated Quantitative Fluorescence Angiography

    DEFF Research Database (Denmark)

    Nerup, Nikolaj; Knudsen, Kristine Bach Korsholm; Ambrus, Rikard

    2017-01-01

    INTRODUCTION: When using fluorescence angiography (FA) in perioperative perfusion assessment, repeated measures with re-injections of fluorescent dye (ICG) may be required. However, repeated injections may cause saturation of dye in the tissue, exceeding the limit of fluorescence intensity...... that the camera can detect. As the emission of fluorescence is dependent of the excitatory light intensity, reduction of this may solve the problem. The aim of the present study was to investigate the reproducibility and reliability of repeated quantitative FA during a reduction of excitatory light....

  4. Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

    Science.gov (United States)

    Seacor, Taylor; Howell, Carina

    2013-03-01

    Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

  5. Observation of self-assembled fluorescent beads by scanning near-field optical microscopy and atomic force microscopy

    International Nuclear Information System (INIS)

    Oh, Y.J.; Jo, W.; Kim, Min-Gon; Kyu Park, Hyun; Hyun Chung, Bong

    2006-01-01

    Optical response and topography of fluorescent latex beads both on flat self-assembled monolayer and on a micron-patterned surface with poly(dimethylsiloxane) are studied. Scanning near-field optical microscopy and atomic force microscopy were utilized together for detecting fluorescence and imaging topography of the patterned latex beads, respectively. As a result, the micro-patterned latex beads where a specific chemical binding occurred show a strong signal, whereas no signals are observed in the case of nonspecific binding. With fluorescein isothiocyanate (FITC), it is convenient to measure fluorescence signal from the patterned beads allowing us to monitor the small balls of fluorescent latex

  6. Molecular recognition of DNA-protein complexes: A straightforward method combining scanning force and fluorescence microscopy

    NARCIS (Netherlands)

    H. Sanchez (Humberto); R. Kanaar (Roland); C. Wyman (Claire)

    2010-01-01

    textabstractCombining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic

  7. Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Molly McQuilken

    2017-05-01

    Full Text Available Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.

  8. Confocal fluorescence microscopy for minimal-invasive tumor diagnosis

    International Nuclear Information System (INIS)

    Zenzinger, M.; Bille, J.

    2000-01-01

    The goal of the project ''stereotactic laser-neurosurgery'' is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis. (orig.)

  9. Rapid analysis and exploration of fluorescence microscopy images.

    Science.gov (United States)

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

    2014-03-19

    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.

  10. Quantitative fluorescence spectroscopy in turbid media using fluorescence differential path length spectroscopy

    NARCIS (Netherlands)

    Amelink, Arjen; Kruijt, Bastiaan; Robinson, Dominic J.; Sterenborg, Henricus J. C. M.

    2008-01-01

    We have developed a new technique, fluorescence differential path length spectroscopy (FDPS), that enables the quantitative investigation of fluorophores in turbid media. FDPS measurements are made with the same probe geometry as differential path length spectroscopy (DPS) measurements. Phantom

  11. Quantitative immunofluorescence microscopy of renal glomeruli from mice exhibiting murien lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, R H [Lawrence Livermore Lab., CA; Greenspan, J S; Moore, D II; Talal, N; Roubinian, J R

    1981-01-01

    Pathologic changes in renal glomeruli of mice with systemic murine lupus erythematosus were quantified using microfluorophotometry. Cryostat sections were taken from kidneys of affected mice, stained with fluorescein-conjugated anti-mouse immunoglobulin, and the extent of immune complex glomerulonephritis was determined. A subjective microscopic examination procedure, which has been used previously, was compared with quantitative microfluorophotometry and a close correlation between the results using each of the two methods was found. Since the microfluorometric procedure measures the total fluorescence per glomerulus, subjective microscopy must estimate that same quantity in a linear fashion. The present advance in measuring capability indicates good potential for rapid, quantitive measurements for further studies on systemic lupus erythematosus, and on other tissue sections stained with fluorescent antibodies.

  12. 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.

    2004-01-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10 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.)

  13. Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong Yongpeng [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China)], E-mail: yongpengt@yahoo.com.cn; Li Changming [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Liang Feng [Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen Jianmin [Shenzhen Municipal Hospital for Chronic Disease Control and Prevention, Guangdong 518020 (China); Zhang Hong; Liu Guoqing; Sun Huibin [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China); Luong, John H.T. [Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, H4P 2R2 (Canada)

    2008-12-15

    Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al{sub 2}O{sub 3} and TiO{sub 2}) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl{sub 2}) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al{sub 2}O{sub 3} and TiO{sub 2} nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe{sub 2}O{sub 3} nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

  14. Nonlinear excitation fluorescence microscopy: source considerations for biological applications

    Science.gov (United States)

    Wokosin, David L.

    2008-02-01

    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.

  15. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    Science.gov (United States)

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  16. Comparison of segmentation algorithms for fluorescence microscopy images of cells.

    Science.gov (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

    2011-07-01

    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.

  17. Fast Segmentation From Blurred Data in 3D Fluorescence Microscopy.

    Science.gov (United States)

    Storath, Martin; Rickert, Dennis; Unser, Michael; Weinmann, Andreas

    2017-10-01

    We develop a fast algorithm for segmenting 3D images from linear measurements based on the Potts model (or piecewise constant Mumford-Shah model). To that end, we first derive suitable space discretizations of the 3D Potts model, which are capable of dealing with 3D images defined on non-cubic grids. Our discretization allows us to utilize a specific splitting approach, which results in decoupled subproblems of moderate size. The crucial point in the 3D setup is that the number of independent subproblems is so large that we can reasonably exploit the parallel processing capabilities of the graphics processing units (GPUs). Our GPU implementation is up to 18 times faster than the sequential CPU version. This allows to process even large volumes in acceptable runtimes. As a further contribution, we extend the algorithm in order to deal with non-negativity constraints. We demonstrate the efficiency of our method for combined image deconvolution and segmentation on simulated data and on real 3D wide field fluorescence microscopy data.

  18. Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments.

    Science.gov (United States)

    LaGraff, John R; Chu-LaGraff, Quynh

    2006-05-09

    Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.

  19. A method for improved clustering and classification of microscopy images using quantitative co-localization coefficients

    LENUS (Irish Health Repository)

    Singan, Vasanth R

    2012-06-08

    AbstractBackgroundThe localization of proteins to specific subcellular structures in eukaryotic cells provides important information with respect to their function. Fluorescence microscopy approaches to determine localization distribution have proved to be an essential tool in the characterization of unknown proteins, and are now particularly pertinent as a result of the wide availability of fluorescently-tagged constructs and antibodies. However, there are currently very few image analysis options able to effectively discriminate proteins with apparently similar distributions in cells, despite this information being important for protein characterization.FindingsWe have developed a novel method for combining two existing image analysis approaches, which results in highly efficient and accurate discrimination of proteins with seemingly similar distributions. We have combined image texture-based analysis with quantitative co-localization coefficients, a method that has traditionally only been used to study the spatial overlap between two populations of molecules. Here we describe and present a novel application for quantitative co-localization, as applied to the study of Rab family small GTP binding proteins localizing to the endomembrane system of cultured cells.ConclusionsWe show how quantitative co-localization can be used alongside texture feature analysis, resulting in improved clustering of microscopy images. The use of co-localization as an additional clustering parameter is non-biased and highly applicable to high-throughput image data sets.

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

    Science.gov (United States)

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

    2016-01-01

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

  1. 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.

  2. An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy

    Science.gov (United States)

    Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

    2017-03-01

    While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in

  3. Quantification of confocal fluorescence microscopy for the detection of cervical intraepithelial neoplasia.

    Science.gov (United States)

    Sheikhzadeh, Fahime; Ward, Rabab K; Carraro, Anita; Chen, Zhao Yang; van Niekerk, Dirk; Miller, Dianne; Ehlen, Tom; MacAulay, Calum E; Follen, Michele; Lane, Pierre M; Guillaud, Martial

    2015-10-24

    Cervical cancer remains a major health problem, especially in developing countries. Colposcopic examination is used to detect high-grade lesions in patients with a history of abnormal pap smears. New technologies are needed to improve the sensitivity and specificity of this technique. We propose to test the potential of fluorescence confocal microscopy to identify high-grade lesions. We examined the quantification of ex vivo confocal fluorescence microscopy to differentiate among normal cervical tissue, low-grade Cervical Intraepithelial Neoplasia (CIN), and high-grade CIN. We sought to (1) quantify nuclear morphology and tissue architecture features by analyzing images of cervical biopsies; and (2) determine the accuracy of high-grade CIN detection via confocal microscopy relative to the accuracy of detection by colposcopic impression. Forty-six biopsies obtained from colposcopically normal and abnormal cervical sites were evaluated. Confocal images were acquired at different depths from the epithelial surface and histological images were analyzed using in-house software. The features calculated from the confocal images compared well with those features obtained from the histological images and histopathological reviews of the specimens (obtained by a gynecologic pathologist). The correlations between two of these features (the nuclear-cytoplasmic ratio and the average of three nearest Delaunay-neighbors distance) and the grade of dysplasia were higher than that of colposcopic impression. The sensitivity of detecting high-grade dysplasia by analysing images collected at the surface of the epithelium, and at 15 and 30 μm below the epithelial surface were respectively 100, 100, and 92 %. Quantitative analysis of confocal fluorescence images showed its capacity for discriminating high-grade CIN lesions vs. low-grade CIN lesions and normal tissues, at different depth of imaging. This approach could be used to help clinicians identify high-grade CIN in clinical

  4. Single beam Fourier transform digital holographic quantitative phase microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Anand, A., E-mail: arun-nair-in@yahoo.com; Chhaniwal, V. K.; Mahajan, S.; Trivedi, V. [Optics Laboratory, Applied Physics Department, Faculty of Technology and Engineering, M.S. University of Baroda, Vadodara 390001 (India); Faridian, A.; Pedrini, G.; Osten, W. [Institut für Technische Optik, Universität Stuttgart, Pfaffenwaldring 9, 70569 Stuttgart (Germany); Dubey, S. K. [Siemens Technology and Services Pvt. Ltd, Corporate Technology—Research and Technology Centre, Bangalore 560100 (India); Javidi, B. [Department of Electrical and Computer Engineering, U-4157, University of Connecticut, Storrs, Connecticut 06269-2157 (United States)

    2014-03-10

    Quantitative phase contrast microscopy reveals thickness or height information of a biological or technical micro-object under investigation. The information obtained from this process provides a means to study their dynamics. Digital holographic (DH) microscopy is one of the most used, state of the art single-shot quantitative techniques for three dimensional imaging of living cells. Conventional off axis DH microscopy directly provides phase contrast images of the objects. However, this process requires two separate beams and their ratio adjustment for high contrast interference fringes. Also the use of two separate beams may make the system more vulnerable to vibrations. Single beam techniques can overcome these hurdles while remaining compact as well. Here, we describe the development of a single beam DH microscope providing whole field imaging of micro-objects. A hologram of the magnified object projected on to a diffuser co-located with a pinhole is recorded with the use of a commercially available diode laser and an arrayed sensor. A Fourier transform of the recorded hologram directly yields the complex amplitude at the image plane. The method proposed was investigated using various phase objects. It was also used to image the dynamics of human red blood cells in which sub-micrometer level thickness variation were measurable.

  5. Single-molecule fluorescence microscopy review: shedding new light on old problems.

    Science.gov (United States)

    Shashkova, Sviatlana; Leake, Mark C

    2017-08-31

    Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. © 2017 The Author(s).

  6. Automated quantitative cytological analysis using portable microfluidic microscopy.

    Science.gov (United States)

    Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

    2016-06-01

    In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Shedding quantitative fluorescence light on novel regulatory mechanisms in skeletal biomedicine and biodentistry.

    Science.gov (United States)

    Lee, Ji-Won; Iimura, Tadahiro

    2017-02-01

    Digitalized fluorescence images contain numerical information such as color (wavelength), fluorescence intensity and spatial position. However, quantitative analyses of acquired data and their validation remained to be established. Our research group has applied quantitative fluorescence imaging on tissue sections and uncovered novel findings in skeletal biomedicine and biodentistry. This review paper includes a brief background of quantitative fluorescence imaging and discusses practical applications by introducing our previous research. Finally, the future perspectives of quantitative fluorescence imaging are discussed.

  8. Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Kim Myung K

    2011-09-01

    Full Text Available Abstract Background Total internal reflection fluorescence microscopy (TIRFM is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein and clathrin light chain (a vesicle coat protein support our theoretical analysis. Conclusions These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

  9. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy

    NARCIS (Netherlands)

    Faas, F.G.A.; Bárcena, M.A.; Agronskaia, A.V.; Gerritsen, H.C.; Moscicka, K.B.; Diebolder, C.A.; Driel, L.F.; Limpens, R.W.A.L.; Bos, E.; Ravelli, R.B.G.; Koning, R.I.; Koster, A.J.

    2013-01-01

    Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain

  10. Activated sludge characterization through microscopy: A review on quantitative image analysis and chemometric techniques

    Energy Technology Data Exchange (ETDEWEB)

    Mesquita, Daniela P. [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Amaral, A. Luís [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Instituto Politécnico de Coimbra, ISEC, DEQB, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra (Portugal); Ferreira, Eugénio C., E-mail: ecferreira@deb.uminho.pt [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal)

    2013-11-13

    Graphical abstract: -- Highlights: •Quantitative image analysis shows potential to monitor activated sludge systems. •Staining techniques increase the potential for detection of operational problems. •Chemometrics combined with quantitative image analysis is valuable for process monitoring. -- Abstract: In wastewater treatment processes, and particularly in activated sludge systems, efficiency is quite dependent on the operating conditions, and a number of problems may arise due to sludge structure and proliferation of specific microorganisms. In fact, bacterial communities and protozoa identification by microscopy inspection is already routinely employed in a considerable number of cases. Furthermore, quantitative image analysis techniques have been increasingly used throughout the years for the assessment of aggregates and filamentous bacteria properties. These procedures are able to provide an ever growing amount of data for wastewater treatment processes in which chemometric techniques can be a valuable tool. However, the determination of microbial communities’ properties remains a current challenge in spite of the great diversity of microscopy techniques applied. In this review, activated sludge characterization is discussed highlighting the aggregates structure and filamentous bacteria determination by image analysis on bright-field, phase-contrast, and fluorescence microscopy. An in-depth analysis is performed to summarize the many new findings that have been obtained, and future developments for these biological processes are further discussed.

  11. New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.

    Science.gov (United States)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-02-15

    Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.

    Directory of Open Access Journals (Sweden)

    Robert P J Nieuwenhuizen

    Full Text Available Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.

  13. Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

    Science.gov (United States)

    Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

    2014-05-01

    The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

  14. Towards single molecule biosensors using super-resolution fluorescence microscopy.

    Science.gov (United States)

    Lu, Xun; Nicovich, Philip R; Gaus, Katharina; Gooding, J Justin

    2017-07-15

    Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers

    Energy Technology Data Exchange (ETDEWEB)

    Schellenberger, Pascale [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Kaufmann, Rainer [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU (United Kingdom); Siebert, C. Alistair; Hagen, Christoph [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Wodrich, Harald [Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, University of Bordeaux SEGALEN, 146 rue Leo Seignat, 33076 Bordeaux (France); Grünewald, Kay, E-mail: kay@strubi.ox.ac.uk [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2014-08-01

    Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. - Highlights: • Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. • TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. • FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. • Adenovirus particles were localised with an average correlation precision of 63 nm.

  16. High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers

    International Nuclear Information System (INIS)

    Schellenberger, Pascale; Kaufmann, Rainer; Siebert, C. Alistair; Hagen, Christoph; Wodrich, Harald; Grünewald, Kay

    2014-01-01

    Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. - Highlights: • Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. • TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. • FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. • Adenovirus particles were localised with an average correlation precision of 63 nm

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

    Directory of Open Access Journals (Sweden)

    Shilpa Dilipkumar

    2015-03-01

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

  18. Automated Quantitative Rare Earth Elements Mineralogy by Scanning Electron Microscopy

    Science.gov (United States)

    Sindern, Sven; Meyer, F. Michael

    2016-09-01

    Increasing industrial demand of rare earth elements (REEs) stems from the central role they play for advanced technologies and the accelerating move away from carbon-based fuels. However, REE production is often hampered by the chemical, mineralogical as well as textural complexity of the ores with a need for better understanding of their salient properties. This is not only essential for in-depth genetic interpretations but also for a robust assessment of ore quality and economic viability. The design of energy and cost-efficient processing of REE ores depends heavily on information about REE element deportment that can be made available employing automated quantitative process mineralogy. Quantitative mineralogy assigns numeric values to compositional and textural properties of mineral matter. Scanning electron microscopy (SEM) combined with a suitable software package for acquisition of backscatter electron and X-ray signals, phase assignment and image analysis is one of the most efficient tools for quantitative mineralogy. The four different SEM-based automated quantitative mineralogy systems, i.e. FEI QEMSCAN and MLA, Tescan TIMA and Zeiss Mineralogic Mining, which are commercially available, are briefly characterized. Using examples of quantitative REE mineralogy, this chapter illustrates capabilities and limitations of automated SEM-based systems. Chemical variability of REE minerals and analytical uncertainty can reduce performance of phase assignment. This is shown for the REE phases parisite and synchysite. In another example from a monazite REE deposit, the quantitative mineralogical parameters surface roughness and mineral association derived from image analysis are applied for automated discrimination of apatite formed in a breakdown reaction of monazite and apatite formed by metamorphism prior to monazite breakdown. SEM-based automated mineralogy fulfils all requirements for characterization of complex unconventional REE ores that will become

  19. Quantitative x-ray fluorescent analysis using fundamental parameters

    International Nuclear Information System (INIS)

    Sparks, C.J. Jr.

    1976-01-01

    A monochromatic source of x-rays for sample excitation permits the use of pure elemental standards and relatively simple calculations to convert the measured fluorescent intensities to an absolute basis of weight per unit weight of sample. Only the mass absorption coefficients of the sample for the exciting and the fluorescent radiation need be determined. Besides the direct measurement of these absorption coefficients in the sample, other techniques are considered which require fewer sample manipulations and measurements. These fundamental parameters methods permit quantitative analysis without recourse to the time-consuming process of preparing nearly identical standards

  20. Quantitative analysis with energy dispersive X-ray fluorescence analyser

    International Nuclear Information System (INIS)

    Kataria, S.K.; Kapoor, S.S.; Lal, M.; Rao, B.V.N.

    1977-01-01

    Quantitative analysis of samples using radioisotope excited energy dispersive x-ray fluorescence system is described. The complete set-up is built around a locally made Si(Li) detector x-ray spectrometer with an energy resolution of 220 eV at 5.94 KeV. The photopeaks observed in the x-ray fluorescence spectra are fitted with a Gaussian function and the intensities of the characteristic x-ray lines are extracted, which in turn are used for calculating the elemental concentrations. The results for a few typical cases are presented. (author)

  1. Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy

    International Nuclear Information System (INIS)

    Vanga, Sudheer Kumar; Mi, Zhaohong; Koh, Long Cheng; Tao, Ye; Bettiol, Andrew A.; Watt, Frank

    2015-01-01

    Ion beam induced fluorescence microscopy is a new imaging technique which has the potential to achieve sub-50 nm spatial resolution fluorescence images. Currently the resolution of the technique has been limited to around 150 nm mainly because of inefficient collection and detection of emitted photons from the sample. To overcome this limitation, a new light collection system based on a custom made parabolic mirror is employed to enhance the fluorescence collection. The custom made mirror is designed so as to obtain both structural (scanning transmission ion microscopy) and ion beam induced fluorescence imaging simultaneously. The design and characterization of the parabolic mirror is discussed in detail

  2. Imaging subsurface damage of grinded fused silica optics by confocal fluorescence microscopy

    International Nuclear Information System (INIS)

    Neauport, J.; Cormont, P.; Destribats, J.; Legros, P.; Ambard, C.

    2009-01-01

    We report an experimental investigation of fluorescence confocal microscopy as a tool to measure subsurface damage on grinded fused silica optics. Confocal fluorescence microscopy was performed with an excitation at the wavelength of 405 nm on fixed abrasive diamond grinded fused silica samples. We detail the measured fluorescence spectrums and compare them to those of oil based coolants and grinding slurries. We evidence that oil based coolant used in diamond grinding induces a fluorescence that marks the subsurface damages and eases its observation. Such residual traces might also be involved in the laser damage process. (authors)

  3. Following Intracellular Cholesterol Transport by Linear and Non-Linear Optical Microscopy of Intrinsically Fluorescent Sterols

    DEFF Research Database (Denmark)

    Wustner, D.

    2012-01-01

    Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow...... is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image...... analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented....

  4. Assessment of LED fluorescence microscopy for the diagnosis of Plasmodium falciparum infections in Gabon

    Directory of Open Access Journals (Sweden)

    Biallas Barbara

    2011-07-01

    Full Text Available Abstract Background Rapid and accurate diagnosis of malaria is central to clinical management and the prevention of drug-overuse, which may lead to resistance development, toxicity and economic losses. So far, light microscopy (LM of Giemsa-stained thick blood smears is the gold standard. Under optimal conditions the procedure is fast and reliable; nevertheless a gain in speed would be a great advantage. Rapid diagnosis tests are an alternative, although they cost more and give qualitative instead of quantitative results. Light-emitting diode (LED fluorescence microscopy (ledFM 400 ×, 1000 × may offer a reliable and cheap alternative, which can be used at the point of care. Methods LedFM and conventional fluorescence microscopy (uvFM were compared to LM in 210 samples from patients with history of fever in the last 24 hours admitted to the Albert Schweitzer Hospital in Lambaréné, Gabon. Results Sensitivities were 99.1% for ledFM and 97.0% for uvFM, specificities 90.7% for ledFM 400 × and 92.6% for ledFM 1000 × and uvFM. High agreement was found in Bland-Altman-plot and Kappa coefficient (ledFM 1000 ×: 0.914, ledFM 400 × and uvFM: 0.895. The time to diagnosis for both FM methods was shorter compared to LM (LM: 43 min, uvFM: 16 min, ledFM 1000 ×: 14 min, ledFM 400 ×: 10 min. Conclusion ledFM is a reliable, accurate, fast and inexpensive tool for daily routine malaria diagnosis and may be used as a point of care diagnostic tool.

  5. Raman and fluorescence microscopy to study the internalization and dissolution of photosensitizer nanoparticles into living cells

    Science.gov (United States)

    Scalfi-Happ, Claudia; Steiner, Rudolf; Wittig, Rainer; Graefe, Susanna; Ryabova, Anastasia; Loschenov, Victor

    2015-07-01

    In this present study we applied Raman and fluorescence microscopy to investigate the internalisation, cellular distribution and effects on cell metabolism of photosensitizer nanoparticles for photodynamic therapy in fibroblasts and macrophages.

  6. Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

    Science.gov (United States)

    Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

    The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

  7. Fluorescent foci quantitation for high-throughput analysis

    Directory of Open Access Journals (Sweden)

    Elena Ledesma-Fernández

    2015-06-01

    Full Text Available A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this ‘FociQuant’ tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells.

  8. Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

    Science.gov (United States)

    Tung, Oi-Hong; Lee, Shyh-Yuan; Lai, Yu-Lin; Chen, How-Foo

    2011-06-01

    Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

  9. On the mobility of biomolecules : a fluorescence microscopy approach

    NARCIS (Netherlands)

    Bogaart, Geert van den

    2008-01-01

    This thesis describes the development and application of a number of fluorescence spectroscopy related techniques (FCS, FRAP, DCFBA) to measure diffusion of biomolecules in cells, in membranes and through membrane pores.

  10. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

  11. Solid-immersion fluorescence microscopy with increased emission and super resolution

    Energy Technology Data Exchange (ETDEWEB)

    Liau, Z. L.; Porter, J. M. [Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, Massachusetts 02420 (United States); Liau, A. A.; Chen, J. J. [Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Salmon, W. C. [Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Sheu, S. S. [Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107 (United States)

    2015-01-07

    We investigate solid-immersion fluorescence microscopy suitable for super-resolution nanotechnology and biological imaging, and have observed limit of resolution as small as 15 nm with microspheres, mitochondria, and chromatin fibers. We have further observed that fluorescence efficiency increases with excitation power density, implicating appreciable stimulated emission and increased resolution. We discuss potential advantages of the solid-immersion microscopy, including combined use with previously established super-resolution techniques for reaching deeper beyond the conventional diffraction limit.

  12. Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

    International Nuclear Information System (INIS)

    Spoeri, Udo; Failla, Antonio Virgilio; Cremer, Christoph

    2004-01-01

    Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of 'colocalization volumes' between SMI nanosizing and conventional confocal laser scanning microscopy

  13. Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

    Science.gov (United States)

    Bergeest, Jan-Philip; Rohr, Karl

    2012-10-01

    In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets 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 consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Quantitative tracking of tumor cells in phase-contrast microscopy exploiting halo artifact pattern

    Science.gov (United States)

    Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

    2012-03-01

    Tumor cell morphology is closely related to its invasiveness characteristics and migratory behaviors. An invasive tumor cell has a highly irregular shape, whereas a spherical cell is non-metastatic. Thus, quantitative analysis of cell features is crucial to determine tumor malignancy or to test the efficacy of anticancer treatment. We use phase-contrast microscopy to analyze single cell morphology and to monitor its change because it enables observation of long-term activity of living cells without photobleaching and phototoxicity, which is common in other fluorescence-labeled microscopy. Despite this advantage, there are image-level drawbacks to phase-contrast microscopy, such as local light effect and contrast interference ring, among others. Thus, we first applied a local filter to compensate for non-uniform illumination. Then, we used intensity distribution information to detect the cell boundary. In phase-contrast microscopy images, the cell normally appears as a dark region surrounded by a bright halo. As the halo artifact around the cell body is minimal and has an asymmetric diffusion pattern, we calculated the cross-sectional plane that intersected the center of each cell and was orthogonal to the first principal axis. Then, we extracted the dark cell region by level set. However, a dense population of cultured cells still rendered single-cell analysis difficult. Finally, we measured roundness and size to classify tumor cells into malignant and benign groups. We validated segmentation accuracy by comparing our findings with manually obtained results.

  15. Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

    DEFF Research Database (Denmark)

    R. Carro-Temboury, Miguel; Arppe, Riikka Matleena; Hempel, Casper

    2017-01-01

    The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance...

  16. Hybrid Rayleigh, Raman and TPE fluorescence spectral confocal microscopy of living cells

    NARCIS (Netherlands)

    Pully, V.V.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2010-01-01

    A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low-wavenumber-resolution Raman imaging, Rayleigh scatter imaging and two-photon fluorescence (TPE) spectral imaging, fast ‘amplitude-only’ TPE-fluorescence imaging and high-spectral-resolution Raman imaging.

  17. Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

    NARCIS (Netherlands)

    Moers, M.H.P.; Moers, M.H.P.; Kalle, W.H.J.; Kalle, W.H.J.; Ruiter, A.G.T.; Wiegant, J.C.A.G.; Raap, A.K.; Greve, Jan; de Grooth, B.G.; van Hulst, N.F.

    1996-01-01

    Fluorescence in situ hybridization o­n human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments o­n a single

  18. Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

    1998-03-01

    Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

  19. Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

    Science.gov (United States)

    Braaf, Boy; de Boer, Johannes F

    2017-03-20

    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.

  20. Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

    Science.gov (United States)

    Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

    2012-05-01

    Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  1. Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas.

    Science.gov (United States)

    Sanai, Nader; Snyder, Laura A; Honea, Norissa J; Coons, Stephen W; Eschbacher, Jennifer M; Smith, Kris A; Spetzler, Robert F

    2011-10-01

    Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

  2. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  3. Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Rufeng Li

    2017-11-01

    Full Text Available This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1 Gaussian filtering to remove the noise of overall fluorescent targets, (2 a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3 an red maximizing inter-class variance thresholding method (OTSU to segment the enhanced image for getting the binary map of the overall micro-droplets, (4 a circular Hough transform (CHT method to detect overall micro-droplets and (5 an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

  4. Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

    Science.gov (United States)

    Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

    2017-11-21

    This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

  5. Quantitative micro x-ray fluorescence analyses without reference standard material; Referenzprobenfreie quantitative Mikro-Roentgenfluoreszenzanalyse

    Energy Technology Data Exchange (ETDEWEB)

    Wolff, Timo

    2009-07-15

    X-ray fluorescence analysis (XRF) is a standard method for non-destructive investigations. Due to the development of polycapillary optics and SDDdetectors requiring no cooling with liquid nitrogen, XRF becomes a suitable method for a large number of applications, e. g. for the analysis of objects in arts and archaeology. Spectrometers developed for those purposes allow investigations outside of laboratories und provide excitation areas with diameters of 10-70 {mu}m. In most applications, quantification of XRF data is realized by the usage of standard reference materials. Due to absorption processes in the samples the accuracy of the results depends strongly on the similarity of the sample and the reference standard. In cases where no suitable references are available, quantification can be done based on the ''fundamental parameter (fp) method''. This quantification procedure is based on a set of equations describing the fluorescence production and detection mathematical. The cross sections for the interaction of x-rays with matter can be taken from different databases. During an iteration process the element concentrations can be determined. Quantitative XRF based on fundamental parameters requires an accurate knowledge of the excitation spectrum. In case of a conventional setup this spectrum is given by the X-ray tube spectrum and can be calculated. The use of polycapillary optics in micro-XRF spectrometers changes the spectral distribution of the excitation radiation. For this reason it is necessary to access the transmission function of the used optic. The aim of this work is to find a procedure to describe this function for routine quantification based on fundamental parameters. Most of the measurements have been carried out using a commercial spectrometer developed for applications in arts and archaeology. On the one hand the parameters of the lens, used in the spectrometer, have been investigated by different experimental characterization

  6. Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

    Science.gov (United States)

    Baumann, Gerd; Place, Robert F; Földes-Papp, Zeno

    2010-08-01

    In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent

  7. Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

    Science.gov (United States)

    Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

    2017-05-01

    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.

  8. Boundary fitting based segmentation of fluorescence microscopy images

    Science.gov (United States)

    Lee, Soonam; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

    2015-03-01

    Segmentation is a fundamental step in quantifying characteristics, such as volume, shape, and orientation of cells and/or tissue. However, quantification of these characteristics still poses a challenge due to the unique properties of microscopy volumes. This paper proposes a 2D segmentation method that utilizes a combination of adaptive and global thresholding, potentials, z direction refinement, branch pruning, end point matching, and boundary fitting methods to delineate tubular objects in microscopy volumes. Experimental results demonstrate that the proposed method achieves better performance than an active contours based scheme.

  9. Fast and accurate automated cell boundary determination for fluorescence microscopy

    Science.gov (United States)

    Arce, Stephen Hugo; Wu, Pei-Hsun; Tseng, Yiider

    2013-07-01

    Detailed measurement of cell phenotype information from digital fluorescence images has the potential to greatly advance biomedicine in various disciplines such as patient diagnostics or drug screening. Yet, the complexity of cell conformations presents a major barrier preventing effective determination of cell boundaries, and introduces measurement error that propagates throughout subsequent assessment of cellular parameters and statistical analysis. State-of-the-art image segmentation techniques that require user-interaction, prolonged computation time and specialized training cannot adequately provide the support for high content platforms, which often sacrifice resolution to foster the speedy collection of massive amounts of cellular data. This work introduces a strategy that allows us to rapidly obtain accurate cell boundaries from digital fluorescent images in an automated format. Hence, this new method has broad applicability to promote biotechnology.

  10. Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

    Directory of Open Access Journals (Sweden)

    Morales Andrea

    2008-05-01

    Full Text Available Abstract Background The isolation of green fluorescent protein (GFP and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types. Results We generated a transgenic Arabidopsis plant that contains four spectrally different fluorescent proteins. Nuclei, plastids, mitochondria and plasma membranes were genetically tagged with cyan, red, yellow and green fluorescent proteins, respectively. In addition, methods to track nuclei, mitochondria and chloroplasts and quantify the interaction between these organelles at a submicron resolution were developed. These analyzes revealed that N-ethylmaleimide disrupts nuclear-mitochondrial but not nuclear-plastids interactions in root epidermal cells of live Arabidopsis seedlings. Conclusion We developed a tool and associated methods for analyzing the complex dynamic of organelle-organelle interactions in real time in planta. Homozygous transgenic Arabidopsis (Kaleidocell is available through Arabidopsis Biological Resource Center.

  11. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    Science.gov (United States)

    Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

    2008-02-01

    Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

  12. Local Delivery of Fluorescent Dye For Fiber-Optics Confocal Microscopy of the Living Heart

    Directory of Open Access Journals (Sweden)

    Chao eHuang

    2014-09-01

    Full Text Available Fiber-optics confocal microscopy (FCM is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release versus foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  13. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    Science.gov (United States)

    Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  14. Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)

    Science.gov (United States)

    Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-02-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

  15. Quantitative analysis by computer controlled X-ray fluorescence spectrometer

    International Nuclear Information System (INIS)

    Balasubramanian, T.V.; Angelo, P.C.

    1981-01-01

    X-ray fluorescence spectroscopy has become a widely accepted method in the metallurgical field for analysis of both minor and major elements. As encountered in many other analytical techniques, the problem of matrix effect generally known as the interelemental effects is to be dealt with effectively in order to make the analysis accurate. There are several methods by which the effects of matrix on the analyte are minimised or corrected for and the mathematical correction is one among them. In this method the characteristic secondary X-ray intensities are measured from standard samples and correction coefficients. If any, for interelemental effects are evaluated by mathematical calculations. This paper describes attempts to evaluate the correction coefficients for interelemental effects by multiple linear regression programmes using a computer for the quantitative analysis of stainless steel and a nickel base cast alloy. The quantitative results obtained using this method for a standard stainless steel sample are compared with the given certified values. (author)

  16. 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

    2010-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-19

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

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies

    Energy Technology Data Exchange (ETDEWEB)

    Cazaux, Séverine; Sadoun, Anaïs; Biarnes-Pelicot, Martine; Martinez, Manuel; Obeid, Sameh [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Bongrand, Pierre [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); APHM, Hôpital de la Conception, Laboratoire d’Immunologie, Marseille F-13385 (France); Limozin, Laurent [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Puech, Pierre-Henri, E-mail: pierre-henri.puech@inserm.fr [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France)

    2016-01-15

    A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand. - Highlights: • A signal coupling AFM and fluorescence microscopy was characterized for soft cantilevers. • It can be used as an intrinsic timer to synchronize images and forces. • Mechanical stimulation of single immune cells while recording calcium fluxes was detailed. • Light-induced mechanical modifications of lymphocytes using a PA-Rac protein were demonstrated. • The precautions and limitations of use of this effect were presented.

  20. Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

    Science.gov (United States)

    Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

    2014-02-01

    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.

  1. Absorption correction factor in X-ray fluorescent quantitative analysis

    International Nuclear Information System (INIS)

    Pimjun, S.

    1994-01-01

    An experiment on absorption correction factor in X-ray fluorescent quantitative analysis were carried out. Standard samples were prepared from the mixture of Fe 2 O 3 and tapioca flour at various concentration of Fe 2 O 3 ranging from 5% to 25%. Unknown samples were kaolin containing 3.5% to-50% of Fe 2 O 3 Kaolin samples were diluted with tapioca flour in order to reduce the absorption of FeK α and make them easy to prepare. Pressed samples with 0.150 /cm 2 and 2.76 cm in diameter, were used in the experiment. Absorption correction factor is related to total mass absorption coefficient (χ) which varied with sample composition. In known sample, χ can be calculated by conveniently the formula. However in unknown sample, χ can be determined by Emission-Transmission method. It was found that the relationship between corrected FeK α intensity and contents of Fe 2 O 3 in these samples was linear. This result indicate that this correction factor can be used to adjust the accuracy of X-ray intensity. Therefore, this correction factor is essential in quantitative analysis of elements comprising in any sample by X-ray fluorescent technique

  2. Fused oblique incidence reflectometry and confocal fluorescence microscopy

    Science.gov (United States)

    Risi, Matthew D.; Rouse, Andrew R.; Gmitro, Arthur F.

    2011-03-01

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure, but relies on exogenous fluorophores, has a relatively limited penetration depth (100 μm) and field of view (700 μm), and produces a high rate of detailed information to the user. A new catheter based multi-modal system has been designed that combines confocal imaging and oblique incidence reflectometry (OIR), which is a non-invasive method capable of rapidly extracting tissue absorption, μa, and reduced scattering, μ's, spectra from tissue. The system builds on previous developments of a custom slit-scan multi-spectral confocal microendoscope and is designed to rapidly switch between diffuse spectroscopy and confocal fluorescence imaging modes of operation. An experimental proof-of-principle catheter has been developed that consists of a fiber bundle for traditional confocal fluorescence imaging and a single OIR source fiber which is manually redirected at +/- 26 degrees. Diffusely scattered light from each orientation of the source fiber is collected via the fiber bundle, with a frame of data representing spectra collected at a range of distances from the OIR source point. Initial results with intralipid phantoms show good agreement to published data over the 550-650 nm spectral range. We successfully imaged and measured the optical properties of rodent cardiac muscle.

  3. Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

    International Nuclear Information System (INIS)

    Jiang Dafeng; Liu Chunxia; Wang Lei; Jiang Wei

    2010-01-01

    A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 x 10 -14 -3.0 x 10 -12 mol L -1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

  4. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  5. Practical guidelines for implementing adaptive optics in fluorescence microscopy

    Science.gov (United States)

    Wilding, Dean; Pozzi, Paolo; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

    2018-02-01

    In life sciences, interest in the microscopic imaging of increasingly complex three dimensional samples, such as cell spheroids, zebrafish embryos, and in vivo applications in small animals, is growing quickly. Due to the increasing complexity of samples, more and more life scientists are considering the implementation of adaptive optics in their experimental setups. While several approaches to adaptive optics in microscopy have been reported, it is often difficult and confusing for the microscopist to choose from the array of techniques and equipment. In this poster presentation we offer a small guide to adaptive optics providing general guidelines for successful adaptive optics implementation.

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

    Directory of Open Access Journals (Sweden)

    Stefania Seidenari

    2012-01-01

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

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

    Science.gov (United States)

    Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

    2017-01-01

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

    OpenAIRE

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

    2004-01-01

    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...

  9. Segmentation and morphometric analysis of cells from fluorescence microscopy images of cytoskeletons.

    Science.gov (United States)

    Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

    2013-01-01

    We developed a method to reconstruct cell geometry from confocal fluorescence microscopy images of the cytoskeleton. In the method, region growing was implemented twice. First, it was applied to the extracellular regions to differentiate them from intracellular noncytoskeletal regions, which both appear black in fluorescence microscopy imagery, and then to cell regions for cell identification. Analysis of morphological parameters revealed significant changes in cell shape associated with cytoskeleton disruption, which offered insight into the mechanical role of the cytoskeleton in maintaining cell shape. The proposed segmentation method is promising for investigations on cell morphological changes with respect to internal cytoskeletal structures.

  10. Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy.

    Directory of Open Access Journals (Sweden)

    Rainer Kaufmann

    Full Text Available Tight Junctions (TJ regulate paracellular permeability of tissue barriers. Claudins (Cld form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity. To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy.TJ-strands were reconstituted by stable transfection of HEK293 cells with the barrier-forming Cld3 or Cld5. Strands were investigated at cell-cell contacts by Spectral Position Determination Microscopy (SPDM, a method of localization microscopy using standard fluorophores. Extended TJ-networks of Cld3-YFP and Cld5-YFP were observed. For each network, 200,000 to 1,100,000 individual molecules were detected with a mean localization accuracy of ∼20 nm, yielding a mean structural resolution of ∼50 nm. Compared to conventional fluorescence microscopy, this strongly improved the visualization of strand networks and enabled quantitative morphometric analysis. Two populations of elliptic meshes (mean diameter <100 nm and 300-600 nm, respectively were revealed. For Cld5 the two populations were more separated than for Cld3. Discrimination of non-polymeric molecules and molecules within polymeric strands was achieved. For both subtypes of claudins the mean density of detected molecules was similar and estimated to be ∼24 times higher within the strands than outside the strands.The morphometry and single molecule information provided advances the mechanistic analysis of paracellular barriers. Applying this novel method to different TJ-proteins is expected to significantly improve the understanding of TJ on the molecular level.

  11. Multiplexed phase-space imaging for 3D fluorescence microscopy.

    Science.gov (United States)

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

    2017-06-26

    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.

  12. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Jennifer R. McCall

    2014-09-01

    Full Text Available Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/ veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

  13. Resonance fluorescence microscopy via three-dimensional atom localization

    Science.gov (United States)

    Panchadhyayee, Pradipta; Dutta, Bibhas Kumar; Das, Nityananda; Mahapatra, Prasanta Kumar

    2018-02-01

    A scheme is proposed to realize three-dimensional (3D) atom localization in a driven two-level atomic system via resonance fluorescence. The field arrangement for the atom localization involves the application of three mutually orthogonal standing-wave fields and an additional traveling-wave coupling field. We have shown the efficacy of such field arrangement in tuning the spatially modulated resonance in all directions. Under different parametric conditions, the 3D localization patterns originate with various shapes such as sphere, sheets, disk, bowling pin, snake flute, flower vase. High-precision localization is achieved when the radiation field detuning equals twice the combined Rabi frequencies of the standing-wave fields. Application of a traveling-wave field of suitable amplitude at optimum radiation field detuning under symmetric standing-wave configuration leads to 100% detection probability even in sub-wavelength domain. Asymmetric field configuration is also taken into consideration to exhibit atom localization with appreciable precision compared to that of the symmetric case. The momentum distribution of the localized atoms is found to follow the Heisenberg uncertainty principle under the validity of Raman-Nath approximation. The proposed field configuration is suitable for application in the study of atom localization in an optical lattice arrangement.

  14. Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

    DEFF Research Database (Denmark)

    R. Carro-Temboury, Miguel; Arppe, Riikka Matleena; Hempel, Casper

    2017-01-01

    The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance...... the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods......, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging....

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

    Directory of Open Access Journals (Sweden)

    Alexander Boreham

    2016-12-01

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

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

    Science.gov (United States)

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

    2016-12-24

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

  17. Joint volumetric extraction and enhancement of vasculature from low-SNR 3-D fluorescence microscopy images.

    Science.gov (United States)

    Almasi, Sepideh; Ben-Zvi, Ayal; Lacoste, Baptiste; Gu, Chenghua; Miller, Eric L; Xu, Xiaoyin

    2017-03-01

    To simultaneously overcome the challenges imposed by the nature of optical imaging characterized by a range of artifacts including space-varying signal to noise ratio (SNR), scattered light, and non-uniform illumination, we developed a novel method that segments the 3-D vasculature directly from original fluorescence microscopy images eliminating the need for employing pre- and post-processing steps such as noise removal and segmentation refinement as used with the majority of segmentation techniques. Our method comprises two initialization and constrained recovery and enhancement stages. The initialization approach is fully automated using features derived from bi-scale statistical measures and produces seed points robust to non-uniform illumination, low SNR, and local structural variations. This algorithm achieves the goal of segmentation via design of an iterative approach that extracts the structure through voting of feature vectors formed by distance, local intensity gradient, and median measures. Qualitative and quantitative analysis of the experimental results obtained from synthetic and real data prove the effcacy of this method in comparison to the state-of-the-art enhancing-segmenting methods. The algorithmic simplicity, freedom from having a priori probabilistic information about the noise, and structural definition gives this algorithm a wide potential range of applications where i.e. structural complexity significantly complicates the segmentation problem.

  18. Identification of Proliferative and Apoptotic Sertoli Cells Using Fluorescence and Confocal Microscopy.

    Science.gov (United States)

    Martínez-Hernández, Jesús; Seco-Rovira, Vicente; Beltrán-Frutos, Ester; Quesada-Cubo, Victor; Ferrer, Concepción; Pastor, Luis Miguel

    2018-01-01

    Sertoli cells, the testicular somatic cells of the seminiferous epithelium, are vital for the survival of the epithelium. They undergo proliferation and apoptosis during fetal, neonatal, and prepubertal development. Apoptosis is increased in certain situations such as exposure to many substances, for example, toxics, or short photoperiod in the non-breeding season of some mammals. Therefore, it has always been considered that Sertoli cells that reach adulthood are quiescent cells, that is to say, nonproliferative, do not die, are terminally differentiated, and whose numbers remain constant. Recently, a degree of both proliferation and apoptosis has been observed in normal adult conditions, suggesting that consideration of this cell as quiescent may be subject to change. All this make it necessary to use histochemical techniques to demonstrate whether Sertoli cells are undergoing proliferation or apoptosis in histological sections and to allow the qualitative and quantitative study of these. In this chapter, we present two double-staining techniques that can be used for identifying Sertoli cells in proliferation or apoptosis by fluorescence microscopy. In both, the Sertoli cells are identified by an immunohistochemistry for vimentin followed by an immunohistochemistry for PCNA or a TUNEL histochemistry.

  19. Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

    Science.gov (United States)

    Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

    2014-01-01

    Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

  20. Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

    Directory of Open Access Journals (Sweden)

    Xiangdong Kong

    Full Text Available The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR, for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36; trisomy 18 (n = 6; trisomy 13 (n = 4; 45, X (n = 5; 47, XXX (n = 3; 48, XXYY (n = 2; and unaffected controls (n = 40. We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

  1. Circular dichroism probed by two-photon fluorescence microscopy in enantiopure chiral polyfluorene thin films

    NARCIS (Netherlands)

    Savoini, M.; Wu, Xiaofei; Celebrano, M.; Ziegler, J.; Biagioni, P.; Meskers, S.C.J.; Duo, L.; Hecht, B.; Finazzi, M.

    2012-01-01

    Two-photon fluorescence scanning confocal microscopy sensitive to circular dichroism with a diffraction-limited resolution well below 500 nm is demonstrated. With this method, the spatial variation of the circular dichroism of thermally annealed chiral polyfluorene thin films has been imaged. We

  2. Accessibility, Structure and Reactivity of Individual Catalyst Particles Studied by Fluorescence Microscopy

    NARCIS (Netherlands)

    Hendriks, F.C.|info:eu-repo/dai/nl/412642697

    2017-01-01

    This PhD thesis is aimed at using fluorescence microscopy to study accessibility, structure and reactivity of two types of systems. The first part of this thesis is focused on model zeolite crystals. Fundamental insights into the accessibility and internal structure of zeolite powders and crystals

  3. Characterization of tissue autofluorescence in Barrett's esophagus by confocal fluorescence microscopy

    NARCIS (Netherlands)

    Kara, M. A.; DaCosta, R. S.; Streutker, C. J.; Marcon, N. E.; Bergman, J. J. G. H. M.; Wilson, B. C.

    2007-01-01

    High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and

  4. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

    Science.gov (United States)

    Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-06-07

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

  5. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

    Science.gov (United States)

    Faas, F G A; Bárcena, M; Agronskaia, A V; Gerritsen, H C; Moscicka, K B; Diebolder, C A; van Driel, L F; Limpens, R W A L; Bos, E; Ravelli, R B G; Koning, R I; Koster, A J

    2013-03-01

    Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

    Directory of Open Access Journals (Sweden)

    François Vromman

    Full Text Available Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

  7. Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

    Science.gov (United States)

    Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

    2011-03-01

    Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

  8. Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

    Science.gov (United States)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

    Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

  9. Discrimination of Dendrobium officinale and Its Common Adulterants by Combination of Normal Light and Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Chu Chu

    2014-03-01

    Full Text Available The stems of Dendrobium officinale Kimura et Migo, named Tie-pi-shi-hu, is one of the most endangered and precious species in China. Because of its various pharmacodynamic effects, D. officinale is widely recognized as a high-quality health food in China and other countries in south and south-east Asia. With the rising interest of D. officinale, its products have a high price due to a limited supply. This high price has led to the proliferation of adulterants in the market. To ensure the safe use of D. officinale, a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. officinale from three commonly used adulterants including Zi-pi-shi-hu (D. devonianum, Shui-cao-shi-hu (D. aphyllum, Guang-jie-shi-hu (D. gratiosissimum. The result demonstrated that D. officinale could be identified by the characteristic “two hat-shaped” vascular bundle sheath observed under the fluorescence microscopy and the distribution of raphides under normal light microscopy. The other three adulterants could be discriminated by the vascular bundle differences and the distribution of raphides under normal light microscopy. This work indicated that combination of normal light and fluorescence microscopy is a fast and efficient technique to scientifically distinguish D. officinale from the commonly confused species.

  10. 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.

  11. Identification of powdered Chinese herbal medicines by fluorescence microscopy, Part 1: Fluorescent characteristics of mechanical tissues, conducting tissues, and ergastic substances.

    Science.gov (United States)

    Wang, Ya-Qiong; Liang, Zhi-Tao; Li, Qin; Yang, Hua; Chen, Hu-Biao; Zhao, Zhong-Zhen; Li, Ping

    2011-03-01

    The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known. Copyright © 2010 Wiley-Liss, Inc.

  12. Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

    Science.gov (United States)

    Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

    2016-08-16

    The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

  13. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

    Science.gov (United States)

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For man...

  14. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR CALIBRATION, QUANTITATION AND SPECTROSCOPY

    Science.gov (United States)

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  15. Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

    Science.gov (United States)

    Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

    2015-09-01

    Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

  16. Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

    Science.gov (United States)

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

    2017-06-01

    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.

  17. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

    Science.gov (United States)

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  18. Charting Monosynaptic Connectivity Maps by Two-Color Light-Sheet Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Christian J. Niedworok

    2012-11-01

    Full Text Available Cellular resolution three-dimensional (3D visualization of defined, fluorescently labeled long-range neuronal networks in the uncut adult mouse brain has been elusive. Here, a virus-based strategy is described that allowed fluorescent labeling of centrifugally projecting neuronal populations in the ventral forebrain and their directly, monosynaptically connected bulbar interneurons upon a single stereotaxic injection into select neuronal populations. Implementation of improved tissue clearing combined with light-sheet fluorescence microscopy permitted imaging of the resulting connectivity maps in a single whole-brain scan. Subsequent 3D reconstructions revealed the exact distribution of the diverse neuronal ensembles monosynaptically connected with distinct bulbar interneuron populations. Moreover, rehydratation of brains after light-sheet fluorescence imaging enabled the immunohistochemical identification of synaptically connected neurons. Thus, this study describes a method for identifying monosynaptic connectivity maps from distinct, virally labeled neuronal populations that helps in better understanding of information flow in neural systems.

  19. Structural and dynamical aspects of skin studied by multiphoton excitation fluorescence microscopy-based methods

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Brewer, Jonathan R.; Bagatolli, Luis

    2013-01-01

    ' parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe--2,7-bis-(2......-carboxyethyl)-5-(and-6)-carboxyfluorescein) and diffusion coefficients of distinct fluorescence probes (raster imaging correlation spectroscopy) can be obtained from different regions of the tissue. Comparative studies of different tissue strata, but also between equivalent regions of normal and abnormal......This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes...

  20. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

    Science.gov (United States)

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

    2016-08-24

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.

  1. Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

    Science.gov (United States)

    Wolenski, Joseph S.; Julich, Doerthe

    2014-01-01

    Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

  2. Quantitative laser-induced fluorescence measurements of nitric oxide in a heavy-duty Diesel engine

    NARCIS (Netherlands)

    Verbiezen, K.; Klein-Douwel, R. J. H.; van Viet, A. P.; Donkerbroek, A. J.; Meerts, W. L.; Dam, N. J.; ter Meulen, J. J.

    2007-01-01

    We present quantitative, in-cylinder, UV-laser-induced fluorescence measurements of nitric oxide in a heavy-duty Diesel engine. Processing of the raw fluorescence signals includes a detailed correction, based on additional measurements, for the effect of laser beam and fluorescence attenuation, and

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

    International Nuclear Information System (INIS)

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

    2003-01-01

    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

  4. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

    Science.gov (United States)

    Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H; Wouters, Fred S; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

    2013-12-24

    We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

  5. Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine.

    Science.gov (United States)

    Alexiev, Ulrike; Volz, Pierre; Boreham, Alexander; Brodwolf, Robert

    2017-07-01

    The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug-loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Applications of two-photon fluorescence microscopy in deep-tissue imaging

    Science.gov (United States)

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

    2000-07-01

    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.

  7. Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

    Science.gov (United States)

    Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

    2018-04-26

    Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

  8. Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

    Science.gov (United States)

    Hackley, Paul C.; Kus, Jolanta

    2015-01-01

    We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

  9. Fast and accurate three-dimensional point spread function computation for fluorescence microscopy.

    Science.gov (United States)

    Li, Jizhou; Xue, Feng; Blu, Thierry

    2017-06-01

    The point spread function (PSF) plays a fundamental role in fluorescence microscopy. A realistic and accurately calculated PSF model can significantly improve the performance in 3D deconvolution microscopy and also the localization accuracy in single-molecule microscopy. In this work, we propose a fast and accurate approximation of the Gibson-Lanni model, which has been shown to represent the PSF suitably under a variety of imaging conditions. We express the Kirchhoff's integral in this model as a linear combination of rescaled Bessel functions, thus providing an integral-free way for the calculation. The explicit approximation error in terms of parameters is given numerically. Experiments demonstrate that the proposed approach results in a significantly smaller computational time compared with current state-of-the-art techniques to achieve the same accuracy. This approach can also be extended to other microscopy PSF models.

  10. Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

    Directory of Open Access Journals (Sweden)

    Attila Bokros

    2013-08-01

    Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

  11. Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

    Science.gov (United States)

    Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

    2013-05-01

    We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

  12. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    William L Rice

    Full Text Available BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF and second harmonic generation (SHG as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(PH, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(PH and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool

  13. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  14. Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

    Science.gov (United States)

    Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

    2015-03-01

    The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

  15. Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

    Science.gov (United States)

    Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

    2013-07-01

    Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

  16. Three-dimensional super-resolution imaging for fluorescence emission difference microscopy

    Energy Technology Data Exchange (ETDEWEB)

    You, Shangting; Kuang, Cuifang, E-mail: cfkuang@zju.edu.cn; Li, Shuai; Liu, Xu; Ding, Zhihua [State key laboratory of modern optical instrumentations, Zhejiang University, Hangzhou 310027 (China)

    2015-08-15

    We propose a method theoretically to break the diffraction limit and to improve the resolution in all three dimensions for fluorescence emission difference microscopy. We produce two kinds of hollow focal spot by phase modulation. By incoherent superposition, these two kinds of focal spot yield a 3D hollow focal spot. The optimal proportion of these two kinds of spot is given in the paper. By employing 3D hollow focal spot, super-resolution image can be yielded by means of fluorescence emission difference microscopy, with resolution enhanced both laterally and axially. According to computation result, size of point spread function of three-dimensional super-resolution imaging is reduced by about 40% in all three spatial directions with respect to confocal imaging.

  17. Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

    Science.gov (United States)

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

    2015-01-27

    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.

  18. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

    Science.gov (United States)

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  19. Contrast Induced by a Static Magnetic Field for Improved Detection in Nanodiamond Fluorescence Microscopy

    Science.gov (United States)

    Singam, Shashi K. R.; Motylewski, Jaroslaw; Monaco, Antonina; Gjorgievska, Elena; Bourgeois, Emilie; Nesládek, Milos; Giugliano, Michele; Goovaerts, Etienne

    2016-12-01

    Diamond nanoparticles with negatively charged nitrogen-vacancy (NV) centers are highly efficient nonblinking emitters that exhibit spin-dependent intensity. An attractive application of these emitters is background-free fluorescence microscopy exploiting the fluorescence quenching induced either by resonant microwaves (RMWs) or by an applied static magnetic field (SMF). Here, we compare RMW- and SMF-induced contrast measurements over a wide range of optical excitation rates for fluorescent nanodiamonds (FNDs) and for NV centers shallowly buried under the (100)-oriented surface of a diamond single crystal (SC). Contrast levels are found to be systematically lower in the FNDs than in the SC. At low excitation rates, the RMW contrast initially rises to a maximum (up to 7% in FNDs and 13% in the SC) but then decreases steadily at higher intensities. Conversely, the SMF contrast increases from approximately 12% at low excitation rates to high values of 20% and 38% for the FNDs and SC, respectively. These observations are well described in a rate-equations model for the charged NV defect using parameters in good agreement with the literature. The SMF approach yields higher induced contrast in image collection under commonly applied optical excitation. Unlike the RMW method, there is no thermal load exerted on the aqueous media in biological samples in the SMF approach. We demonstrate imaging by SMF-induced contrast in neuronal cultures incorporating FNDs (i) in a setup for patch-clamp experiments in parallel with differential-interference-contrast microscopy, (ii) after a commonly used staining procedure as an illustration of the high selectivity against background fluorescence, and (iii) in a confocal fluorescence microscope in combination with bright-field microscopy.

  20. Optical coherent tomography and fluorescent microscopy for the study of meningeal lymphatic systems

    Science.gov (United States)

    Semyachkina-Glushkovskaya, O.; Abdurashitov, A.; Namykin, A.; Fedosov, I.; Pavlov, A.; Karavaev, A.; Sindeeva, O.; Shirokov, A.; Ulanova, M.; Shushunova, N.; Khorovodov, A.; Agranovich, I.; Bodrova, A.; Sagatova, M.; Shareef, Ali Esmat; Saranceva, E.; Dvoryatkina, M.; Tuchin, V.

    2018-04-01

    The development of novel technologies for the imaging of meningeal lymphatic vessels is one of the amazing trends of biophotonics thanks to discovery of brain lymphatics over several years ago. However, there is the limited technologies exist for the study of lymphatics in vivo because lymphatic vessels are transparent with a low speed flow of lymph. Here we demonstrate the successful application of fluorescent microscopy for the imaging of lymphatic system in the mouse brain in vivo.

  1. X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

    OpenAIRE

    Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2009-01-01

    Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this techn...

  2. Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

    OpenAIRE

    Hovhannisyan, V.; Guo, H. W.; Hovhannisyan, A.; Ghukasyan, V.; Buryakina, T.; Chen, Y. F.; Dong, C. Y.

    2014-01-01

    Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the ...

  3. Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Wu, LinPing; Andersen, Helene

    2014-01-01

    A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requir...... of performance and intracellular trafficking of polyplexes as well as for assessing cell functionality. This review highlights the application of some of the most promising fluorescent microscopy platforms in relation to polyplex-mediated transfection processes....

  4. A new approach to dual-color two-photon microscopy with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Rebane Aleks

    2010-02-01

    Full Text Available Abstract Background Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA efficiency. Results Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. Conclusion Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

  5. Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis.

    Science.gov (United States)

    Peretz, Avi; Geffen, Yuval; Socea, Soergiu D; Pastukh, Nina; Graffi, Shmuel

    2015-08-01

    Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media-non-nutrient agar (NNA), culture on liquid growth media-peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. © The American Society of Tropical Medicine and Hygiene.

  6. Development of confocal X-ray fluorescence (XRF) microscopy at the Cornell high energy synchrotron source

    International Nuclear Information System (INIS)

    Woll, A.R.; Huang, R.; Mass, J.; Bisulca, C.; Bilderback, D.H.; Gruner, S.; Gao, N.

    2006-01-01

    A confocal X-ray fluorescence microscope was built at the Cornell High Energy Synchrotron Source (CHESS) to obtain compositional depth profiles of historic paintings. The microscope consists of a single-bounce, borosilicate monocapillary optic to focus the incident beam onto the painting and a commercial borosilicate polycapillary lens to collect the fluorescent X-rays. The resolution of the microscope was measured by scanning a variety of thin metal films through this confocal volume while monitoring the fluorescence signal. The capabilities of the technique were then probed using test paint microstructures with up to four distinct layers, each having a thickness in the range of 10-80 microns. Results from confocal XRF were compared with those from stand-alone XRF and visible light microscopy of the paint cross-sections. A large area, high-resolution scanner is currently being built to perform 3D scans on moderately sized paintings. (orig.)

  7. Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

    Science.gov (United States)

    Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

    Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

  8. Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

    Science.gov (United States)

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

    2014-03-01

    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.

  9. Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

    Science.gov (United States)

    Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

    2013-07-02

    Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.

    Science.gov (United States)

    Boehm, E M; Subramanyam, S; Ghoneim, M; Washington, M Todd; Spies, M

    2016-01-01

    Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today. © 2016 Elsevier Inc. All rights reserved.

  11. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  12. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation

    Science.gov (United States)

    Quinn, Kyle P.; Sridharan, Gautham V.; Hayden, Rebecca S.; Kaplan, David L.; Lee, Kyongbum; Georgakoudi, Irene

    2013-12-01

    The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda

    2014-08-01

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

  15. Quantitative Scanning Transmission Electron Microscopy of Electronic and Nanostructured Materials

    Science.gov (United States)

    Yankovich, Andrew B.

    Electronic and nanostructured materials have been investigated using advanced scanning transmission electron microscopy (STEM) techniques. The first topic is the microstructure of Ga and Sb-doped ZnO. Ga-doped ZnO is a candidate transparent conducting oxide material. The microstructure of GZO thin films grown by MBE under different growth conditions and different substrates were examined using various electron microscopy (EM) techniques. The microstructure, prevalent defects, and polarity in these films strongly depend on the growth conditions and substrate. Sb-doped ZnO nanowires have been shown to be the first route to stable p-type ZnO. Using Z-contrast STEM, I have showed that an unusual microstructure of Sb-decorated head-to-head inversion domain boundaries and internal voids contain all the Sb in the nanowires and cause the p-type conduction. InGaN thin films and InGaN / GaN quantum wells (QW) for light emitting diodes are the second topic. Low-dose Z-contrast STEM, PACBED, and EDS on InGaN QW LED structures grown by MOCVD show no evidence for nanoscale composition variations, contradicting previous reports. In addition, a new extended defect in GaN and InGaN was discovered. The defect consists of a faceted pyramid-shaped void that produces a threading dislocation along the [0001] growth direction, and is likely caused by carbon contamination during growth. Non-rigid registration (NRR) and high-precision STEM of nanoparticles is the final topic. NRR is a new image processing technique that corrects distortions arising from the serial nature of STEM acquisition that previously limited the precision of locating atomic columns and counting the number of atoms in images. NRR was used to demonstrate sub-picometer precision in STEM images of single crystal Si and GaN, the best achieved in EM. NRR was used to measure the atomic surface structure of Pt nanoacatalysts and Au nanoparticles, which revealed new bond length variation phenomenon of surface atoms. In

  16. Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

    Science.gov (United States)

    Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

    2017-06-01

    We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

  17. Quantitative DIC microscopy using an off-axis self-interference approach.

    Science.gov (United States)

    Fu, Dan; Oh, Seungeun; Choi, Wonshik; Yamauchi, Toyohiko; Dorn, August; Yaqoob, Zahid; Dasari, Ramachandra R; Feld, Michael S

    2010-07-15

    Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

  18. Quantitative microwave impedance microscopy with effective medium approximations

    Directory of Open Access Journals (Sweden)

    T. S. Jones

    2017-02-01

    Full Text Available Microwave impedance microscopy (MIM is a scanning probe technique to measure local changes in tip-sample admittance. The imaginary part of the reported change is calibrated with finite element simulations and physical measurements of a standard capacitive sample, and thereafter the output ΔY is given a reference value in siemens. Simulations also provide a means of extracting sample conductivity and permittivity from admittance, a procedure verified by comparing the estimated permittivity of polytetrafluoroethlyene (PTFE to the accepted value. Simulations published by others have investigated the tip-sample system for permittivity at a given conductivity, or conversely conductivity and a given permittivity; here we supply the full behavior for multiple values of both parameters. Finally, the well-known effective medium approximation of Bruggeman is considered as a means of estimating the volume fractions of the constituents in inhomogeneous two-phase systems. Specifically, we consider the estimation of porosity in carbide-derived carbon, a nanostructured material known for its use in energy storage devices.

  19. Light-sheet microscopy for quantitative ovarian folliculometry

    Science.gov (United States)

    Lin, Hsiao-Chun Amy; Dutta, Rahul; Mandal, Subhamoy; Kind, Alexander; Schnieke, Angelika; Razansky, Daniel

    2017-02-01

    Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. Owing to the large field of view and fast acquisition speed of our newly developed SPIM system, volumetric image stacks from entire intact samples of pig ovaries have been rendered demonstrating clearly discernible follicular features like follicle diameters (70 μm - 2.5 mm), size of developing Cumulus oophorus complexes (COC ) (40 μm - 110 μm), and follicular wall thicknesses (90 μm-120 μm). The observation of clearly distinguishable COCs protruding into the follicular antrum was also shown possible, and correlation with the developmental stage of the follicles was determined. Follicles of all developmental stages were identified, and even the small primordial follicle clusters forming the egg nest could be observed. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

  20. The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

    Science.gov (United States)

    Blackman, S M; Cobb, C E; Beth, A H; Piston, D W

    1996-01-01

    The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms. Images FIGURE 4 FIGURE 8 FIGURE 9 PMID:8804603

  1. A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation.

    Science.gov (United States)

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

    2004-10-01

    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

  2. Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

    Science.gov (United States)

    Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

    2015-09-01

    A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  3. Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue.

    Science.gov (United States)

    Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M; Koch, Edmund

    2012-07-01

    Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

  4. Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2

    International Nuclear Information System (INIS)

    McConnell, Gail; Riis, Erling

    2004-01-01

    We report on a novel and compact reliable laser source capable of short-wavelength two-photon laser scanning fluorescence microscopy based on soliton self-frequency shift effects in photonic crystal fibre. We demonstrate the function of the system by performing two-photon microscopy of smooth muscle cells and cardiac myocytes from the rat pulmonary vein and Chinese hamster ovary cells loaded with the fluorescent calcium indicator fura-2/AM

  5. HIV taken by STORM: Super-resolution fluorescence microscopy of a viral infection

    Directory of Open Access Journals (Sweden)

    Pereira Cândida F

    2012-05-01

    Full Text Available Abstract Background The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. Results Using direct stochastic optical reconstruction microscopy (dSTORM to achieve a lateral resolution of 15–20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1 before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. Conclusions This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.

  6. In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver

    Science.gov (United States)

    Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.

    2018-02-01

    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. Rapid diagnosis of malaria by fluorescent microscopy with light microscope and interface filter

    International Nuclear Information System (INIS)

    Hussain, I.; Tayyib, M.; Farooq, M.; Ahmed, N.

    2008-01-01

    The present study is planned to compare acridine orange (A.O) staining with Giemsa staining by using light microscopy with IF and also with fluorescent microscopy for detection of parasites in peripheral blood of patients suffering from clinically suspected cases of malaria. 200 patients with fever and shivering were included. General investigations like Hb, TLC and platelets were done by sysmex K-1000. Thin and thick blood films were made and stained according to protocol given i.e. by Giemsa and AO stains and slides were examined by different microscopes i.e. light microscope, light microscope with IFS and fluorescent microscope. Out of 200 subjects, 170 (85%) patients showed positive parasitaemia and 30 (15%) subjects were negative for malaria parasites. fib, TLC and platelets were reduced when comparing with MP negative cases. IFS microscope with acridine orange staining showed early detection of malaria parasites by counting fewer fields as compared to light microscopy with Giemsa stains. Time consumed for detection of parasites was also significantly reduced in IFS microscope by using AO stains. (author)

  8. 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

  9. Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

    Science.gov (United States)

    Zhang, Ying; Huang, Tao; Jorgens, Danielle M; Nickerson, Andrew; Lin, Li-Jung; Pelz, Joshua; Gray, Joe W; López, Claudia S; Nan, Xiaolin

    2017-01-01

    Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

  10. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Novak, Ivana

    2001-01-01

    of this reaction in confocal microscopy, we monitored luciferin fluorescence as a sign of ATP release by single acini. In addition we used quinacrine to mark ATP stores, which were similar to those marked with fluorescent ATP, 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate, but only partially...

  11. Single-Molecule Fluorescence Microscopy Reveals Local Diffusion Coefficients in the Pore Network of an Individual Catalyst Particle

    NARCIS (Netherlands)

    Hendriks, Frank|info:eu-repo/dai/nl/412642697; Meirer, Florian; Kubarev, Alexey V.; Ristanovic, Zoran|info:eu-repo/dai/nl/328233005; Roeffaers, Maarten B J; Vogt, Eelco T. C.|info:eu-repo/dai/nl/073717398; Bruijnincx, Pieter C. A.|info:eu-repo/dai/nl/33799529X; Weckhuysen, Bert M.|info:eu-repo/dai/nl/285484397

    2017-01-01

    We used single-molecule fluorescence microscopy to study self-diffusion of a feedstock-like probe molecule with nanometer accuracy in the macropores of a micrometer-sized, real-life fluid catalytic cracking (FCC) particle. Movies of single fluorescent molecules allowed their movement through the

  12. Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

    Science.gov (United States)

    Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

    2015-01-01

    Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

  13. Fluorescent nanoscale detection of biotin-streptavidin interaction using near-field scanning optical microscopy

    International Nuclear Information System (INIS)

    Park, Hyun Kyu; Chung, Bong Hyun; Gokarna, Anisha; Hulme, John P; Park, Hyun Gyu

    2008-01-01

    We describe a nanoscale strategy for detecting biotin-streptavidin binding using near-field scanning optical microscopy (NSOM) that exploits the fluorescence properties of single polydiacetylene (PDA) liposomes. NSOM is more useful to observe nanomaterials having optical properties with the help of topological information. We synthesized amine-terminated 10,12-pentacosadiynoic acid (PCDA) monomer (PCDA-NH 2 ) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH 2 liposomes were immobilized on an aldehyde-functionalized glass surface followed by photopolymerization by using a 254 nm light source. To measure the biotin-streptavidin binding, we conjugated photoactivatable biotin to immobilized PCDA-NH 2 liposomes by UV irradiation (365 nm) and subsequently allowed them to interact with streptavidin. We analyzed the fluorescence using a fluorescence scanner and observed single liposomes using NSOM. The average height and NSOM signal observed in a single liposome after binding were ∼31.3 to 8.5 ± 0.5 nm and 0.37 to 0.16 ± 0.6 kHz, respectively. This approach, which has the advantage of not requiring a fluorescent label, could prove highly beneficial for single molecule detection technology

  14. How to Characterize Individual Nano-Size Liposomes with Simple Self-Calibrating Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Mortensen, Kim I.; Tassone, Chiara; Ehrlich, Nicky

    2018-01-01

    and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, respectively. A vesicle then images as two spots, one in each color....... They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Non...... channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles...

  15. Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking

    Science.gov (United States)

    Ryu, Inkeon; Kim, Daekeun

    2018-04-01

    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.

  16. Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

    2016-04-01

    Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

  17. The Identification of Aluminum in Human Brain Tissue Using Lumogallion and Fluorescence Microscopy

    Science.gov (United States)

    Mirza, Ambreen; King, Andrew; Troakes, Claire; Exley, Christopher

    2016-01-01

    Aluminum in human brain tissue is implicated in the etiologies of neurodegenerative diseases including Alzheimer’s disease. While methods for the accurate and precise measurement of aluminum in human brain tissue are widely acknowledged, the same cannot be said for the visualization of aluminum. Herein we have used transversely-heated graphite furnace atomic absorption spectrometry to measure aluminum in the brain of a donor with Alzheimer’s disease, and we have developed and validated fluorescence microscopy and the fluor lumogallion to show the presence of aluminum in the same tissue. Aluminum is observed as characteristic orange fluorescence that is neither reproduced by other metals nor explained by autofluorescence. This new and relatively simple method to visualize aluminum in human brain tissue should enable more rigorous testing of the aluminum hypothesis of Alzheimer’s disease (and other neurological conditions) in the future. PMID:27472886

  18. Characterization of LiF-based soft X-ray imaging detectors by confocal fluorescence microscopy

    International Nuclear Information System (INIS)

    Bonfigli, F; Gaudio, P; Lupelli, I; Nichelatti, E; Richetta, M; Vincenti, M A; Montereali, R M

    2010-01-01

    X-ray microscopy represents a powerful tool to obtain images of samples with very high spatial resolution. The main limitation of this technique is represented by the poor spatial resolution of standard imaging detectors. We proposed an innovative high-performance X-ray imaging detector based on the visible photoluminescence of colour centres in lithium fluoride. In this work, a confocal microscope in fluorescence mode was used to characterize LiF-based imaging detectors measuring CC integrated visible fluorescence signals of LiF crystals and films (grown on several kinds of substrates) irradiated by soft X-rays produced by a laser plasma source in different exposure conditions. The results are compared with the CC photoluminescence spectra measured on the same samples and discussed.

  19. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

    Science.gov (United States)

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-05-05

    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.

  20. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee

    2015-01-01

    BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

  1. Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy.

    Science.gov (United States)

    Hovhannisyan, V; Guo, H W; Hovhannisyan, A; Ghukasyan, V; Buryakina, T; Chen, Y F; Dong, C Y

    2014-05-01

    Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin-mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.

  2. Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

    Science.gov (United States)

    Kuzmenko, Anton; Tankov, Stoyan; English, Brian P.; Tarassov, Ivan; Tenson, Tanel; Kamenski, Piotr; Elf, Johan; Hauryliuk, Vasili

    2011-12-01

    Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

  3. Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells

    International Nuclear Information System (INIS)

    Kim, Kyujung; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun; Kim, Dong Jun

    2009-01-01

    We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  4. Single cell adhesion strength assessed with variable-angle total internal reflection fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Marcelina Cardoso Dos Santos

    2017-06-01

    Full Text Available We propose a new strategy to evaluate adhesion strength at the single cell level. This approach involves variable-angle total internal reflection fluorescence microscopy to monitor in real time the topography of cell membranes, i.e. a map of the membrane/substrate separation distance. According to the Boltzmann distribution, both potential energy profile and dissociation energy related to the interactions between the cell membrane and the substrate were determined from the membrane topography. We have highlighted on glass substrates coated with poly-L-lysine and fibronectin, that the dissociation energy is a reliable parameter to quantify the adhesion strength of MDA-MB-231 motile cells.

  5. Occlusal overload investigations by noninvasive technology: fluorescence microscopy and en-face optical coherence tomography

    Science.gov (United States)

    Marcauteanu, Corina; Negrutiu, Meda; Sinescu, Cosmin; Demjan, Enikö; Hughes, Michael; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2009-07-01

    The aim of this study is the early detection and monitoring of occlusal overload in bruxing patients. En-Face Optical coherence tomography (eF-OCT) and fluorescence microscopy (FM) were used for the imaging of several anterior teeth extracted from patients with light active bruxism. We found a characteristic pattern of enamel cracks, that reached the tooth surface. We concluded that the combination of the en-Face OCT and FM is a promising non-invasive alternative technique for reliable monitoring of occlusal overload.

  6. Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy

    DEFF Research Database (Denmark)

    Norlén, Lars; Plasencia Gil, Maria Inés; Bagatolli, Luis

    2008-01-01

    -related biophysical techniques (e.g. atomic force microscopy and confocal/two-photon excitation fluorescence microscopy), it was recently shown that reconstituted membranes composed of extracted decontaminated human stratum corneum lipids do not form a fluid phase, but exclusively a single-gel phase that segregates...

  7. Integrated Transmission Electron and Single‐Molecule Fluorescence Microscopy Correlates Reactivity with Ultrastructure in a Single Catalyst Particle

    OpenAIRE

    Hendriks, Frank C.; Mohammadian, Sajjad; Ristanović, Zoran; Kalirai, Sam; Meirer, Florian; Vogt, Eelco T. C.; Bruijnincx, Pieter C. A.; Gerritsen, Hans C.; Weckhuysen, Bert M.

    2017-01-01

    Abstract Establishing structure–activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single‐molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure–reactivity information was obtained for 100 nm thin, microtomed secti...

  8. Integrated Transmission Electron and Single-Molecule Fluorescence Microscopy Correlates Reactivity with Ultrastructure in a Single Catalyst Particle

    OpenAIRE

    Hendriks, Frank C.; Mohammadian, Sajjad; Ristanovic, Zoran; Kalirai, Samanbir; Meirer, Florian; Vogt, Eelco T. C.; Bruijnincx, Pieter C. A.; Gerritsen, Hans; Weckhuysen, Bert M.

    2018-01-01

    Establishing structure–activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single-molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure–reactivity information was obtained for 100 nm thin, microtomed sections of a ...

  9. Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging

    Science.gov (United States)

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

    2017-02-01

    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.

  10. Characterization and quantitative determination of calcium aluminate clinker phases through reflected light microscopy

    International Nuclear Information System (INIS)

    Marciano Junior, E.; Cunha Munhoz, F.A. da; Splettstoser Junior, J.; Placido, W.F.

    1989-01-01

    The identification and quantitative determination of phases in calcium aluminate clinker is of great importance to the producer, as it enables a better understanding of the cement and concrete properties, specially those concerning setting time and compressive strenght. Polished sections of three electrofused clinkers, one experimental and two industrial, were used to select the most suitable etchings in order to identify by microscopy the main phases (Ca, CA 2 , C 2 AS, C 12 A 7 , α-Al 2 O 3 ). Quantitative phases determinations by reflected light microscopy showed good results when compared to X-ray diffractometry measurements [pt

  11. Quantitative wavelenght-resolved fluorescence detection for microchip capillary electrophoresis

    NARCIS (Netherlands)

    Götz, S.

    2006-01-01

    This thesis describes the development and application of a new wavelengthresolved CCD-based fluorescence detector for microchip separations. In recent years, miniaturization has been one of the major trends in the development of new analytical separation systems. As the manipulated sample amounts

  12. Improved detection of soma location and morphology in fluorescence microscopy images of neurons.

    Science.gov (United States)

    Kayasandik, Cihan Bilge; Labate, Demetrio

    2016-12-01

    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.

  13. Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

    Science.gov (United States)

    Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

    2011-01-01

    Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

  14. Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting.

    Directory of Open Access Journals (Sweden)

    Jessica Minion

    Full Text Available INTRODUCTION: Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. METHODS: In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS. Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. RESULTS: There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. CONCLUSIONS: Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used.

  15. NicoLase-An open-source diode laser combiner, fiber launch, and sequencing controller for fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Philip R Nicovich

    Full Text Available Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available.

  16. Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality.

    Science.gov (United States)

    Hermann, Martin; Nussbaumer, Oliver; Knöfler, Ralf; Hengster, Paul; Nussbaumer, Walter; Streif, Werner

    2010-01-01

    BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.

  17. Adaptive platform for fluorescence microscopy-based high-content screening

    Science.gov (United States)

    Geisbauer, Matthias; Röder, Thorsten; Chen, Yang; Knoll, Alois; Uhl, Rainer

    2010-04-01

    Fluorescence microscopy has become a widely used tool for the study of medically relevant intra- and intercellular processes. Extracting meaningful information out of a bulk of acquired images is usually performed during a separate post-processing task. Thus capturing raw data results in an unnecessary huge number of images, whereas usually only a few images really show the particular information that is searched for. Here we propose a novel automated high-content microscope system, which enables experiments to be carried out with only a minimum of human interaction. It facilitates a huge speed-increase for cell biology research and its applications compared to the widely performed workflows. Our fluorescence microscopy system can automatically execute application-dependent data processing algorithms during the actual experiment. They are used for image contrast enhancement, cell segmentation and/or cell property evaluation. On-the-fly retrieved information is used to reduce data and concomitantly control the experiment process in real-time. Resulting in a closed loop of perception and action the system can greatly decrease the amount of stored data on one hand and increases the relative valuable data content on the other hand. We demonstrate our approach by addressing the problem of automatically finding cells with a particular combination of labeled receptors and then selectively stimulate them with antagonists or agonists. The results are then compared against the results of traditional, static systems.

  18. New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

    Science.gov (United States)

    Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

    2015-05-01

    Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  19. Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

    Science.gov (United States)

    Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

    2014-09-01

    Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

  20. Systematic study of alginate-based microcapsules by micropipette aspiration and confocal fluorescence microscopy.

    Science.gov (United States)

    Kleinberger, Rachelle M; Burke, Nicholas A D; Dalnoki-Veress, Kari; Stöver, Harald D H

    2013-10-01

    Micropipette aspiration and confocal fluorescence microscopy were used to study the structure and mechanical properties of calcium alginate hydrogel beads (A beads), as well as A beads that were additionally coated with poly-L-lysine (P) and sodium alginate (A) to form, respectively, AP and APA hydrogels. A beads were found to continue curing for up to 500 h during storage in saline, due to residual calcium chloride carried over from the gelling bath. In subsequent saline washes, micropipette aspiration proved to be a sensitive indicator of gel weakening and calcium loss. Aspiration tests were used to compare capsule stiffness before and after citrate extraction of calcium. They showed that the initial gel strength is largely due to the calcium alginate gel cores, while the long term strength is solely due to the poly-L-lysine-alginate polyelectrolyte complex (PEC) shells. Confocal fluorescence microscopy showed that calcium chloride exposure after PLL deposition led to PLL redistribution into the hydrogel bead, resulting in thicker but more diffuse and weaker PEC shells. Adding a final alginate coating to form APA capsules did not significantly change the PEC membrane thickness and stiffness, but did speed the loss of calcium from the bead core. © 2013.

  1. Development of new photon-counting detectors for single-molecule fluorescence microscopy

    Science.gov (United States)

    Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

    2013-01-01

    Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

  2. Analysis of archaeological ceramics by total-reflection X-ray fluorescence: Quantitative approaches

    International Nuclear Information System (INIS)

    Fernandez-Ruiz, R.; Garcia-Heras, M.

    2008-01-01

    This paper reports the quantitative methodologies developed for the compositional characterization of archaeological ceramics by total-reflection X-ray fluorescence at two levels. A first quantitative level which comprises an acid leaching procedure, and a second selective level, which seeks to increase the number of detectable elements by eliminating the iron present in the acid leaching procedure. Total-reflection X-ray fluorescence spectrometry has been compared, at a quantitative level, with Instrumental Neutron Activation Analysis in order to test its applicability to the study of this kind of materials. The combination of a solid chemical homogenization procedure previously reported with the quantitative methodologies here presented allows the total-reflection X-ray fluorescence to analyze 29 elements with acceptable analytical recoveries and accuracies

  3. Analysis of archaeological ceramics by total-reflection X-ray fluorescence: Quantitative approaches

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez-Ruiz, R. [Servicio Interdepartamental de Investigacion, Facultad de Ciencias, Universidad Autonoma de Madrid, Modulo C-9, Laboratorio de TXRF, Crta. Colmenar, Km 15, Cantoblanco, E-28049, Madrid (Spain)], E-mail: ramon.fernandez@uam.es; Garcia-Heras, M. [Grupo de Arqueometria de Vidrios y Materiales Ceramicos, Instituto de Historia, Centro de Ciencias Humanas y Sociales, CSIC, C/ Albasanz, 26-28, 28037 Madrid (Spain)

    2008-09-15

    This paper reports the quantitative methodologies developed for the compositional characterization of archaeological ceramics by total-reflection X-ray fluorescence at two levels. A first quantitative level which comprises an acid leaching procedure, and a second selective level, which seeks to increase the number of detectable elements by eliminating the iron present in the acid leaching procedure. Total-reflection X-ray fluorescence spectrometry has been compared, at a quantitative level, with Instrumental Neutron Activation Analysis in order to test its applicability to the study of this kind of materials. The combination of a solid chemical homogenization procedure previously reported with the quantitative methodologies here presented allows the total-reflection X-ray fluorescence to analyze 29 elements with acceptable analytical recoveries and accuracies.

  4. Modeling optical behavior of birefringent biological tissues for evaluation of quantitative polarized light microscopy

    NARCIS (Netherlands)

    Turnhout, van M.C.; Kranenbarg, S.; Leeuwen, van J.L.

    2009-01-01

    Quantitative polarized light microscopy (qPLM) is a popular tool for the investigation of birefringent architectures in biological tissues. Collagen, the most abundant protein in mammals, is such a birefringent material. Interpretation of results of qPLM in terms of collagen network architecture and

  5. Controlled initiation and quantitative visualization of cell interaction dynamics - a novel hybrid microscopy method -

    NARCIS (Netherlands)

    Snijder-van As, M.I.

    2010-01-01

    This thesis describes the development, validation, and application of a hybrid microscopy technique to study cell-substrate and cell-cell interactions in a controlled and quantitative manner. We studied the spatial and temporal dynamics of the selected membrane molecules CD6 and the activated

  6. Supramolecular assembly affording a ratiometric two-photon fluorescent nanoprobe for quantitative detection and bioimaging.

    Science.gov (United States)

    Wang, Peng; Zhang, Cheng; Liu, Hong-Wen; Xiong, Mengyi; Yin, Sheng-Yan; Yang, Yue; Hu, Xiao-Xiao; Yin, Xia; Zhang, Xiao-Bing; Tan, Weihong

    2017-12-01

    Fluorescence quantitative analyses for vital biomolecules are in great demand in biomedical science owing to their unique detection advantages with rapid, sensitive, non-damaging and specific identification. However, available fluorescence strategies for quantitative detection are usually hard to design and achieve. Inspired by supramolecular chemistry, a two-photon-excited fluorescent supramolecular nanoplatform ( TPSNP ) was designed for quantitative analysis with three parts: host molecules (β-CD polymers), a guest fluorophore of sensing probes (Np-Ad) and a guest internal reference (NpRh-Ad). In this strategy, the TPSNP possesses the merits of (i) improved water-solubility and biocompatibility; (ii) increased tissue penetration depth for bioimaging by two-photon excitation; (iii) quantitative and tunable assembly of functional guest molecules to obtain optimized detection conditions; (iv) a common approach to avoid the limitation of complicated design by adjustment of sensing probes; and (v) accurate quantitative analysis by virtue of reference molecules. As a proof-of-concept, we utilized the two-photon fluorescent probe NHS-Ad-based TPSNP-1 to realize accurate quantitative analysis of hydrogen sulfide (H 2 S), with high sensitivity and good selectivity in live cells, deep tissues and ex vivo -dissected organs, suggesting that the TPSNP is an ideal quantitative indicator for clinical samples. What's more, TPSNP will pave the way for designing and preparing advanced supramolecular sensors for biosensing and biomedicine.

  7. Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan

    2016-09-19

    Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. X-ray optics for scanning fluorescence microscopy and other applications

    International Nuclear Information System (INIS)

    Ryon, R.W.; Warburton, W.K.

    1992-05-01

    Scanning x-ray fluorescence microscopy is analogous to scanning electron microscopy. Maps of chemical element distribution are produced by scanning with a very small x-ray beam. Goal is to perform such scanning microscopy with resolution in the range of <1 to 10 μm, using standard laboratory x-ray tubes. We are investigating mirror optics in the Kirkpatrick-Baez (K-B) configuration. K-B optics uses two curved mirrors mounted orthogonally along the optical axis. The first mirror provides vertical focus, the second mirror provides horizontal focus. We have used two types of mirrors: synthetic multilayers and crystals. Multilayer mirrors are used with lower energy radiation such as Cu Kα. At higher energies such as Ag Kα, silicon wafers are used in order to increase the incidence angles and thereby the photon collection efficiency. In order to increase the surface area of multilayers which reflects x-rays at the Bragg angle, we have designed mirrors with the spacing between layers graded along the optic axis in order to compensate for the changing angle of incidence. Likewise, to achieve a large reflecting surface with silicon, the wafers are placed on a specially designed lever arm which is bent into a log spiral by applying force at one end. In this way, the same diffracting angle is maintained over the entire surface of the wafer, providing a large solid angle for photon collection

  9. Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination

    International Nuclear Information System (INIS)

    Kim, Taejoong; Gweon, DaeGab; Lee, Jun-Hee

    2009-01-01

    Confocal microscopy is an optical imaging technique used to reconstruct three-dimensional images without physical sectioning. As with other optical microscopes, the lateral resolution of the confocal microscope cannot surpass the diffraction limit. This paper presents a novel imaging system, structured illumination confocal scanning microscopy (SICSM), that uses structured illumination to improve the lateral resolution of the confocal microscope. The SICSM can easily be implemented by introducing a structured illumination generating optics to conventional line-scanning fluorescence confocal microscopy. In this paper, we report our analysis of the lateral and axial resolutions of the SICSM by use of mathematical imaging theory. Numerical simulation results show that the lateral resolution of the SICSM is 1.43-fold better than that of the confocal microscope. In the axial direction, however, the resolution of the SICSM is ∼15% poorer than that of the confocal microscope. This deterioration arises because of a decrease in the axial cut-off frequency caused by the process of generating structured illumination. We propose the use of imaging conditions under which a compromise between the axial and lateral resolutions is chosen. Finally, we show simulated images of diversely shaped test objects to demonstrate the lateral and axial resolution performance of the SICSM

  10. Context based mixture model for cell phase identification in automated fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Zhou Xiaobo

    2007-01-01

    Full Text Available Abstract Background Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task. Results The data is generated from Hela H2B GFP cells imaged during a 2-day period with images acquired 15 minutes apart using an automated time-lapse fluorescence microscopy. The patterns are described with four kinds of features, including twelve general features, Haralick texture features, Zernike moment features, and wavelet features. To generate a new set of features with more discriminate power, the commonly used feature reduction techniques are used, which include Principle Component Analysis (PCA, Linear Discriminant Analysis (LDA, Maximum Margin Criterion (MMC, Stepwise Discriminate Analysis based Feature Selection (SDAFS, and Genetic Algorithm based Feature Selection (GAFS. Then, we propose a Context Based Mixture Model (CBMM for dealing with the time-series cell sequence information and compare it to other traditional classifiers: Support Vector Machine (SVM, Neural Network (NN, and K-Nearest Neighbor (KNN. Being a standard practice in machine learning, we systematically compare the performance of a number of common feature reduction techniques and classifiers to select an optimal combination of a feature reduction technique and a classifier. A cellular database containing 100 manually labelled subsequence is built for evaluating the performance of the classifiers. The generalization error is estimated using the cross validation technique. The

  11. Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

    Science.gov (United States)

    Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

    2008-09-01

    Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

  12. Automated Image Analysis for Quantitative Fluorescence In Situ Hybridization with Environmental Samples▿ †

    OpenAIRE

    Zhou, Zhi; Pons, Marie Noëlle; Raskin, Lutgarde; Zilles, Julie L.

    2007-01-01

    When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An au...

  13. The nanosizing of fluorescent objects by 458 nm spatially modulated illumination microscopy using a simplified size evaluation algorithm

    Energy Technology Data Exchange (ETDEWEB)

    Schweitzer, Andreas; Wagner, Christian; Cremer, Christoph [Kirchhoff-Institute for Physics of the University, Im Neuenheimer Feld 227, 69120 Heidelberg (Germany)

    2004-07-07

    In fluorescent light microscopy, structured illumination approaches have emerged as a novel tool to analyse subwavelength sized objects in thick transparent specimens. In this report, new size measurements ('nanosizing') of small subwavelength sized fluorescent objects applying spatially modulated illumination (SMI) microscopy with an excitation wavelength of {lambda}{sub ex} 458 nm are presented. These measurements were made using fluorescent particles with a given diameter. From the SMI data achieved, the size (diameter) was determined using special calibration curves derived from analytical considerations assuming a Gaussian dye distribution within the object. The results showed that with SMI microscopy combined with suitable calibration, size measurements of objects considerably smaller than the epifluorescent optical resolution at {lambda}{sub ex} = 458 nm are feasible.

  14. Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

    Science.gov (United States)

    1994-01-01

    A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate. PMID:7519623

  15. Comparison of nanoparticle diffusion using fluorescence correlation spectroscopy and differential dynamic microscopy within concentrated polymer solutions

    Science.gov (United States)

    Shokeen, Namita; Issa, Christopher; Mukhopadhyay, Ashis

    2017-12-01

    We studied the diffusion of nanoparticles (NPs) within aqueous entangled solutions of polyethylene oxide (PEO) by using two different optical techniques. Fluorescence correlation spectroscopy, a method widely used to investigate nanoparticle dynamics in polymer solution, was used to measure the long-time diffusion coefficient (D) of 25 nm radius particles within high molecular weight, Mw = 600 kg/mol PEO in water solutions. Differential dynamic microscopy (DDM) was used to determine the wave-vector dependent dynamics of NPs within the same polymer solutions. Our results showed good agreement between the two methods, including demonstration of normal diffusion and almost identical diffusion coefficients obtained by both techniques. The research extends the scope of DDM to study the dynamics and rheological properties of soft matter at a nanoscale. The measured diffusion coefficients followed a scaling theory, which can be explained by the coupling between polymer dynamics and NP motion.

  16. The lymphatic mechanisms of brain cleaning: application of optical coherence tomography and fluorescence microscopy

    Science.gov (United States)

    Glushkovskaya-Semyachkina, O.; Abdurashitov, A.; Fedosov, I.; Namykin, A.; Pavlov, A.; Shirokov, A.; Shushunova, N.; Sindeeva, O.; Khorovodov, A.; Ulanova, M.; Sagatova, V.; Agranovich, I.; Bodrova, A.; Kurths, J.

    2018-04-01

    Here we studied the role of cerebral lymphatic system in the brain clearing using intraparenchymal injection of Evans Blue and gold nanorods assessed by optical coherent tomography and fluorescence microscopy. Our data clearly show that the cerebral lymphatic system plays an important role in the brain cleaning via meningeal lymphatic vessels but not cerebral veins. Meningeal lymphatic vessels transport fluid from the brain into the deep cervical node, which is the first anatomical "station" for lymph outflow from the brain. The lymphatic processes underlying brain clearing are more slowly vs. peripheral lymphatics. These results shed light on the lymphatic mechanisms responsible for brain clearing as well as interaction between the intra- and extracranial lymphatic compartment.

  17. Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy.

    Science.gov (United States)

    Dreier, Roland Felix; Santos, José Carlos; Broz, Petr

    2018-01-01

    Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

  18. Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors

    Science.gov (United States)

    Chen, Youhua; Cao, Ruizhi; Liu, Wenjie; Zhu, Dazhao; Zhang, Zhiming; Kuang, Cuifang; Liu, Xu

    2018-04-01

    We present an alternative approach to realize structured illumination microscopy (SIM), which is capable for live cell imaging. The prototype utilizes two sets of scanning galvo mirrors, a polarization converter and a piezo-platform to generate a fast shifted, s-polarization interfered and periodic variable illumination patterns. By changing the angle of the scanning galvanometer, we can change the position of the spots at the pupil plane of the objective lens arbitrarily, making it easy to switch between widefield and total internal reflection fluorescent-SIM mode and adapting the penetration depth in the sample. Also, a twofold resolution improvement is achieved in our experiments. The prototype offers more flexibility of pattern period and illumination orientation changing than previous systems.

  19. Active Appearance Segmentation for Intensity Inhomogeneity in Light Sheet Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Jensen, Casper Bo; Lyksborg, Mark; Hecksher-Sørensen, J.

    2016-01-01

    inhomogeneities which are often seen in Light Sheet Fluorescence Microscopy (LSFM) images. This robustness is achieved by modelling the appearance of an image as a regularized Normalized Gradient Field (rNGF). We perform two experiments to challenge the model. First it is tested using a repeated leave......Active Appearance Models (AAM) are used for annotating or segmenting shapes in biomedical images. Performance relies heavily on the image data used to train the AAM. In this paper we improve the generalization properties of the model by making it robust to slowly varying spatial intensity......-one-out approach on images with minimal imperfections where the left out images are corrupted by a simulated bias field and segmented using the AAM. Secondly we test the model on LSFM images with common acquisition problems. In both experiments the proposed approach outperforms the often used AAM implementation...

  20. Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Youling Gu

    2011-01-01

    Full Text Available Sindbis virus (SINV is an alphavirus that has a broad host range and has been widely used as a vector for recombinant gene transduction, DNA-based vaccine production, and oncolytic cancer therapy. The mechanism of SINV entry into host cells has yet to be fully understood. In this paper, we used single virus tracking under total internal reflection fluorescence microscopy (TIRFM to investigate SINV attachment to cell surface. Biotinylated viral particles were labeled with quantum dots, which retained viral viability and infectivity. By time-lapse imaging, we showed that the SINV exhibited a heterogeneous dynamics on the surface of the host cells. Analysis of SINV motility demonstrated a two-step attachment reaction. Moreover, dual color TIRFM of GFP-Rab5 and SINV suggested that the virus was targeted to the early endosomes after endocytosis. These findings demonstrate the utility of quantum dot labeling in studying the early steps and behavior of SINV infection.

  1. Combining reflectometry and fluorescence microscopy: an assay for the investigation of leakage processes across lipid membranes.

    Science.gov (United States)

    Stephan, Milena; Mey, Ingo; Steinem, Claudia; Janshoff, Andreas

    2014-02-04

    The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated.

  2. Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Dylan Myers Owen

    2013-12-01

    Full Text Available The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes.

  3. Measurement of buried undercut structures in microfluidic devices by laser fluorescent confocal microscopy

    International Nuclear Information System (INIS)

    Li Shiguang; Liu Jing; Nguyen, Nam-Trung; Fang Zhongping; Yoon, Soon Fatt

    2009-01-01

    Measuring buried, undercut microstructures is a challenging task in metrology. These structures are usually characterized by measuring their cross sections after physically cutting the samples. This method is destructive and the obtained information is incomplete. The distortion due to cutting also affects the measurement accuracy. In this paper, we first apply the laser fluorescent confocal microscopy and intensity differentiation algorithm to obtain the complete three-dimensional profile of the buried, undercut structures in microfluidic devices, which are made by the soft lithography technique and bonded by the oxygen plasma method. The impact of material wettability and the refractive index (n) mismatch among the liquid, samples, cover layer, and objective on the measurement accuracy are experimentally investigated.

  4. CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

    Science.gov (United States)

    Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

    2015-02-07

    FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

  5. Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

    Science.gov (United States)

    Zhu, Dazhao; Chen, Youhua; Fang, Yue; Hussain, Anwar; Kuang, Cuifang; Zhou, Xiaoxu; Xu, Yingke; Liu, Xu

    2017-12-01

    A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.

  6. Visual Understanding of Light Absorption and Waveguiding in Standing Nanowires with 3D Fluorescence Confocal Microscopy.

    Science.gov (United States)

    Frederiksen, Rune; Tutuncuoglu, Gozde; Matteini, Federico; Martinez, Karen L; Fontcuberta I Morral, Anna; Alarcon-Llado, Esther

    2017-09-20

    Semiconductor nanowires are promising building blocks for next-generation photonics. Indirect proofs of large absorption cross sections have been reported in nanostructures with subwavelength diameters, an effect that is even more prominent in vertically standing nanowires. In this work we provide a three-dimensional map of the light around vertical GaAs nanowires standing on a substrate by using fluorescence confocal microscopy, where the strong long-range disruption of the light path along the nanowire is illustrated. We find that the actual long-distance perturbation is much larger in size than calculated extinction cross sections. While the size of the perturbation remains similar, the intensity of the interaction changes dramatically over the visible spectrum. Numerical simulations allow us to distinguish the effects of scattering and absorption in the nanowire leading to these phenomena. This work provides a visual understanding of light absorption in semiconductor nanowire structures, which is of high interest for solar energy conversion applications.

  7. Fluorescent chemosensor for detection and quantitation of carbon dioxide gas.

    Science.gov (United States)

    Liu, Yang; Tang, Youhong; Barashkov, Nikolay N; Irgibaeva, Irina S; Lam, Jacky W Y; Hu, Rongrong; Birimzhanova, Dinara; Yu, Yong; Tang, Ben Zhong

    2010-10-13

    CO(2) sensing is of great societal implications, as CO(2) is a component of gas mixtures from many natural and anthropogenic processes with huge impacts on globe climate and human well-being. Herein we report a CO(2) assay scheme over a wide concentration range, utilizing a fluorogen with an aggregation-induced emission feature and a liquid with tunable polarity and viscosity. The CO(2) sensing process is specific, quantitative, and interferent tolerant.

  8. Synthesis, quantitative structure-property relationship study of novel fluorescence active 2-pyrazolines and application

    Science.gov (United States)

    Girgis, Adel S.; Basta, Altaf H.; El-Saied, Houssni; Mohamed, Mohamed A.; Bedair, Ahmad H.; Salim, Ahmad S.

    2018-03-01

    A variety of fluorescence-active fluorinated pyrazolines 13-33 was synthesized in good yields through cyclocondensation reaction of propenones 1-9 with aryl hydrazines 10-12. Some of the synthesized compounds provided promising fluorescence properties with quantum yield (Φ) higher than that of quinine sulfate (standard reference). Quantitative structure-property relationship studies were undertaken supporting the exhibited fluorescence properties and estimating the parameters governing properties. Five synthesized fluorescence-active pyrazolines (13, 15, 18, 19 and 23) with variable Φ were selected for treating two types of paper sheets (Fabriano and Bible paper). These investigated fluorescence compounds, especially compounds 19 and 23, provide improvements in strength properties of paper sheets. Based on the observed performance they can be used as markers in security documents.

  9. Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

    Science.gov (United States)

    Hwang, Wonsang; Seo, JinWon; Song, Jun ho; Kim, DongEun; Won, YoungJae; Choi, In-Hong; Yoo, Kyung-Hwa; Kim, Dug Young

    2018-02-01

    A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

  10. Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

    Science.gov (United States)

    Puchkov, Evgeny O

    2010-06-01

    In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

  11. Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast.

    Science.gov (United States)

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

    2012-08-01

    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.

  12. Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window

    Science.gov (United States)

    Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.

    2012-03-01

    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.

  13. Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

    Science.gov (United States)

    Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

    2017-02-01

    Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

  14. Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

    Science.gov (United States)

    Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

    2016-05-01

    Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Koshari, Stijn H S; Ross, Jean L; Nayak, Purnendu K; Zarraga, Isidro E; Rajagopal, Karthikan; Wagner, Norman J; Lenhoff, Abraham M

    2017-02-06

    Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

  16. Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Tardif, Pier-Luc; Bertrand, Marie-Jeanne; Abran, Maxime; Castonguay, Alexandre; Lefebvre, Joël; Stähli, Barbara E; Merlet, Nolwenn; Mihalache-Avram, Teodora; Geoffroy, Pascale; Mecteau, Mélanie; Busseuil, David; Ni, Feng; Abulrob, Abedelnasser; Rhéaume, Éric; L'Allier, Philippe; Tardif, Jean-Claude; Lesage, Frédéric

    2016-12-15

    Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

  17. Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

    Science.gov (United States)

    Dörr, Jonas M; van Coevorden-Hameete, Marleen H; Hoogenraad, Casper C; Killian, J Antoinette

    2017-11-01

    Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Effects of hyperthermia on intracellular CA/sup 2+/ monitored by digitized video image fluorescence microscopy

    International Nuclear Information System (INIS)

    Asher, C.R.; Mikkelsen, R.B.

    1987-01-01

    With digitized video image fluorescence microscopy and the fluorescent Ca/sup 2+/ dye, fuca-2, the authors examined heat effects on intracellular free Ca/sup 2+/, [Ca/sup 2/]/sub f/. HT-29 human colon cancer cells grown on coverslip were equilibrated with 2.0 μM fura-2 in RPMI 1540 (20 0 , 15 min), washed three times and incubated at 20 0 for 1 h. Coverslips were mounted in a Dvorok perfusion chamber sitting within a temperature controlled microscope stage. Fluorescence was monitored at 500 nm by epi-illumination at 385 nm, excitation maximum for free dye, and 340 nm, maximum for Ca/sup 2+/ complexed dye, with a computer controlled filter wheel. The emission intensity ratio, I/sub 340//I/sub 385/, which corrects for dye leakage, photo-bleaching and cell thickness was used to calculate [Ca/sup 2+/]/sub f/. Measurements of 200 cells at 37 0 using a bit pad and mouse to select 0.6 x 0.6 μ cytoplasmi areas indicated 3 populations of cells in terms of [Ca/sup 2+/]/sub f/ (70%, 40-60nM; 15% 70-110nM; 15%, 120-200 nM). Heating to 43 0 for 1 h resulted in an overall decrease in [Ca/sup 2+/]/sub f/ with greater than 90% cells within 30-50 nM. Not all cells responded to heat. Post-incubation for 3 h at 37 0 showed the identical cell distribution; at 24 h, cell distribution was that of non-heated cells. The relationship of these results to cell killing and thermotolerance are not understood, but these results indicated the importance of cell heterogeneity in response to heat

  19. Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

    Science.gov (United States)

    Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

    2017-01-01

    Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. PMID:28956810

  20. Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

    Directory of Open Access Journals (Sweden)

    Michael Hausmann

    2017-09-01

    Full Text Available Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2 in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

  1. Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes

    Directory of Open Access Journals (Sweden)

    David Llères

    2017-02-01

    Full Text Available How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer-based fluorescence lifetime imaging microscopy (FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

  2. Attributed relational graphs for cell nucleus segmentation in fluorescence microscopy images.

    Science.gov (United States)

    Arslan, Salim; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

    2013-06-01

    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.

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

    Science.gov (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

    2010-08-17

    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.

  4. 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.

  5. Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy.

    Science.gov (United States)

    Spedden, Elise; White, James D; Naumova, Elena N; Kaplan, David L; Staii, Cristian

    2012-09-05

    Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1-2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1-8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

    Science.gov (United States)

    Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

    2014-02-01

    Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

  7. Iron filled carbon nanotubes as novel monopole-like sensors for quantitative magnetic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wolny, F; Muehl, T; Weissker, U; Lipert, K; Schumann, J; Leonhardt, A; Buechner, B, E-mail: f.wolny@ifw-dresden.de, E-mail: t.muehl@ifw-dresden.de [Leibniz Institute for Solid State and Materials Research (IFW) Dresden, Helmholtzstrasse 20, 01069 Dresden (Germany)

    2010-10-29

    We present a novel ultrahigh stability sensor for quantitative magnetic force microscopy (MFM) based on an iron filled carbon nanotube. In contrast to the complex magnetic structure of conventional MFM probes, this sensor constitutes a nanomagnet with defined properties. The long iron nanowire can be regarded as an extended dipole of which only the monopole close to the sample surface is involved in the imaging process. We demonstrate its potential for high resolution imaging. Moreover, we present an easy routine to determine its monopole moment and prove that this calibration, unlike other approaches, is universally applicable. For the first time this enables straightforward quantitative MFM measurements.

  8. Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

    Directory of Open Access Journals (Sweden)

    Kwang-Ho Hur

    Full Text Available The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

  9. Flipping interferometry and its application for quantitative phase microscopy in a micro-channel.

    Science.gov (United States)

    Roitshtain, Darina; Turko, Nir A; Javidi, Bahram; Shaked, Natan T

    2016-05-15

    We present a portable, off-axis interferometric module for quantitative phase microscopy of live cells, positioned at the exit port of a coherently illuminated inverted microscope. The module creates on the digital camera an interference pattern between the image of the sample and its flipped version. The proposed simplified module is based on a retro-reflector modification in an external Michelson interferometer. The module does not contain any lenses, pinholes, or gratings and its alignment is straightforward. Still, it allows full control of the off-axis angle and does not suffer from ghost images. As experimentally demonstrated, the module is useful for quantitative phase microscopy of live cells rapidly flowing in a micro-channel.

  10. HAADF-STEM atom counting in atom probe tomography specimens: Towards quantitative correlative microscopy.

    Science.gov (United States)

    Lefebvre, W; Hernandez-Maldonado, D; Moyon, F; Cuvilly, F; Vaudolon, C; Shinde, D; Vurpillot, F

    2015-12-01

    The geometry of atom probe tomography tips strongly differs from standard scanning transmission electron microscopy foils. Whereas the later are rather flat and thin (atom probe tomography specimens. Based on simulations (electron probe propagation and image simulations), the possibility to apply quantitative high angle annular dark field scanning transmission electron microscopy to of atom probe tomography specimens has been tested. The influence of electron probe convergence and the benefice of deconvolution of electron probe point spread function electron have been established. Atom counting in atom probe tomography specimens is for the first time reported in this present work. It is demonstrated that, based on single projections of high angle annular dark field imaging, significant quantitative information can be used as additional input for refining the data obtained by correlative analysis of the specimen in APT, therefore opening new perspectives in the field of atomic scale tomography. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Multimodal microscopy and the stepwise multi-photon activation fluorescence of melanin

    Science.gov (United States)

    Lai, Zhenhua

    The author's work is divided into three aspects: multimodal microscopy, stepwise multi-photon activation fluorescence (SMPAF) of melanin, and customized-profile lenses (CPL) for on-axis laser scanners, which will be introduced respectively. A multimodal microscope provides the ability to image samples with multiple modalities on the same stage, which incorporates the benefits of all modalities. The multimodal microscopes developed in this dissertation are the Keck 3D fusion multimodal microscope 2.0 (3DFM 2.0), upgraded from the old 3DFM with improved performance and flexibility, and the multimodal microscope for targeting small particles (the "Target" system). The control systems developed for both microscopes are low-cost and easy-to-build, with all components off-the-shelf. The control system have not only significantly decreased the complexity and size of the microscope, but also increased the pixel resolution and flexibility. The SMPAF of melanin, activated by a continuous-wave (CW) mode near-infrared (NIR) laser, has potential applications for a low-cost and reliable method of detecting melanin. The photophysics of melanin SMPAF has been studied by theoretical analysis of the excitation process and investigation of the spectra, activation threshold, and photon number absorption of melanin SMPAF. SMPAF images of melanin in mouse hair and skin, mouse melanoma, and human black and white hairs are compared with images taken by conventional multi-photon fluorescence microscopy (MPFM) and confocal reflectance microscopy (CRM). SMPAF images significantly increase specificity and demonstrate the potential to increase sensitivity for melanin detection compared to MPFM images and CRM images. Employing melanin SMPAF imaging to detect melanin inside human skin in vivo has been demonstrated, which proves the effectiveness of melanin detection using SMPAF for medical purposes. Selective melanin ablation with micrometer resolution has been presented using the Target system

  12. A novel method for identifying a graph-based representation of 3-D microvascular networks from fluorescence microscopy image stacks.

    Science.gov (United States)

    Almasi, Sepideh; Xu, Xiaoyin; Ben-Zvi, Ayal; Lacoste, Baptiste; Gu, Chenghua; Miller, Eric L

    2015-02-01

    A novel approach to determine the global topological structure of a microvasculature network from noisy and low-resolution fluorescence microscopy data that does not require the detailed segmentation of the vessel structure is proposed here. The method is most appropriate for problems where the tortuosity of the network is relatively low and proceeds by directly computing a piecewise linear approximation to the vasculature skeleton through the construction of a graph in three dimensions whose edges represent the skeletal approximation and vertices are located at Critical Points (CPs) on the microvasculature. The CPs are defined as vessel junctions or locations of relatively large curvature along the centerline of a vessel. Our method consists of two phases. First, we provide a CP detection technique that, for junctions in particular, does not require any a priori geometric information such as direction or degree. Second, connectivity between detected nodes is determined via the solution of a Binary Integer Program (BIP) whose variables determine whether a potential edge between nodes is or is not included in the final graph. The utility function in this problem reflects both intensity-based and structural information along the path connecting the two nodes. Qualitative and quantitative results confirm the usefulness and accuracy of this method. This approach provides a mean of correctly capturing the connectivity patterns in vessels that are missed by more traditional segmentation and binarization schemes because of imperfections in the images which manifest as dim or broken vessels. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

    Science.gov (United States)

    Du, Yuncheng; Budman, Hector M; Duever, Thomas A

    2016-06-01

    Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

  14. Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

    Science.gov (United States)

    Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

    2015-08-01

    In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  15. Fluorescence quantitative PCR in detection of HBV DNA

    International Nuclear Information System (INIS)

    Shen Zheng; Li Ming; Shen Xia

    2003-01-01

    Objective: To study the relationship between the serum content of HBV-DNA and expression of serological markers with HBV infection patients. Methods: Serum samples from 375 hepatitis B patients with different clinical status and 70 normal persons were quantitated for HBV-DNA by FQ-PCR. Results: The average of HBV-DNA contents in the patient in the groups of HBsAg (+) and of HBeAg(+) were significantly higher than those in the group of HBsAg(-) and of HBeAg(-). Even in the group of HBeAg negative, high HBV-DNA contents might still be present in both the HBeAg(+) and HBeAg(-) groups. Conclusion: FQ-PCR can be used to monitor the real state of HBV infection, replication and the course of disease

  16. Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

    Science.gov (United States)

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

    2010-01-01

    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.

  17. Improved cancer risk stratification and diagnosis via quantitative phase microscopy (Conference Presentation)

    Science.gov (United States)

    Liu, Yang; Uttam, Shikhar; Pham, Hoa V.; Hartman, Douglas J.

    2017-02-01

    Pathology remains the gold standard for cancer diagnosis and in some cases prognosis, in which trained pathologists examine abnormality in tissue architecture and cell morphology characteristic of cancer cells with a bright-field microscope. The limited resolution of conventional microscope can result in intra-observer variation, missed early-stage cancers, and indeterminate cases that often result in unnecessary invasive procedures in the absence of cancer. Assessment of nanoscale structural characteristics via quantitative phase represents a promising strategy for identifying pre-cancerous or cancerous cells, due to its nanoscale sensitivity to optical path length, simple sample preparation (i.e., label-free) and low cost. I will present the development of quantitative phase microscopy system in transmission and reflection configuration to detect the structural changes in nuclear architecture, not be easily identifiable by conventional pathology. Specifically, we will present the use of transmission-mode quantitative phase imaging to improve diagnostic accuracy of urine cytology and the nuclear dry mass is progressively correlate with negative, atypical, suspicious and positive cytological diagnosis. In a second application, we will present the use of reflection-mode quantitative phase microscopy for depth-resolved nanoscale nuclear architecture mapping (nanoNAM) of clinically prepared formalin-fixed, paraffin-embedded tissue sections. We demonstrated that the quantitative phase microscopy system detects a gradual increase in the density alteration of nuclear architecture during malignant transformation in animal models of colon carcinogenesis and in human patients with ulcerative colitis, even in tissue that appears histologically normal according to pathologists. We evaluated the ability of nanoNAM to predict "future" cancer progression in patients with ulcerative colitis.

  18. Quantitative liquid and vapor distribution measurements in evaporating fuel sprays using laser-induced exciplex fluorescence

    International Nuclear Information System (INIS)

    Fansler, Todd D; Drake, Michael C; Gajdeczko, Boguslaw; Düwel, Isabell; Koban, Wieland; Zimmermann, Frank P; Schulz, Christof

    2009-01-01

    Fully quantitative two-dimensional measurements of liquid- and vapor-phase fuel distributions (mass per unit volume) from high-pressure direct-injection gasoline injectors are reported for conditions of both slow and rapid vaporization in a heated, high-pressure spray chamber. The measurements employ the coevaporative gasoline-like fluorobenzene (FB)/diethylmethylamine (DEMA)/hexane exciplex tracer/fuel system. In contrast to most previous laser-induced exciplex-fluorescence (LIEF) experiments, the quantitative results here include regions in which liquid and vapor fuel coexist (e.g. near the injector exit). A unique aspect is evaluation of both vapor- and liquid-phase distributions at varying temperature and pressure using only in situ vapor-phase fluorescence calibration measurements at room temperature and atmospheric pressure. This approach draws on recent extensive measurements of the temperature-dependent spectroscopic properties of the FB–DEMA exciplex system, in particular on knowledge of the quantum efficiencies of the vapor-phase and liquid-phase (exciplex) fluorescence. In addition to procedures necessary for quantitative measurements, we discuss corrections for liquid–vapor crosstalk (liquid fluorescence that overlaps the vapor-fluorescence bandpass), the unknown local temperature due to vaporization-induced cooling, and laser-sheet attenuation by scattering and absorption

  19. Wide-field spectrally resolved quantitative fluorescence imaging system: toward neurosurgical guidance in glioma resection

    Science.gov (United States)

    Xie, Yijing; Thom, Maria; Ebner, Michael; Wykes, Victoria; Desjardins, Adrien; Miserocchi, Anna; Ourselin, Sebastien; McEvoy, Andrew W.; Vercauteren, Tom

    2017-11-01

    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.

  20. In vivo imaging of the airway wall in asthma: fibered confocal fluorescence microscopy in relation to histology and lung function

    Directory of Open Access Journals (Sweden)

    Bel Elisabeth H

    2011-06-01

    Full Text Available Abstract Background Airway remodelling is a feature of asthma including fragmentation of elastic fibres observed in the superficial elastin network of the airway wall. Fibered confocal fluorescence microscopy (FCFM is a new and non-invasive imaging technique performed during bronchoscopy that may visualize elastic fibres, as shown by in vitro spectral analysis of elastin powder. We hypothesized that FCFM images capture in vivo elastic fibre patterns within the airway wall and that such patterns correspond with airway histology. We aimed to establish the concordance between the bronchial elastic fibre pattern in histology and FCFM. Second, we examined whether elastic fibre patterns in histology and FCFM were different between asthmatic subjects and healthy controls. Finally, the association between these patterns and lung function parameters was investigated. Methods In a cross-sectional study comprising 16 subjects (8 atopic asthmatic patients with controlled disease and 8 healthy controls spirometry and bronchoscopy were performed, with recording of FCFM images followed by endobronchial biopsy at the airway main carina. Elastic fibre patterns in histological sections and FCFM images were scored semi-quantitatively. Agreement between histology and FCFM was analysed using linearly weighted kappa κw. Results The patterns observed in histological sections and FCFM images could be divided into 3 distinct groups. There was good agreement between elastic fibre patterns in histology and FCFM patterns (κw 0.744. The semi-quantitative pattern scores were not different between asthmatic patients and controls. Notably, there was a significant difference in post-bronchodilator FEV1 %predicted between the different patterns by histology (p = 0.001 and FCFM (p = 0.048, regardless of asthma or atopy. Conclusion FCFM captures the elastic fibre pattern within the airway wall in humans in vivo. The association between post-bronchodilator FEV1 %predicted and

  1. Fluorescence, Absorption, and Excitation Spectra of Polycyclic Aromatic Hydrocarbons as a Tool for Quantitative Analysis

    Science.gov (United States)

    Rivera-Figueroa, A. M.; Ramazan, K. A.; Finlayson-Pitts, B. J.

    2004-01-01

    A quantitative and qualitative study of the interplay between absorption, fluorescence, and excitation spectra of pollutants called polycyclic aromatic hydrocarbons (PAHs) is conducted. The study of five PAH displays the correlation of the above-mentioned properties along with the associated molecular changes.

  2. Time-Gated Raman Spectroscopy for Quantitative Determination of Solid-State Forms of Fluorescent Pharmaceuticals.

    Science.gov (United States)

    Lipiäinen, Tiina; Pessi, Jenni; Movahedi, Parisa; Koivistoinen, Juha; Kurki, Lauri; Tenhunen, Mari; Yliruusi, Jouko; Juppo, Anne M; Heikkonen, Jukka; Pahikkala, Tapio; Strachan, Clare J

    2018-04-03

    Raman spectroscopy is widely used for quantitative pharmaceutical analysis, but a common obstacle to its use is sample fluorescence masking the Raman signal. Time-gating provides an instrument-based method for rejecting fluorescence through temporal resolution of the spectral signal and allows Raman spectra of fluorescent materials to be obtained. An additional practical advantage is that analysis is possible in ambient lighting. This study assesses the efficacy of time-gated Raman spectroscopy for the quantitative measurement of fluorescent pharmaceuticals. Time-gated Raman spectroscopy with a 128 × (2) × 4 CMOS SPAD detector was applied for quantitative analysis of ternary mixtures of solid-state forms of the model drug, piroxicam (PRX). Partial least-squares (PLS) regression allowed quantification, with Raman-active time domain selection (based on visual inspection) improving performance. Model performance was further improved by using kernel-based regularized least-squares (RLS) regression with greedy feature selection in which the data use in both the Raman shift and time dimensions was statistically optimized. Overall, time-gated Raman spectroscopy, especially with optimized data analysis in both the spectral and time dimensions, shows potential for sensitive and relatively routine quantitative analysis of photoluminescent pharmaceuticals during drug development and manufacturing.

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

    Science.gov (United States)

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

  4. Detection of chromosome abnormalities by quantitative fluorescent PCR in ectopic pregnancies

    NARCIS (Netherlands)

    Goddijn, Mariette; van Stralen, Marja; Schuring-Blom, Heleen; Redeker, Bert; van Leeuwen, Liesbeth; Repping, Sjoerd; Leschot, Nico; van der Veen, Fulco

    2005-01-01

    Objective: To evaluate the potential value of quantitative fluorescent polymerase chain reaction (QF-PCR) in the detection of chromosome abnormalities in ectopic pregnancies. Methods: Seventy chorionic villi samples of ectopic pregnancies were studied by QF-PCR. Primers for chromosomes 16, 21, X and

  5. Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

    Science.gov (United States)

    Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

    2018-05-01

    We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

  6. Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope.

    Science.gov (United States)

    Blancett, Candace D; Fetterer, David P; Koistinen, Keith A; Morazzani, Elaine M; Monninger, Mitchell K; Piper, Ashley E; Kuehl, Kathleen A; Kearney, Brian J; Norris, Sarah L; Rossi, Cynthia A; Glass, Pamela J; Sun, Mei G

    2017-10-01

    A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

    Directory of Open Access Journals (Sweden)

    Ma Mingxiao

    Full Text Available Sacbrood virus (SBV is a picorna-like virus that affects honey bees (Apis mellifera and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

  8. Direct evaluation of thyroid 127I and iodine overload: in vivo study by X-ray fluorescence and in vitro by SIMS microscopy

    International Nuclear Information System (INIS)

    Briancon, C.; Jeusset, J.; Halpern, S.; Fragu, P.

    1992-01-01

    This review describes the two methods which allow direct estimation of stable iodine ( 127 I) within thyroid gland either in vivo by X-ray fluorescence or in vitro by secondary ion mass spectrometry (SIMS) microscopy on tissue section. Although the measurement of thyroid iodine content (TIC) by X-ray fluorescence has little relevance for routine explorations of thyroid function, this is a valuable method for understanding complex pathophysiological conditions such as the thyroid adaptation to iodine overload. On the other hand, SIMS microscopy which is able to characterize the functional activity of thyroid tissue by measuring 127 I concentration within the thyroid follicles, can be used to determine the extent to which exogeneous iodine affects the regulation of iodine within the thyroid follicles. Both methods were used to evaluate the quantitative changes in thyroid 127 I induced by amiodarone iodine overload. TIC measurements shows that hyperthyroidism occured only in patients who increased their iodine stores, while the patients who developed hypothyroidism has low iodine stores. The SIMS microscopy data obtained in mice demonstrated that the thyroid response to amiodarone is related to dietary iodine intake leading to an increase in local iodine concentration in iodine deficient mice and to a decrease in iodine supplemented mice. This response is specific and different from that induced by an iodine overload. These results could explain that hyperthyroidism with high thyroid iodine content occured in areas with low thyroid iodine content in areas with a supplemented iodine diet

  9. Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy

    Science.gov (United States)

    Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; D'Souza-Li, Lilia; Assunção, Maria do Carmo; Bottcher-Luiz, Fátima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

    2012-08-01

    We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.

  10. Determination of lead in clay enameled by X-ray fluorescence technique in Total reflection and by Scanning Electron Microscopy

    International Nuclear Information System (INIS)

    Zarazua O, G.; Carapia M, L.

    2000-01-01

    This work has the objective of determining lead free in the glazed commercial stewing pans using the X-ray fluorescence technique in Total reflection (FRX) and the observation and semiquantitative determination of lead by Analytical Scanning Electron Microscopy (ASEM). (Author)

  11. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tittmann, B. R. [Penn State; Xi, X. [Penn State

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  12. Quantitative fluorescence lifetime spectroscopy in turbid media: comparison of theoretical, experimental and computational methods

    International Nuclear Information System (INIS)

    Vishwanath, Karthik; Mycek, Mary-Ann; Pogue, Brian

    2002-01-01

    A Monte Carlo model developed to simulate time-resolved fluorescence propagation in a semi-infinite turbid medium was validated against previously reported theoretical and computational results. Model simulations were compared to experimental measurements of fluorescence spectra and lifetimes on tissue-simulating phantoms for single and dual fibre-optic probe geometries. Experiments and simulations using a single probe revealed that scattering-induced artefacts appeared in fluorescence emission spectra, while fluorescence lifetimes were unchanged. Although fluorescence lifetime measurements are generally more robust to scattering artefacts than are measurements of fluorescence spectra, in the dual-probe geometry scattering-induced changes in apparent lifetime were predicted both from diffusion theory and via Monte Carlo simulation, as well as measured experimentally. In all cases, the recovered apparent lifetime increased with increasing scattering and increasing source-detector separation. Diffusion theory consistently underestimated the magnitude of these increases in apparent lifetime (predicting a maximum increase of ∼15%), while Monte Carlo simulations and experiment were closely matched (showing increases as large as 30%). These results indicate that quantitative simulations of time-resolved fluorescence propagation in turbid media will be important for accurate recovery of fluorophore lifetimes in biological spectroscopy and imaging applications. (author)

  13. Quantitative analysis by X-ray fluorescence using first principles for matrix correction

    International Nuclear Information System (INIS)

    Hulett, L.D.; Dunn, H.W.; Tarter, J.G.

    1978-01-01

    The quantitative interpretation of X-ray fluorescence (XRF) data is often difficult because of matrix effects. The intensity of fluorescence measured for a given element is not only dependent on the element's concentration, but also on the mass absorption coefficients of the sample for the excitation and fluorescence radiation. Also, there are interelement effects in which high-energy fluorescence from heavier elements is absorbed by lighter elements with a resulting enhancement of their fluorescence. Recent theoretical treatments of this problem have shown that X-ray fluorescence data can be corrected for these matrix effects by calculations based on first principles. Fundamental constants, available in atomic physics data tables, are the only parameters needed. It is not necessary to make empirical calibrations. The application of this correctional procedure to alloys and alumina-supported catalysts is described. A description is given of a low-background spectrometer which uses monochromatic Ag Ksub(α) radiation for excitation. Matrix corrections by first principles can be easily applied to data from instruments of this type because fluorescence excitation cross-sections and mass absorption coefficients can be accurately defined for monochromatic radiation. (author)

  14. Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

    Science.gov (United States)

    Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira

    2016-08-01

    To evaluate the potential use of fluorescence confocal microscopy (FCM) for ex vivo diagnosis and excision margin assessment of conjunctival neoplasms. Validity study. setting: Single institution. Consecutive patients with clinically suspicious conjunctival lesions. Conjunctival lesions were excised in toto using a standard "no-touch technique" by a single surgeon (A.I.). Collected specimens were examined with a commercially available laser scanning fluorescence confocal microscope after immersion in a 0.6 mM solution of acridine orange dye for 10-20 seconds. Specimens were subsequently processed with standard histologic analysis. FCM diagnosis of the nature and extension of conjunctival lesions. Sixteen consecutive patients were included in the study (11 male, 5 female; mean age 58.1 ± 26.1 years, range 10-90 years). The median time needed to process and analyze a sample with FCM was 15 minutes. Eleven of 16 lesions were identified by FCM as squamous (2 benign papillomas, 2 grade 2 conjunctival intraepithelial neoplasias, 7 in situ squamous carcinomas) and 5 as nonsquamous (1 pingueculum, 1 dermolipoma, 2 melanocytic nevi, 1 melanoma). In all cases FCM was able to detect horizontal and vertical extension of the lesion. All FCM findings were confirmed by corresponding subsequent histologic examination. FCM provides a fast ex vivo preliminary diagnosis of suspicious conjunctival lesions with good histologic details and margin assessment, and may represent a novel tool for intraoperative and postsurgical management of conjunctival tumors. This is the first study to investigate ex vivo FCM application in ophthalmology. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Quantitative schemes in energy dispersive X-ray fluorescence implemented in AXIL

    International Nuclear Information System (INIS)

    Tchantchane, A.; Benamar, M.A.; Tobbeche, S.

    1995-01-01

    E.D.X.R.F (Energy Dispersive X-ray Fluorescence) has long been used for quantitative analysis of many types of samples including environment samples. the software package AXIL (Analysis of x-ray spectra by iterative least quares) is extensively used for the spectra analysis and the quantification of x-ray spectra. It includes several methods of quantitative schemes for evaluating element concentrations. We present the general theory behind each scheme implemented into the software package. The spectra of the performance of each of these quantitative schemes. We have also investigated their performance relative to the uncertainties in the experimental parameters and sample description

  16. Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

    Science.gov (United States)

    Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

    2013-02-01

    Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

  17. 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

    2015-01-01

    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)

  18. Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

    Science.gov (United States)

    Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

    2017-10-20

    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.

  19. Single particle transfer for quantitative analysis with total-reflection X-ray fluorescence spectrometry

    International Nuclear Information System (INIS)

    Esaka, Fumitaka; Esaka, Konomi T.; Magara, Masaaki; Sakurai, Satoshi; Usuda, Shigekazu; Watanabe, Kazuo

    2006-01-01

    The technique of single particle transfer was applied to quantitative analysis with total-reflection X-ray fluorescence (TXRF) spectrometry. The technique was evaluated by performing quantitative analysis of individual Cu particles with diameters between 3.9 and 13.2 μm. The direct quantitative analysis of the Cu particle transferred onto a Si carrier gave a discrepancy between measured and calculated Cu amounts due to the absorption effects of incident and fluorescent X-rays within the particle. By the correction for the absorption effects, the Cu amounts in individual particles could be determined with the deviation within 10.5%. When the Cu particles were dissolved with HNO 3 solution prior to the TXRF analysis, the deviation was improved to be within 3.8%. In this case, no correction for the absorption effects was needed for quantification

  20. Comparison of supervised machine learning algorithms for waterborne pathogen detection using mobile phone fluorescence microscopy

    Science.gov (United States)

    Ceylan Koydemir, Hatice; Feng, Steve; Liang, Kyle; Nadkarni, Rohan; Benien, Parul; Ozcan, Aydogan

    2017-06-01

    Giardia lamblia is a waterborne parasite that affects millions of people every year worldwide, causing a diarrheal illness known as giardiasis. Timely detection of the presence of the cysts of this parasite in drinking water is important to prevent the spread of the disease, especially in resource-limited settings. Here we provide extended experimental testing and evaluation of the performance and repeatability of a field-portable and cost-effective microscopy platform for automated detection and counting of Giardia cysts in water samples, including tap water, non-potable water, and pond water. This compact platform is based on our previous work, and is composed of a smartphone-based fluorescence microscope, a disposable sample processing cassette, and a custom-developed smartphone application. Our mobile phone microscope has a large field of view of 0.8 cm2 and weighs only 180 g, excluding the phone. A custom-developed smartphone application provides a user-friendly graphical interface, guiding the users to capture a fluorescence image of the sample filter membrane and analyze it automatically at our servers using an image processing algorithm and training data, consisting of >30,000 images of cysts and >100,000 images of other fluorescent particles that are captured, including, e.g. dust. The total time that it takes from sample preparation to automated cyst counting is less than an hour for each 10 ml of water sample that is tested. We compared the sensitivity and the specificity of our platform using multiple supervised classification models, including support vector machines and nearest neighbors, and demonstrated that a bootstrap aggregating (i.e. bagging) approach using raw image file format provides the best performance for automated detection of Giardia cysts. We evaluated the performance of this machine learning enabled pathogen detection device with water samples taken from different sources (e.g. tap water, non-potable water, pond water) and achieved a

  1. Comparison of supervised machine learning algorithms for waterborne pathogen detection using mobile phone fluorescence microscopy

    KAUST Repository

    Ceylan Koydemir, Hatice

    2017-06-14

    Giardia lamblia is a waterborne parasite that affects millions of people every year worldwide, causing a diarrheal illness known as giardiasis. Timely detection of the presence of the cysts of this parasite in drinking water is important to prevent the spread of the disease, especially in resource-limited settings. Here we provide extended experimental testing and evaluation of the performance and repeatability of a field-portable and cost-effective microscopy platform for automated detection and counting of Giardia cysts in water samples, including tap water, non-potable water, and pond water. This compact platform is based on our previous work, and is composed of a smartphone-based fluorescence microscope, a disposable sample processing cassette, and a custom-developed smartphone application. Our mobile phone microscope has a large field of view of ~0.8 cm2 and weighs only ~180 g, excluding the phone. A custom-developed smartphone application provides a user-friendly graphical interface, guiding the users to capture a fluorescence image of the sample filter membrane and analyze it automatically at our servers using an image processing algorithm and training data, consisting of >30,000 images of cysts and >100,000 images of other fluorescent particles that are captured, including, e.g. dust. The total time that it takes from sample preparation to automated cyst counting is less than an hour for each 10 ml of water sample that is tested. We compared the sensitivity and the specificity of our platform using multiple supervised classification models, including support vector machines and nearest neighbors, and demonstrated that a bootstrap aggregating (i.e. bagging) approach using raw image file format provides the best performance for automated detection of Giardia cysts. We evaluated the performance of this machine learning enabled pathogen detection device with water samples taken from different sources (e.g. tap water, non-potable water, pond water) and achieved

  2. Comparison of supervised machine learning algorithms for waterborne pathogen detection using mobile phone fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Ceylan Koydemir Hatice

    2017-06-01

    Full Text Available Giardia lamblia is a waterborne parasite that affects millions of people every year worldwide, causing a diarrheal illness known as giardiasis. Timely detection of the presence of the cysts of this parasite in drinking water is important to prevent the spread of the disease, especially in resource-limited settings. Here we provide extended experimental testing and evaluation of the performance and repeatability of a field-portable and cost-effective microscopy platform for automated detection and counting of Giardia cysts in water samples, including tap water, non-potable water, and pond water. This compact platform is based on our previous work, and is composed of a smartphone-based fluorescence microscope, a disposable sample processing cassette, and a custom-developed smartphone application. Our mobile phone microscope has a large field of view of ~0.8 cm2 and weighs only ~180 g, excluding the phone. A custom-developed smartphone application provides a user-friendly graphical interface, guiding the users to capture a fluorescence image of the sample filter membrane and analyze it automatically at our servers using an image processing algorithm and training data, consisting of >30,000 images of cysts and >100,000 images of other fluorescent particles that are captured, including, e.g. dust. The total time that it takes from sample preparation to automated cyst counting is less than an hour for each 10 ml of water sample that is tested. We compared the sensitivity and the specificity of our platform using multiple supervised classification models, including support vector machines and nearest neighbors, and demonstrated that a bootstrap aggregating (i.e. bagging approach using raw image file format provides the best performance for automated detection of Giardia cysts. We evaluated the performance of this machine learning enabled pathogen detection device with water samples taken from different sources (e.g. tap water, non-potable water, pond

  3. Comparison of supervised machine learning algorithms for waterborne pathogen detection using mobile phone fluorescence microscopy

    KAUST Repository

    Ceylan Koydemir, Hatice; Feng, Steve; Liang, Kyle; Nadkarni, Rohan; Benien, Parul; Ozcan, Aydogan

    2017-01-01

    Giardia lamblia is a waterborne parasite that affects millions of people every year worldwide, causing a diarrheal illness known as giardiasis. Timely detection of the presence of the cysts of this parasite in drinking water is important to prevent the spread of the disease, especially in resource-limited settings. Here we provide extended experimental testing and evaluation of the performance and repeatability of a field-portable and cost-effective microscopy platform for automated detection and counting of Giardia cysts in water samples, including tap water, non-potable water, and pond water. This compact platform is based on our previous work, and is composed of a smartphone-based fluorescence microscope, a disposable sample processing cassette, and a custom-developed smartphone application. Our mobile phone microscope has a large field of view of ~0.8 cm2 and weighs only ~180 g, excluding the phone. A custom-developed smartphone application provides a user-friendly graphical interface, guiding the users to capture a fluorescence image of the sample filter membrane and analyze it automatically at our servers using an image processing algorithm and training data, consisting of >30,000 images of cysts and >100,000 images of other fluorescent particles that are captured, including, e.g. dust. The total time that it takes from sample preparation to automated cyst counting is less than an hour for each 10 ml of water sample that is tested. We compared the sensitivity and the specificity of our platform using multiple supervised classification models, including support vector machines and nearest neighbors, and demonstrated that a bootstrap aggregating (i.e. bagging) approach using raw image file format provides the best performance for automated detection of Giardia cysts. We evaluated the performance of this machine learning enabled pathogen detection device with water samples taken from different sources (e.g. tap water, non-potable water, pond water) and achieved

  4. Isotropic differential phase contrast microscopy for quantitative phase bio-imaging.

    Science.gov (United States)

    Chen, Hsi-Hsun; Lin, Yu-Zi; Luo, Yuan

    2018-05-16

    Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical studies. In recent examples, differential phase contrast (DPC) microscopy can recover phase image of thin sample under multi-axis intensity measurements in wide-field scheme. Unlike conventional DPC, based on theoretical approach under partially coherent condition, we propose a new method to achieve isotropic differential phase contrast (iDPC) with high accuracy and stability for phase recovery in simple and high-speed fashion. The iDPC is simply implemented with a partially coherent microscopy and a programmable thin-film transistor (TFT) shield to digitally modulate structured illumination patterns for QPI. In this article, simulation results show consistency of our theoretical approach for iDPC under partial coherence. In addition, we further demonstrate experiments of quantitative phase images of a standard micro-lens array, as well as label-free live human cell samples. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Quantitative assessment of cancer cell morphology and motility using telecentric digital holographic microscopy and machine learning.

    Science.gov (United States)

    Lam, Van K; Nguyen, Thanh C; Chung, Byung M; Nehmetallah, George; Raub, Christopher B

    2018-03-01

    The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two-dimensional surfaces in vitro, MDA-MB-231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA-MB-231 cell line were cultured on glass, tissue-culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F-actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F-actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA-MB-231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  6. Whole-slide imaging is a robust alternative to traditional fluorescent microscopy for fluorescence in situ hybridization imaging using break-apart DNA probes.

    Science.gov (United States)

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

    2013-08-01

    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.

  7. Groping for Quantitative Digital 3-D Image Analysis: An Approach to Quantitative Fluorescence In Situ Hybridization in Thick Tissue Sections of Prostate Carcinoma

    Directory of Open Access Journals (Sweden)

    Karsten Rodenacker

    1997-01-01

    Full Text Available In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH. The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA can be estimated by counting the number of FISH signals per cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI to a software package for display, inspection, count and (semi‐automatic analysis of 3‐D images for pathologists is outlined including the underlying methods of 3‐D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer‐aided analysis of large 3‐D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3‐D data is not in sight. A semi‐automatic segmentation method is thus presented here.

  8. Multicolor fluorescent intravital live microscopy (FILM) for surgical tumor resection in a mouse xenograft model.

    Science.gov (United States)

    Thurber, Greg M; Figueiredo, Jose L; Weissleder, Ralph

    2009-11-30

    Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM) where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time. Theoretical simulations of imaging probe localization were carried out for three agents with specificity for cancer cells, stromal host response, or vascular perfusion. This transport analysis gave insight into the probe pharmacokinetics and tissue distribution, facilitating the experimental design and allowing predictions to be made about the localization of the probes in other animal models and in the clinic. The imaging probes were administered systemically at optimal time points based on the simulations, and the multicolor FILM images obtained in vivo were then compared to conventional pathological sections. Our data show the feasibility of real time in vivo pathology at cellular resolution and molecular specificity with excellent agreement between intravital and traditional in vitro immunohistochemistry. Multicolor FILM is an accurate method for identifying malignant tissue and cells in vivo. The imaging probes distributed in a manner similar to predictions based on transport principles, and these models can be used to design future probes and experiments. FILM can provide critical real time feedback and should be a useful tool for more effective and complete cancer resection.

  9. Multicolor fluorescent intravital live microscopy (FILM for surgical tumor resection in a mouse xenograft model.

    Directory of Open Access Journals (Sweden)

    Greg M Thurber

    2009-11-01

    Full Text Available Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time.Theoretical simulations of imaging probe localization were carried out for three agents with specificity for cancer cells, stromal host response, or vascular perfusion. This transport analysis gave insight into the probe pharmacokinetics and tissue distribution, facilitating the experimental design and allowing predictions to be made about the localization of the probes in other animal models and in the clinic. The imaging probes were administered systemically at optimal time points based on the simulations, and the multicolor FILM images obtained in vivo were then compared to conventional pathological sections. Our data show the feasibility of real time in vivo pathology at cellular resolution and molecular specificity with excellent agreement between intravital and traditional in vitro immunohistochemistry.Multicolor FILM is an accurate method for identifying malignant tissue and cells in vivo. The imaging probes distributed in a manner similar to predictions based on transport principles, and these models can be used to design future probes and experiments. FILM can provide critical real time feedback and should be a useful tool for more effective and complete cancer resection.

  10. X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

    Science.gov (United States)

    Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2009-01-01

    Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis. PMID:19379757

  11. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

    Science.gov (United States)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-02

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

  12. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    Science.gov (United States)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  13. Development of an automated asbestos counting software based on fluorescence microscopy.

    Science.gov (United States)

    Alexandrov, Maxym; Ichida, Etsuko; Nishimura, Tomoki; Aoki, Kousuke; Ishida, Takenori; Hirota, Ryuichi; Ikeda, Takeshi; Kawasaki, Tetsuo; Kuroda, Akio

    2015-01-01

    An emerging alternative to the commonly used analytical methods for asbestos analysis is fluorescence microscopy (FM), which relies on highly specific asbestos-binding probes to distinguish asbestos from interfering non-asbestos fibers. However, all types of microscopic asbestos analysis require laborious examination of large number of fields of view and are prone to subjective errors and large variability between asbestos counts by different analysts and laboratories. A possible solution to these problems is automated counting of asbestos fibers by image analysis software, which would lower the cost and increase the reliability of asbestos testing. This study seeks to develop a fiber recognition and counting software for FM-based asbestos analysis. We discuss the main features of the developed software and the results of its testing. Software testing showed good correlation between automated and manual counts for the samples with medium and high fiber concentrations. At low fiber concentrations, the automated counts were less accurate, leading us to implement correction mode for automated counts. While the full automation of asbestos analysis would require further improvements in accuracy of fiber identification, the developed software could already assist professional asbestos analysts and record detailed fiber dimensions for the use in epidemiological research.

  14. Visualising recalcitrance by colocalisation of cellulase, lignin and cellulose in pretreated pine biomass using fluorescence microscopy

    Science.gov (United States)

    Donaldson, Lloyd; Vaidya, Alankar

    2017-03-01

    Mapping the location of bound cellulase enzymes provides information on the micro-scale distribution of amenable and recalcitrant sites in pretreated woody biomass for biofuel applications. The interaction of a fluorescently labelled cellulase enzyme cocktail with steam-exploded pine (SEW) was quantified using confocal microscopy. The spatial distribution of Dylight labelled cellulase was quantified relative to lignin (autofluorescence) and cellulose (Congo red staining) by measuring their colocalisation using Pearson correlations. Correlations were greater in cellulose-rich secondary cell walls compared to lignin-rich middle lamella but with significant variations among individual biomass particles. The distribution of cellulose in the pretreated biomass accounted for 30% of the variation in the distribution of enzyme after correcting for the correlation between lignin and cellulose. For the first time, colocalisation analysis was able to quantify the spatial distribution of amenable and recalcitrant sites in relation to the histochemistry of cellulose and lignin. This study will contribute to understanding the role of pretreatment in enzymatic hydrolysis of recalcitrant softwood biomass.

  15. Fibred confocal fluorescence microscopy in the diagnosis of interstitial lung diseases.

    Science.gov (United States)

    Meng, Peng; Tan, Gan Liang; Low, Su Ying; Takano, Angela; Ng, Yuen Li; Anantham, Devanand

    2016-12-01

    Accurate diagnosis is critical to both therapeutic decisions and prognostication in interstitial lung diseases (ILD). However, surgical lung biopsies carry high complication rates. Fibred confocal fluorescence microscopy (FCFM) offers an alternative as it can visualize lung tissue in vivo at the cellular level with minimal adverse events. We wanted to investigate the diagnostic utility, and safety of using FCFM for patients with ILD. In patients with suspected ILD, FCFM images were obtained from multiple bronchopulmonary segments using a miniprobe inserted through the working channel of a flexible bronchoscope. The procedure was performed under moderate sedation in an outpatient setting. Morphometric measurements and fibre pattern analyses were co-related with computed tomography (CT) findings and patients' final diagnoses based on multi-disciplinary consensus. One hundred and eighty four segments were imaged in 27 patients (18 males) with a median age of 67 years (range, 24-79 years). They were grouped into chronic fibrosing interstitial pneumonia (16 patients) and other ILDs. Six distinct FCFM patterns were observed: normal, increased fibres, densely packed fibres, hypercellular, thickened fibres and others/non-specific. The pattern resembling densely packed fibres was seen in at least one segment in 68.8% patients with chronic fibrosing interstitial pneumonia, but only 36.4% in other ILD (P=0.097). An association between inflammatory patterns on CT and a hypercellular pattern on FCFM was also found (P<0.001). Our study shows the potential of FCFM in classifying ILD, but its role in further diagnosis remains limited.

  16. Intraoperative diagnosis of nonpigmented nail tumours with ex vivo fluorescence confocal microscopy: 10 cases.

    Science.gov (United States)

    Debarbieux, S; Gaspar, R; Depaepe, L; Dalle, S; Balme, B; Thomas, L

    2015-04-01

    Ex vivo fluorescence confocal microscopy (FCM) permits real-time imaging of freshly excised skin tissues. Its usefulness as a time-sparing alternative to frozen sections in Mohs surgery of basal cell carcinoma is well documented. The purpose of this study was to describe the ex vivo FCM features of a series of benign and malignant nonpigmented tumours of the nail unit, and to correlate them with conventional histopathology. Nail apparatus tumours from 10 patients were imaged during surgical exploration using ex vivo FCM after immersion in acridine orange. Confocal mosaics of the freshly performed biopsies were evaluated in real time and retrospectively compared with haematoxylin and eosin sections. Our series included two invasive epithelial tumours (Group 1), four in situ or minimally invasive squamous cell carcinomas (SCC) (Group 2), three benign epithelial tumours (Group 3) and one nodular melanoma (Group 4). The correlation was excellent for malignant epithelial tumours exhibiting marked cytological and architectural atypias (Bowen disease, invasive SCC and onycholemmal carcinoma). Onychomatricomas exhibited a very peculiar aspect with densely cellular papillae. The correlation was less favourable for minimally invasive well-differentiated SCCs with slight cytological atypias. The correlation was poor for our case of amelanotic invasive subungual melanoma. Ex vivo FCM could be a useful tool to shorten management of nonpigmented nail tumours: in the case of a malignant tumour, it could indeed lead to performing wide excision during the same surgical procedure and possibly assessing the surgical margins. © 2014 British Association of Dermatologists.

  17. Real-time monitoring of magnetic drug targeting using fibered confocal fluorescence microscopy.

    Science.gov (United States)

    Bai, Jie; Wang, Julie Tzu-Wen; Mei, Kuo-Ching; Al-Jamal, Wafa T; Al-Jamal, Khuloud T

    2016-12-28

    Magnetic drug targeting has been proposed as means of concentrating therapeutic agents at a target site and the success of this approach has been demonstrated in a number of studies. However, the behavior of magnetic carriers in blood vessels and tumor microcirculation still remains unclear. In this work, we utilized polymeric magnetic nanocapsules (m-NCs) for magnetic targeting in tumors and dynamically visualized them within blood vessels and tumor tissues before, during and after magnetic field exposure using fibered confocal fluorescence microscopy (FCFM). Our results suggested that the distribution of m-NCs within tumor vasculature changed dramatically, but in a reversible way, upon application and removal of a magnetic field. The m-NCs were concentrated and stayed as clusters near a blood vessel wall when tumors were exposed to a magnetic field but without rupturing the blood vessel. The obtained FCFM images provided in vivo in situ microvascular observations of m-NCs upon magnetic targeting with high spatial resolution but minimally invasive surgical procedures. This proof-of-concept descriptive study in mice is envisaged to track and quantify nanoparticles in vivo in a non-invasive manner at microscopic resolution. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Lithographically-fabricated channel arrays for confocal x-ray fluorescence microscopy and XAFS

    International Nuclear Information System (INIS)

    Woll, Arthur R; Agyeman-Budu, David; Choudhury, Sanjukta; Coulthard, Ian; Hallin, Emil; Finnefrock, Adam C; Gordon, Robert; Mass, Jennifer

    2014-01-01

    Confocal X-ray Fluorescence Microscopy (CXRF) employs overlapping focal regions of two x-ray optics—a condenser and collector—to directly probe a 3D volume. The minimum-achievable size of this probe volume is limited by the collector, for which polycapillaries are generally the optic of choice. Recently, we demonstrated an alternative collection optic for CXRF, consisting of an array of micron-scale collimating channels, etched in silicon, and arranged like spokes of a wheel directed towards a single source position. The optic, while successful, had a working distance of only 0.2 mm and exhibited relatively low total collection efficiency, limiting its practical application. Here, we describe a new design in which the collimating channels are formed by a staggered array of pillars whose side-walls taper away from the channel axis. This approach improves both collection efficiency and working distance, while maintaining excellent spatial resolution. We illustrate these improvements with confocal XRF data obtained at the Cornell High Energy Synchrotron Source (CHESS) and the Advanced Photon Source (APS) beamline 20-ID-B.

  19. A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

    Science.gov (United States)

    Lee, Jacqueline A; Collings, David A; Glover, Chris N

    2016-08-15

    The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. A system architecture for online data interpretation and reduction in fluorescence microscopy

    Science.gov (United States)

    Röder, Thorsten; Geisbauer, Matthias; Chen, Yang; Knoll, Alois; Uhl, Rainer

    2010-01-01

    In this paper we present a high-throughput sample screening system that enables real-time data analysis and reduction for live cell analysis using fluorescence microscopy. We propose a novel system architecture capable of analyzing a large amount of samples during the experiment and thus greatly minimizing the post-analysis phase that is the common practice today. By utilizing data reduction algorithms, relevant information of the target cells is extracted from the online collected data stream, and then used to adjust the experiment parameters in real-time, allowing the system to dynamically react on changing sample properties and to control the microscope setup accordingly. The proposed system consists of an integrated DSP-FPGA hybrid solution to ensure the required real-time constraints, to execute efficiently the underlying computer vision algorithms and to close the perception-action loop. We demonstrate our approach by addressing the selective imaging of cells with a particular combination of markers. With this novel closed-loop system the amount of superfluous collected data is minimized, while at the same time the information entropy increases.

  1. 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

    2016-01-01

    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

  2. Quantitative sub-surface and non-contact imaging using scanning microwave microscopy

    International Nuclear Information System (INIS)

    Gramse, Georg; Kasper, Manuel; Hinterdorfer, Peter; Brinciotti, Enrico; Rankl, Christian; Kienberger, Ferry; Lucibello, Andrea; Marcelli, Romolo; Patil, Samadhan B.; Giridharagopal, Rajiv

    2015-01-01

    The capability of scanning microwave microscopy for calibrated sub-surface and non-contact capacitance imaging of silicon (Si) samples is quantitatively studied at broadband frequencies ranging from 1 to 20 GHz. Calibrated capacitance images of flat Si test samples with varying dopant density (10 15 –10 19 atoms cm −3 ) and covered with dielectric thin films of SiO 2 (100–400 nm thickness) are measured to demonstrate the sensitivity of scanning microwave microscopy (SMM) for sub-surface imaging. Using standard SMM imaging conditions the dopant areas could still be sensed under a 400 nm thick oxide layer. Non-contact SMM imaging in lift-mode and constant height mode is quantitatively demonstrated on a 50 nm thick SiO 2 test pad. The differences between non-contact and contact mode capacitances are studied with respect to the main parameters influencing the imaging contrast, namely the probe tip diameter and the tip–sample distance. Finite element modelling was used to further analyse the influence of the tip radius and the tip–sample distance on the SMM sensitivity. The understanding of how the two key parameters determine the SMM sensitivity and quantitative capacitances represents an important step towards its routine application for non-contact and sub-surface imaging. (paper)

  3. Quantitative in situ magnetization reversal studies in Lorentz microscopy and electron holography

    International Nuclear Information System (INIS)

    Rodríguez, L.A.; Magén, C.; Snoeck, E.; Gatel, C.; Marín, L.; Serrano-Ramón, L.

    2013-01-01

    A generalized procedure for the in situ application of magnetic fields by means of the excitation of the objective lens for magnetic imaging experiments in Lorentz microscopy and electron holography is quantitatively described. A protocol for applying magnetic fields with arbitrary in-plane magnitude and orientation is presented, and a freeware script for Digital Micrograph ™ is provided to assist the operation of the microscope. Moreover, a method to accurately reconstruct hysteresis loops is detailed. We show that the out-of-plane component of the magnetic field cannot be always neglected when performing quantitative measurements of the local magnetization. Several examples are shown to demonstrate the accuracy and functionality of the methods. - Highlights: • Generalized procedure for application of magnetic fields with the TEM objective lens. • Arbitrary in-plane magnetic field magnitude and orientation can be applied. • Method to accurately reconstruct hysteresis loops by electron holography. • Out-of-plane field component should be considered in quantitative measurements. • Examples to illustrate the method in Lorentz microscopy and electron holography

  4. Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

    Science.gov (United States)

    Sharifzadeh, Mohsen; Bernstein, Paul S.; Gellermann, Werner

    2006-10-01

    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.

  5. Atomic-resolution environmental TEM for quantitative in-situ microscopy in materials science.

    Science.gov (United States)

    Takeda, Seiji; Yoshida, Hideto

    2013-02-01

    We have compiled our recent in-situ quantitative environmental transmission electron microscopy (ETEM) studies on typical gold nanoparticulate catalysts for the low-temperature oxidation of CO to describe the issues surrounding the application of ETEM, with a special regard to catalyst chemistry. Thanks to the recent development of high-resolution environmental transmission electron microscopes that can work robustly to accumulate observation data in controlled environments, we can deal with the electron irradiation effects and heterogeneity of real catalysts. We established a structural evolution diagram that summarizes the structure of catalysts under electron irradiation as a function of the electron current density ϕ and the electron dose, D. By extrapolating to ϕ = 0, D = 0, we could deduce the intrinsic catalysis structure (without electron irradiation) in various environments, including reaction environments. By numerically and statistically analyzing a substantial number of ETEM images of gold nanoparticles, we established a morphology phase diagram that summarizes how the majority of gold nanoparticles change their morphology systematically as a function of the partial pressures of CO and O(2). Similar diagrams will be helpful in elucidating the phenomena that directly correlate with the catalytic activity determined from ETEM observations. Using these quantitative analyses, we could analyze Cs-corrected ETEM images of the catalysts. The surfaces of gold nanoparticles were structurally reconstructed under reaction conditions, via interactions with CO molecules. CO molecules were observed on the surfaces of catalysts under reaction conditions using high-resolution ETEM. Finally, we discuss the potential of environmental transmission electron microscopy for quantitative in-situ microscopy at the atomic scale.

  6. Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

    Science.gov (United States)

    Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

    2017-07-01

    Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

  7. Spatially resolved quantitative mapping of thermomechanical properties and phase transition temperatures using scanning probe microscopy

    Science.gov (United States)

    Jesse, Stephen; Kalinin, Sergei V; Nikiforov, Maxim P

    2013-07-09

    An approach for the thermomechanical characterization of phase transitions in polymeric materials (polyethyleneterephthalate) by band excitation acoustic force microscopy is developed. This methodology allows the independent measurement of resonance frequency, Q factor, and oscillation amplitude of a tip-surface contact area as a function of tip temperature, from which the thermal evolution of tip-surface spring constant and mechanical dissipation can be extracted. A heating protocol maintained a constant tip-surface contact area and constant contact force, thereby allowing for reproducible measurements and quantitative extraction of material properties including temperature dependence of indentation-based elastic and loss moduli.

  8. New generation quantitative x-ray microscopy encompassing phase-contrast

    International Nuclear Information System (INIS)

    Wilkins, S.W.; Mayo, S.C.; Gureyev, T.E.; Miller, P.R.; Pogany, A.; Stevenson, A.W.; Gao, D.; Davis, T.J.; Parry, D.J.; Paganin, D.

    2000-01-01

    Full text: We briefly outline a new approach to X-ray ultramicroscopy using projection imaging in a scanning electron microscope (SEM). Compared to earlier approaches, the new approach offers spatial resolution of ≤0.1 micron and includes novel features such as: i) phase contrast to give additional sample information over a wide energy range, rapid phase/amplitude extraction algorithms to enable new real-time modes of microscopic imaging widespread applications are envisaged to fields such as materials science, biomedical research, and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to X-ray projection microscopy using synchrotron sources

  9. Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy.

    Science.gov (United States)

    Rinehart, Matthew T; Drake, Tyler K; Robles, Francisco E; Rohan, Lisa C; Katz, David; Wax, Adam

    2011-12-01

    Quantitative phase microscopy is applied to image temporal changes in the refractive index (RI) distributions of solutions created by microbicidal films undergoing hydration. We present a novel method of using an engineered polydimethylsiloxane structure as a static phase reference to facilitate calibration of the absolute RI across the entire field. We present a study of dynamic structural changes in microbicidal films during hydration and subsequent dissolution. With assumptions about the smoothness of the phase changes induced by these films, we calculate absolute changes in the percentage of film in regions across the field of view.

  10. Nanoscale nuclear architecture for cancer diagnosis by spatial-domain low-coherence quantitative phase microscopy

    Science.gov (United States)

    Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Staton, Kevin D.; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

    2011-03-01

    Alterations in nuclear architecture are the hallmark diagnostic characteristic of cancer cells. In this work, we show that the nuclear architectural characteristics quantified by spatial-domain low-coherence quantitative phase microscopy (SL-QPM), is more sensitive for the identification of cancer cells than conventional cytopathology. We demonstrated the importance of nuclear architectural characteristics in both an animal model of intestinal carcinogenesis - APC/Min mouse model and human cytology specimens with colorectal cancer by identifying cancer from cytologically noncancerous appearing cells. The determination of nanoscale nuclear architecture using this simple and practical optical instrument is a significant advance towards cancer diagnosis.

  11. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peddie, Christopher J.; Blight, Ken; Wilson, Emma [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Melia, Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden (Netherlands); Marrison, Jo [Department of Biology, The University of York, Heslington, York (United Kingdom); Carzaniga, Raffaella [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Domart, Marie-Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); O' Toole, Peter [Department of Biology, The University of York, Heslington, York (United Kingdom); Larijani, Banafshe [Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa (Spain); IKERBASQUE, Basque Foundation for Science, Bilbao (Spain); Collinson, Lucy M. [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom)

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. - Highlights: • GFP and mCherry fluorescence are preserved in heavy-metal stained mammalian cells embedded in resin • Fluorophores are stable and intensity is sufficient for detection in ultrathin sections • Overlay of separate LM and EM images from the same ultrathin section improves CLEM protein localisation precision • GFP is stable and active in the vacuum of an integrated light and scanning EM • Integrated light and electron microscopy shows new subcellular locations of the lipid diacylglycerol.

  12. Quantitative surface topography determination by Nomarski reflection microscopy. 2: Microscope modification, calibration, and planar sample experiments

    International Nuclear Information System (INIS)

    Hartman, J.S.; Gordon, R.L.; Lessor, D.L.

    1980-01-01

    The application of reflective Nomarski differential interference contrast microscopy for the determination of quantitative sample topography data is presented. The discussion includes a review of key theoretical results presented previously plus the experimental implementation of the concepts using a commercial Momarski microscope. The experimental work included the modification and characterization of a commercial microscope to allow its use for obtaining quantitative sample topography data. System usage for the measurement of slopes on flat planar samples is also discussed. The discussion has been designed to provide the theoretical basis, a physical insight, and a cookbook procedure for implementation to allow these results to be of value to both those interested in the microscope theory and its practical usage in the metallography laboratory

  13. New approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy

    Science.gov (United States)

    Pohl, L.; Kaiser, M.; Ketelhut, S.; Pereira, S.; Goycoolea, F.; Kemper, Björn

    2016-03-01

    Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

  14. Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC.

    Directory of Open Access Journals (Sweden)

    Zachary F Phillips

    Full Text Available We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification-an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC, is a single-shot variant of Differential Phase Contrast (DPC, which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps for various in vitro cell samples and c. elegans in a micro-fluidic channel.

  15. Quantitative in situ magnetization reversal studies in Lorentz microscopy and electron holography.

    Science.gov (United States)

    Rodríguez, L A; Magén, C; Snoeck, E; Gatel, C; Marín, L; Serrano-Ramón, L; Prieto, J L; Muñoz, M; Algarabel, P A; Morellon, L; De Teresa, J M; Ibarra, M R

    2013-11-01

    A generalized procedure for the in situ application of magnetic fields by means of the excitation of the objective lens for magnetic imaging experiments in Lorentz microscopy and electron holography is quantitatively described. A protocol for applying magnetic fields with arbitrary in-plane magnitude and orientation is presented, and a freeware script for Digital Micrograph(™) is provided to assist the operation of the microscope. Moreover, a method to accurately reconstruct hysteresis loops is detailed. We show that the out-of-plane component of the magnetic field cannot be always neglected when performing quantitative measurements of the local magnetization. Several examples are shown to demonstrate the accuracy and functionality of the methods. © 2013 Elsevier B.V. All rights reserved.

  16. Dynamic characterization of hydrophobic and hydrophilic solutes in oleic-acid enhanced transdermal delivery using two-photon fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Te-Yu; Yang, Chiu-Sheng; Chen, Yang-Fang [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Tsai, Tsung-Hua [Department of Dermatology, Far Eastern Memorial Hospital, New Taipei City, Taiwan (China); Dong, Chen-Yuan, E-mail: cydong@phys.ntu.edu.tw [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Center for Quantum Science and Engineering, National Taiwan University, Taipei, Taiwan (China); Center for Optoelectronic Biomedicine, National Taiwan University, Taipei, Taiwan (China)

    2014-10-20

    In this letter, we propose an efficient methodology of investigating dynamic properties of sulforhodamine B and rhodamine B hexyl ester molecules transporting across ex-vivo human stratum corneum with and without oleic acid enhancement. Three-dimensional, time-lapse fluorescence images of the stratum corneum can be obtained using two-photon fluorescence microscopy. Furthermore, temporal quantifications of transport enhancements in diffusion parameters can be achieved with the use of Fick's second law. Dynamic characterization of solutes transporting across the stratum corneum is an effective method for understanding transient phenomena in transdermal delivery of probe molecules, leading to improved delivery strategies of molecular species for therapeutic purposes.

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

    Science.gov (United States)

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

    2016-01-01

    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

  18. Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

    Science.gov (United States)

    Hezel, Marcus; Ebrahimi, Fahim; Koch, Marco; Dehghani, Faramarz

    2012-10-01

    Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell

  19. Diagnosis of malaria by acridine orange fluorescent microscopy in an endemic area of Venezuela

    Directory of Open Access Journals (Sweden)

    Irene Bosch

    1996-02-01

    Full Text Available Fluorescent (acridine orange microscopical examination of capillary centrifuged blood (quantitative buffy coat [QBC®] analysis and Giemsa stained thick blood smears (GTS were compared for diagnosis of malaria in blood specimens from adults living in malaria transmission areas of the States of Bolivar and Amazonas in southeastern and south Venezuela, respectively. Of a total of 198 GTS examined, 95 subjects (48% showed parasitaemia. Among the 95 blood films with a positive GTS, 94 were judged positive by the QBC. However, positive QBC tubes were found in 29 out of 103 blood specimens with a negative GTS. Thus, relative to a GTS standard, the sensitivity and specificity of the QBC-test was 99.2% and 72%, respectively. Young trophozoites of Plasmodium vivax and P. falciparum could not be distinguished with certainty. It is confirmed that the QBC offers many advantages compared with the standard diagnosis of malaria parasites, specifically in the speed of staining and ease of interpretation. However, in places where P. falciparum and P. vivax occur, species and stage differentiation should be confirmed with the GTS.

  20. Quantitative dopant profiling in semiconductors. A new approach to Kelvin probe force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Baumgart, Christine

    2012-07-01

    Failure analysis and optimization of semiconducting devices request knowledge of their electrical properties. To meet the demands of today's semiconductor industry, an electrical nanometrology technique is required which provides quantitative information about the doping profile and which enables scans with a lateral resolution in the sub-10 nm range. In the presented work it is shown that Kelvin probe force microscopy (KPFM) is a very promising electrical nanometrology technique to face this challenge. The technical and physical aspects of KPFM measurements on semiconductors required for the correct interpretation of the detected KPFM bias are discussed. A new KPFM model is developed which enables the quantitative correlation between the probed KPFM bias and the dopant concentration in the investigated semiconducting sample. Quantitative dopant profiling by means of the new KPFM model is demonstrated by the example of differently structured, n- and p-type doped silicon. Additionally, the transport of charge carriers during KPFM measurements, in particular in the presence of intrinsic electric fields due to vertical and horizontal pn junctions as well as due to surface space charge regions, is discussed. Detailed investigations show that transport of charge carriers in the semiconducting sample is a crucial aspect and has to be taken into account when aiming for a quantitative evaluation of the probed KPFM bias.

  1. Reducing depth induced spherical aberration in 3D widefield fluorescence microscopy by wavefront coding using the SQUBIC phase mask

    Science.gov (United States)

    Patwary, Nurmohammed; Doblas, Ana; King, Sharon V.; Preza, Chrysanthe

    2014-03-01

    Imaging thick biological samples introduces spherical aberration (SA) due to refractive index (RI) mismatch between specimen and imaging lens immersion medium. SA increases with the increase of either depth or RI mismatch. Therefore, it is difficult to find a static compensator for SA1. Different wavefront coding methods2,3 have been studied to find an optimal way of static wavefront correction to reduce depth-induced SA. Inspired by a recent design of a radially symmetric squared cubic (SQUBIC) phase mask that was tested for scanning confocal microscopy1 we have modified the pupil using the SQUBIC mask to engineer the point spread function (PSF) of a wide field fluorescence microscope. In this study, simulated images of a thick test object were generated using a wavefront encoded engineered PSF (WFEPSF) and were restored using space-invariant (SI) and depth-variant (DV) expectation maximization (EM) algorithms implemented in the COSMOS software4. Quantitative comparisons between restorations obtained with both the conventional and WFE PSFs are presented. Simulations show that, in the presence of SA, the use of the SIEM algorithm and a single SQUBIC encoded WFE-PSF can yield adequate image restoration. In addition, in the presence of a large amount of SA, it is possible to get adequate results using the DVEM with fewer DV-PSFs than would typically be required for processing images acquired with a clear circular aperture (CCA) PSF. This result implies that modification of a widefield system with the SQUBIC mask renders the system less sensitive to depth-induced SA and suitable for imaging samples at larger optical depths.

  2. Probing Local Heterogeneity in the Optoelectronic Properties of Organic-Inorganic Perovskites Using Fluorescence Microscopy

    Science.gov (United States)

    De Quilettes, Dane W.

    rational design of materials and is leveraged to deploy chemical passivation techniques to improve the optoelectronic quality of the material, with the ultimate goal of improving photovoltaic power conversion efficiency. Reducing non-radiative recombination in semiconducting materials is a prerequisite for achieving the highest performance in a host of light-emitting and photovoltaic applications. In the first study described herein, we used confocal fluorescence microscopy correlated with scanning electron microscopy to spatially resolve the photoluminescence (PL) decay dynamics from films of nonstoichiometric organic-inorganic perovskites, CH3NH 3PbI3(Cl). The PL intensities and lifetimes varied between different grains in the same film, even for films that exhibited long bulk lifetimes. The grain boundaries were dimmer and exhibited faster non-radiative decay. Energy-dispersive x-ray spectroscopy showed a positive correlation between chlorine concentration and regions of brighter PL, while PL imaging revealed that chemical treatment with pyridine could activate previously dark grains. Next, to better elucidate the sources of these loss pathways, we performed a systematic study using confocal and widefield fluorescence microscopy to deconvolve the contributions from diffusion and non-radiative recombination which lead to the observed image heterogeneity. We showed that, in addition to local variations in non-radiative loss, carriers diffuse anisotropically due to heterogeneous intergrain connectivity. In addition to non-radiative recombination impeding material performance, we also showed that the materials exhibit a range of complex dynamic phenomena under illumination. We used a unique combination of confocal PL microscopy and chemical imaging to correlate the local changes in photophysics with composition in CH3NH 3PbI3 films under illumination. We demonstrated that the photo-induced "brightening" of the perovskite PL can be attributed to an order

  3. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

    Science.gov (United States)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-01

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

  4. 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

    2015-01-01

    A quantitative method to discriminate caries lesions for a fluorescence imaging system is proposed in this paper. The autofluorescence spectral investigation of 39 teeth samples classified by the International Caries Detection and Assessment System levels was performed at 405 nm excitation. The major differences in the different caries lesions focused on the relative spectral intensity range of 565–750 nm. The spectral parameter, defined as the ratio of wavebands at 565–750 nm to the whole spectral range, was calculated. The image component ratio R/(G + B) of color components was statistically computed by considering the spectral parameters (e.g. autofluorescence, optical filter, and spectral sensitivity) in our fluorescence color imaging system. Results showed that the spectral parameter and image component ratio presented a linear relation. Therefore, the image component ratio was graded as <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)

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

    Science.gov (United States)

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

    2015-08-01

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

  6. Detection of Colorectal Cancer by a Quantitative Fluorescence Determination of DNA Amplification in Stool

    Directory of Open Access Journals (Sweden)

    Daniele Calistri

    2004-09-01

    Full Text Available DNA amplification of exfoliated cells in stool repre sents an inexpensive and rapid test, but has only 50% to 60% sensitivity. A new quantitative method, calle( fluorescence long DNA, was developed and validate( in our laboratory on stool obtained from 86 patient., with primary colorectal cancer and from 62 health individuals. It consists of the amplification of stoo DNA with fluorescence primers and the quantification of the amplification using a standard curve. Results are arbitrarily expressed in nanograms. The potential of thi new method compared to the conventional approact was analyzed in a subgroup of 94 individuals (51 patients and 38 healthy volunteers. In the presen series, DNA amplification analysis showed a specific ity of 97% and a sensitivity of only 50%. Conversely fluorescence DNA evaluation, using the best cutoff o 25 ng, showed a sensitivity of about 76% and a spec ificity of 93%. Similar sensitivity was observed regard less of Dukes stage, tumor location, and size, thu., also permitting the detection of early-stage tumors The present study seems to indicate that quantitative fluorescence DNA determination in stool successfully identifies colorectal cancer patients with a sensitivity comparable, if not superior, to that of multiple gene analysis but at a lower cost and in a shorter time.

  7. Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

    Science.gov (United States)

    Favicchio, Rosy; Psycharakis, Stylianos; Schönig, Kai; Bartsch, Dusan; Mamalaki, Clio; Papamatheakis, Joseph; Ripoll, Jorge; Zacharakis, Giannis

    2016-02-01

    Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.

  8. A methodology for the extraction of quantitative information from electron microscopy images at the atomic level

    International Nuclear Information System (INIS)

    Galindo, P L; Pizarro, J; Guerrero, E; Guerrero-Lebrero, M P; Scavello, G; Yáñez, A; Sales, D L; Herrera, M; Molina, S I; Núñez-Moraleda, B M; Maestre, J M

    2014-01-01

    In this paper we describe a methodology developed at the University of Cadiz (Spain) in the past few years for the extraction of quantitative information from electron microscopy images at the atomic level. This work is based on a coordinated and synergic activity of several research groups that have been working together over the last decade in two different and complementary fields: Materials Science and Computer Science. The aim of our joint research has been to develop innovative high-performance computing techniques and simulation methods in order to address computationally challenging problems in the analysis, modelling and simulation of materials at the atomic scale, providing significant advances with respect to existing techniques. The methodology involves several fundamental areas of research including the analysis of high resolution electron microscopy images, materials modelling, image simulation and 3D reconstruction using quantitative information from experimental images. These techniques for the analysis, modelling and simulation allow optimizing the control and functionality of devices developed using materials under study, and have been tested using data obtained from experimental samples

  9. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    Directory of Open Access Journals (Sweden)

    Sangeeta Nath

    Full Text Available INTRODUCTION: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. METHOD: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. RESULTS: The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii retention and spatial localization of chemical compounds vary within and between each cell line; and (iii the structural similarities of compounds can infer their non-specific binding properties. CONCLUSION: We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  10. Scattering features for lung cancer detection in fibered confocal fluorescence microscopy images.

    Science.gov (United States)

    Rakotomamonjy, Alain; Petitjean, Caroline; Salaün, Mathieu; Thiberville, Luc

    2014-06-01

    To assess the feasibility of lung cancer diagnosis using fibered confocal fluorescence microscopy (FCFM) imaging technique and scattering features for pattern recognition. FCFM imaging technique is a new medical imaging technique for which interest has yet to be established for diagnosis. This paper addresses the problem of lung cancer detection using FCFM images and, as a first contribution, assesses the feasibility of computer-aided diagnosis through these images. Towards this aim, we have built a pattern recognition scheme which involves a feature extraction stage and a classification stage. The second contribution relies on the features used for discrimination. Indeed, we have employed the so-called scattering transform for extracting discriminative features, which are robust to small deformations in the images. We have also compared and combined these features with classical yet powerful features like local binary patterns (LBP) and their variants denoted as local quinary patterns (LQP). We show that scattering features yielded to better recognition performances than classical features like LBP and their LQP variants for the FCFM image classification problems. Another finding is that LBP-based and scattering-based features provide complementary discriminative information and, in some situations, we empirically establish that performance can be improved when jointly using LBP, LQP and scattering features. In this work we analyze the joint capability of FCFM images and scattering features for lung cancer diagnosis. The proposed method achieves a good recognition rate for such a diagnosis problem. It also performs well when used in conjunction with other features for other classical medical imaging classification problems. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Dynamic quantitative analysis of adherent cell cultures by means of lens-free video microscopy

    Science.gov (United States)

    Allier, C.; Vincent, R.; Navarro, F.; Menneteau, M.; Ghenim, L.; Gidrol, X.; Bordy, T.; Hervé, L.; Cioni, O.; Bardin, S.; Bornens, M.; Usson, Y.; Morales, S.

    2018-02-01

    We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view ( 30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

  12. Microscopy

    Science.gov (United States)

    Patricia A. Moss; Les Groom

    2001-01-01

    Microscopy is the study and interpretation of images produced by a microscope. "Interpretation" is the keyword, because the microscope enables one to see structures that are too small or too close together to be resolved by the unaided eye. (The human eye cannot separate two points or lines that are closer together than 0.1 mm.) it is important to...

  13. Reconstitution radicicol containing apolipoprotein B lipoparticle and tracing its cell uptake process by super resolution fluorescent microscopy.

    Science.gov (United States)

    Lin, Chung Ching; Lin, Po-Yen; Chang, Chia-Ching

    Apolipoprotein B (apoB) is the only protein of LDL. LDL delivers cholesterol, triacylglycerides and lipids to the target cells. Reconstitute apoB lipoparticle (rABL) will be an idea drug delivery vehicle for hydrophobic and amphiphilic materials delivery. It is challenged to renature ApoB into its functional state from denatured state. By using modified bile salt and radicicol (Rad) added over-critical refolding process, apoB can be restored into its native like state. The intrinsic fluorescence of apoB increased during the refolding process. Moreover, radicicol (Rad) molecules have been encapsulated into reconstitute rABL (Rad@rABL). To investigate the cell uptake mechanism of Rad@rABL, a super resolution ground state depletion (GSD) microscopy is used in this research. Fluorescence labeled Rad@rABL can be traced within the tumor cell. Key words: LDL, radicicol, protein refolding, super resolution microscopy.

  14. Massively parallel data processing for quantitative total flow imaging with optical coherence microscopy and tomography

    Science.gov (United States)

    Sylwestrzak, Marcin; Szlag, Daniel; Marchand, Paul J.; Kumar, Ashwin S.; Lasser, Theo

    2017-08-01

    We present an application of massively parallel processing of quantitative flow measurements data acquired using spectral optical coherence microscopy (SOCM). The need for massive signal processing of these particular datasets has been a major hurdle for many applications based on SOCM. In view of this difficulty, we implemented and adapted quantitative total flow estimation algorithms on graphics processing units (GPU) and achieved a 150 fold reduction in processing time when compared to a former CPU implementation. As SOCM constitutes the microscopy counterpart to spectral optical coherence tomography (SOCT), the developed processing procedure can be applied to both imaging modalities. We present the developed DLL library integrated in MATLAB (with an example) and have included the source code for adaptations and future improvements. Catalogue identifier: AFBT_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AFBT_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPLv3 No. of lines in distributed program, including test data, etc.: 913552 No. of bytes in distributed program, including test data, etc.: 270876249 Distribution format: tar.gz Programming language: CUDA/C, MATLAB. Computer: Intel x64 CPU, GPU supporting CUDA technology. Operating system: 64-bit Windows 7 Professional. Has the code been vectorized or parallelized?: Yes, CPU code has been vectorized in MATLAB, CUDA code has been parallelized. RAM: Dependent on users parameters, typically between several gigabytes and several tens of gigabytes Classification: 6.5, 18. Nature of problem: Speed up of data processing in optical coherence microscopy Solution method: Utilization of GPU for massively parallel data processing Additional comments: Compiled DLL library with source code and documentation, example of utilization (MATLAB script with raw data) Running time: 1,8 s for one B-scan (150 × faster in comparison to the CPU

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

    Science.gov (United States)

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

    2014-08-01

    Primary biological aerosol particles (PBAPs) can contribute significantly to the coarse particle burden in many environments. PBAPs can thus influence climate and precipitation systems as cloud nuclei and can spread disease to humans, animals, and plants. Measurement data and techniques for PBAPs in natural environments at high time- and size resolution are, however, sparse, and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in southwestern Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of a waveband integrated bioaerosol sensor (WIBS-4) with a ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behavior, with increased fluorescent bioparticle concentrations at night, when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each was correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multimodal distributions turning into a broad featureless single mode after averaging, and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent

  16. Tools for the quantitative analysis of sedimentation boundaries detected by fluorescence optical analytical ultracentrifugation.

    Directory of Open Access Journals (Sweden)

    Huaying Zhao

    Full Text Available Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.

  17. Performance of LED-based fluorescence microscopy to diagnose tuberculosis in a peripheral health centre in Nairobi.

    Directory of Open Access Journals (Sweden)

    Maryline Bonnet

    Full Text Available BACKGROUND: Sputum microscopy is the only tuberculosis (TB diagnostic available at peripheral levels of care in resource limited countries. Its sensitivity is low, particularly in high HIV prevalence settings. Fluorescence microscopy (FM can improve performance of microscopy and with the new light emitting diode (LED technologies could be appropriate for peripheral settings. The study aimed to compare the performance of LED-FM versus Ziehl-Neelsen (ZN microscopy and to assess feasibility of LED-FM at a low level of care in a high HIV prevalence country. METHODS: A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from suspected TB patients. Each sample was processed with Auramine O and ZN methods and a 4(th specimen was collected for TB culture reference standard. Auramine smears were read using the same microscope, equipped with the FluoLED™ fluorescence illuminator. Inter-reader agreement, reading time and technicians' acceptability assessed feasibility. RESULTS: 497 patients were included and 1394 specimens were collected. The detection yields of LED-FM and ZN microscopy were 20.3% and 20.6% (p = 0.64, respectively. Sensitivity was 73.2% for LED-FM and 72% for ZN microscopy, p = 0.32. It was 96.7% and 95.9% for specificity, p = 0.53. Inter-reader agreement was high (kappa = 0.9. Mean reading time was three times faster than ZN microscopy with very good acceptance by technicians. CONCLUSIONS: Although it did not increase sensitivity, the faster reading time combined with very good acceptance and ease of use supports the introduction of LED-FM at the peripheral laboratory level of high TB and HIV burden countries.

  18. Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

    Science.gov (United States)

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

    2017-03-01

    In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of 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.

  19. Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

    Science.gov (United States)

    Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

    2009-01-01

    Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

  20. Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

    International Nuclear Information System (INIS)

    Wang Lei; Xu Guang; Shi Zhikun; Jiang Wei; Jin Wenrui

    2007-01-01

    We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10 -11 to 8.1 x 10 -10 mol L -1

  1. LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular analysis of Her-2/neu oncogene amplification

    Directory of Open Access Journals (Sweden)

    Vollmer Ekkehard

    2008-12-01

    Full Text Available Abstract Light emitting diodes (LED, which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2 gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own

  2. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    Science.gov (United States)

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images

    DEFF Research Database (Denmark)

    Hidayat, Budi J.; Weisskopf, Carmen; Felby, Claus

    2015-01-01

    or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method...

  4. Quantitative cellular uptake of double fluorescent core-shelled model submicronic particles

    Energy Technology Data Exchange (ETDEWEB)

    Leclerc, Lara, E-mail: leclerc@emse.fr [Ecole Nationale Superieure des Mines, CIS-EMSE, LINA (France); Boudard, Delphine [LINA (France); Pourchez, Jeremie; Forest, Valerie [Ecole Nationale Superieure des Mines, CIS-EMSE, LINA (France); Marmuse, Laurence; Louis, Cedric [NANO-H S.A.S (France); Bin, Valerie [LINA (France); Palle, Sabine [Universite Jean Monnet, Centre de Microscopie Confocale Multiphotonique (France); Grosseau, Philippe; Bernache-Assollant, Didier [Ecole Nationale Superieure des Mines, CIS-EMSE, LINA (France); Cottier, Michele [LINA (France)

    2012-11-15

    The relationship between particles' physicochemical parameters, their uptake by cells and their degree of biological toxicity represent a crucial issue, especially for the development of new technologies such as fabrication of micro- and nanoparticles in the promising field of drug delivery systems. This work was aimed at developing a proof-of-concept for a novel model of double fluorescence submicronic particles that could be spotted inside phagolysosomes. Fluorescein isothiocyanate (FITC) particles were synthesized and then conjugated with a fluorescent pHrodo Trade-Mark-Sign probe, red fluorescence of which increases in acidic conditions such as within lysosomes. After validation in acellular conditions by spectral analysis with confocal microscopy and dynamic light scattering, quantification of phagocytosis was conducted on a macrophage cell line in vitro. The biological impact of pHrodo functionalization (cytotoxicity, inflammatory response, and oxidative stress) was also investigated. Results validate the proof-of-concept of double fluorescent particles (FITC + pHrodo), allowing detection of entirely engulfed pHrodo particles (green and red labeling). Moreover incorporation of pHrodo had no major effects on cytotoxicity compared to particles without pHrodo, making them a powerful tool for micro- and nanotechnologies.

  5. Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

    Science.gov (United States)

    Cerussi, Albert Edward

    1999-09-01

    In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of

  6. MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

    Science.gov (United States)

    Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

    2015-01-01

    We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

  7. Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

    Directory of Open Access Journals (Sweden)

    Francis Kevin P

    2011-08-01

    Full Text Available Abstract Background Transient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP-based imaging assay to quantitatively assess suppressor protein activity. Results In a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP P0 or Plum pox virus (PPV HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi and P0 giving higher long term GFP fluorescence (at 21 dpi. Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas. Conclusions Fluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

  8. Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

    Science.gov (United States)

    Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

    2016-08-01

    Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

  9. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

    Directory of Open Access Journals (Sweden)

    Yaser Afshar

    Full Text Available Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10 pixels, but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

  10. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

    Science.gov (United States)

    Afshar, Yaser; Sbalzarini, Ivo F

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

  11. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

    Science.gov (United States)

    Afshar, Yaser; Sbalzarini, Ivo F.

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

  12. Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy

    Science.gov (United States)

    Fallet, Clément; Caron, Julien; Oddos, Stephane; Tinevez, Jean-Yves; Moisan, Lionel; Sirat, Gabriel Y.; Braitbart, Philippe O.; Shorte, Spencer L.

    2014-08-01

    We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon taking place when a polarized beam is diffracted through a biaxial crystal. The illumination patterns generated by conical diffraction are more compact than the classical Gaussian beam; we use them to generate a super-resolution imaging modality. Conical Diffraction Microscopy (CODIM) resolution enhancement can be achieved with any type of objective on any kind of sample preparation and standard fluorophores. Conical diffraction can be used in multiple fashion to create new and disruptive technologies for super-resolution microscopy. This paper will focus on the first one that has been implemented and give a glimpse at what the future of microscopy using conical diffraction could be.

  13. Current limitations in super-resolution fluorescence microscopy for biological specimens: How deep can we go from the cover glass?

    Science.gov (United States)

    Okada, Yasushi

    2017-04-01

    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.

  14. Quantitative X ray analysis system. User's manual and guide to X ray fluorescence technique

    International Nuclear Information System (INIS)

    2009-01-01

    This guide covers trimmed and re-arranged version 3.6 of the Quantitative X ray Analysis System (QXAS) software package that includes the most frequently used methods of quantitative analysis. QXAS is a comprehensive quantitative analysis package that has been developed by the IAEA through research and technical contracts. Additional development has also been carried out in the IAEA Laboratories in Seibersdorf where QXAS was extensively tested. New in this version of the manual are the descriptions of the Voigt-profile peak fitting, the backscatter fundamental parameters' and emission-transmission methods of chemical composition analysis, an expanded chapter on the X ray fluorescence physics, and completely revised and increased number of practical examples of utilization of the QXAS software package. The analytical data accompanying this manual were collected in the IAEA Seibersdorf Laboratories in the years 2006/2007

  15. An FFT-based Method for Attenuation Correction in Fluorescence Confocal Microscopy

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.; Bakker, M.

    1993-01-01

    A problem in three-dimensional imaging by a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. In this paper we propose a new method to correct for these

  16. First identification of the herpes simplex virus by skin-dedicated ex vivo fluorescence confocal microscopy during herpetic skin infections.

    Science.gov (United States)

    Cinotti, E; Perrot, J L; Labeille, B; Campolmi, N; Thuret, G; Naigeon, N; Bourlet, T; Pillet, S; Cambazard, F

    2015-06-01

    Skin-dedicated ex vivo fluorescence confocal microscopy (FCM) has so far been used to identify cutaneous tumours on freshly excised samples using acridine orange as fluorochrome. To use FCM for a new indication, namely, the identification of the herpes simplex virus (HSV) in skin lesions, using fluorescent antibodies. Six roof samples from skin vesicles suspicious for HSV lesions were incubated with anti-HSV-1 and anti-HSV-2 antibodies coupled with fluorescein isothiocyanate, and examined under skin-dedicated ex vivo FCM. The positive controls were swabs taken from the floor of each vesicle and observed under conventional direct fluorescence assay (DFA) and by viral cultures. Roof samples from three bullae of bullous pemphigoid were the negative controls. Using ex vivo FCM, the samples from the lesions clinically suspicious for HSV infection were seen to be fluorescent after incubation with anti-HSV-1, and were negative after incubation with anti-HSV-2 antibodies. Conventional DFA with an optical microscope and cultures confirmed the presence of HSV-1 infection. By using fluorescent antibodies to identify precise structures, ex vivo FCM can be used for indications other than tumour identification. More specifically, it can be an additional diagnostic tool for HSV infection. © 2014 British Association of Dermatologists.

  17. Use of quantitative light-induced fluorescence to monitor tooth whitening

    Science.gov (United States)

    Amaechi, Bennett T.; Higham, Susan M.

    2001-04-01

    The changing of tooth shade by whitening agents occurs gradually. Apart from being subjective and affected by the conditions of the surroundings, visual observation cannot detect a very slight change in tooth color. An electronic method, which can communicate the color change quantitatively, would be more reliable. Quantitative Light- induced Fluorescence (QLF) was developed to detect and assess dental caries based on the phenomenon of change of autofluorescence of a tooth by demineralization. However, stains on the tooth surface exhibit the same phenomenon, and therefore QLF can be used to measure the percentage fluorescence change of stained enamel with respect to surrounding unstained enamel. The present study described a technique of assessing the effect of a tooth-whitening agent using QLF. This was demonstrated in two experiments in which either wholly or partially stained teeth were whitened by intermittent immersion in sodium hypochlorite. Following each immersion, the integrated fluorescence change due to the stain was quantified using QLF. In either situation, the value of (Delta) Q decreased linearly as the tooth regained its natural shade. It was concluded that gradual changing of the shade of discolored teeth by a whitening agent could be quantified using QLF.

  18. Quantitative electron microscopy and spectroscopy of MgB2 wires and tapes

    International Nuclear Information System (INIS)

    Birajdar, B; Peranio, N; Eibl, O

    2008-01-01

    In MgB 2 the correlation of microstructure with superconducting properties, in particular the critical current density, requires powerful analytical tools. Critical current densities and electrical resistivities of different MgB 2 superconductors differ by orders of magnitudes and the current limiting mechanisms have not been fully understood. Granularity of MgB 2 is one significant reason for reduced critical current densities and is introduced intrinsically by the anisotropy of B c2 but also extrinsically by the microstructure of the material. B c2 enhancement by doping is another important challenge for chemical analysis and, at present, doping levels are not well controlled on the sub-μm scale. In this paper the quantitative electron microscopy and spectroscopy methods essential for the microstructural analysis of MgB 2 are described. By quantitative electron microscopy and spectroscopy we mean a combined SEM and TEM analysis that covers various length scales from μm to nm. Contamination-free sample preparation, chemical mapping including B, and advanced chemical quantification using x-ray microanalysis were essential elements of the applied methodology. The methodology was applied to in situ and ex situ MgB 2 wires and tapes with and without SiC additives. Quantitative B analysis by EDX spectroscopy was applied quantitatively in the SEM and TEM, which is a major achievement. Although MgB 2 is a binary system, the thermodynamics of phase formation is complex, and the complexity is dramatically increased if additives like SiC are used. The small, sub-μm grain sizes of the matrix and secondary phases require TEM methods. However, granularity on the μm scale was also identified and underlines the importance of the combined SEM and TEM studies. Significant differences in the microstructure were observed for in situ and ex situ samples. This holds particularly if SiC was added and yielded Mg 2 Si for in situ samples annealed at 600-650 deg. C and Mg-Si-O phases

  19. Quantitative characterization of crosstalk effects for friction force microscopy with scan-by-probe SPMs

    Energy Technology Data Exchange (ETDEWEB)

    Prunici, Pavel [Institute of Physical Chemistry, University of Heidelberg, D-69120 Heidelberg (Germany); Hess, Peter [Institute of Physical Chemistry, University of Heidelberg, D-69120 Heidelberg (Germany)], E-mail: peter.hess@urz.uni-heidelberg.de

    2008-06-15

    If the photodetector and cantilever of an atomic force microscope (AFM) are not properly adjusted, crosstalk effects will appear. These effects disturb measurements of the absolute vertical and horizontal cantilever deflections, which are involved in friction force microscopy (FFM). A straightforward procedure is proposed to study quantitatively crosstalk effects observed in scan-by-probe SPMs. The advantage of this simple, fast, and accurate procedure is that no hardware change or upgrade is needed. The results indicate that crosstalk effects depend not only on the alignment of the detector but also on the cantilever properties, position, and detection conditions. The measurements may provide information on the origin of the crosstalk effect. After determination of its magnitude, simple correction formulas can be applied to correct the crosstalk effects and then the single-load wedge method, using a commercially available grating, can be employed for accurate calibration of the lateral force.

  20. Quantitative characterization of crosstalk effects for friction force microscopy with scan-by-probe SPMs

    International Nuclear Information System (INIS)

    Prunici, Pavel; Hess, Peter

    2008-01-01

    If the photodetector and cantilever of an atomic force microscope (AFM) are not properly adjusted, crosstalk effects will appear. These effects disturb measurements of the absolute vertical and horizontal cantilever deflections, which are involved in friction force microscopy (FFM). A straightforward procedure is proposed to study quantitatively crosstalk effects observed in scan-by-probe SPMs. The advantage of this simple, fast, and accurate procedure is that no hardware change or upgrade is needed. The results indicate that crosstalk effects depend not only on the alignment of the detector but also on the cantilever properties, position, and detection conditions. The measurements may provide information on the origin of the crosstalk effect. After determination of its magnitude, simple correction formulas can be applied to correct the crosstalk effects and then the single-load wedge method, using a commercially available grating, can be employed for accurate calibration of the lateral force

  1. Investigation of shape memory of red blood cells using optical tweezers and quantitative phase microscopy

    Science.gov (United States)

    Cardenas, Nelson; Mohanty, Samarendra K.

    2012-03-01

    RBC has been shown to possess shape memory subsequent to shear-induced shape transformation. However, this property of RBC may not be generalized to all kinds of stresses. Here, we report our observation on the action of radiation pressure forces on RBC's shape memory using optical manipulation and quantitative phase microscopy (OMQPM). QPM, based on Mach-Zehnder interferrometry, allowed measurement of dynamic changes of shape of RBC in optical tweezers at different trapping laser powers. In high power near-infrared optical tweezers (>200mW), the RBC was found to deform significantly due to optical forces. Upon removal of the tweezers, hysteresis in recovering its original resting shape was observed. In very high power tweezers or long-term stretching events, shape memory was almost erased. This irreversibility of the deformation may be due to temperature rise or stress-induced phase transformation of lipids in RBC membrane.

  2. Quantitative measurement of solvation shells using frequency modulated atomic force microscopy

    Science.gov (United States)

    Uchihashi, T.; Higgins, M.; Nakayama, Y.; Sader, J. E.; Jarvis, S. P.

    2005-03-01

    The nanoscale specificity of interaction measurements and additional imaging capability of the atomic force microscope make it an ideal technique for measuring solvation shells in a variety of liquids next to a range of materials. Unfortunately, the widespread use of atomic force microscopy for the measurement of solvation shells has been limited by uncertainties over the dimensions, composition and durability of the tip during the measurements, and problems associated with quantitative force calibration of the most sensitive dynamic measurement techniques. We address both these issues by the combined use of carbon nanotube high aspect ratio probes and quantifying the highly sensitive frequency modulation (FM) detection technique using a recently developed analytical method. Due to the excellent reproducibility of the measurement technique, additional information regarding solvation shell size as a function of proximity to the surface has been obtained for two very different liquids. Further, it has been possible to identify differences between chemical and geometrical effects in the chosen systems.

  3. Quantitative nitric oxide measurements by means of laser-induced fluorescence in a heavy-duty Diesel engine

    NARCIS (Netherlands)

    Verbiezen, K.; Vliet, van A.P.; Klein-Douwel, R.J.H.; Ganippa, L.C.; Bougie, H.J.T.; Meerts, W.L.; Dam, N.J.; Meulen, ter J.J.

    2005-01-01

    Quantitative in-cylinder laser-induced fluorescence measurements ofnitric oxide in a heavy-duty Diesel engine are presented. Special attention is paid to experimental techniques to assess the attenuation of the laser beam and the fluorescence signal by the cylinder contents.This attenuation can be

  4. Quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope.

    Science.gov (United States)

    Phillips, Kevin G; Baker-Groberg, Sandra M; McCarty, Owen J T

    2014-04-07

    We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using "off-the-shelf" microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with

  5. Quantitative comparison of X-ray fluorescence microtomography setups: Standard and confocal collimator apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Chukalina, M. [Institute of Microelectronics Technology RAS, 142432, Chernogolovka, Moscow District (Russian Federation)], E-mail: marina@ipmt-hpm.ac.ru; Simionovici, A. [Laboratoire de Geophysique Interne et Tectonophysique, University of Grenoble, BP 53, 38041, Grenoble (France)], E-mail: alexandre.simionovici@ujf-grenoble.fr; Zaitsev, S. [Institute of Microelectronics Technology RAS, 142432, Chernogolovka, Moscow District (Russian Federation)], E-mail: zaitsev@ipmt-hpm.ac.ru; Vanegas, C.J. [Institute of Microelectronics Technology RAS, 142432, Chernogolovka, Moscow District (Russian Federation)], E-mail: vanegas@ipmt-hpm.ac.ru

    2007-07-15

    Recently, there has been a renewed interest for fluorescence spectroscopy, as provided by modern setups which allow 2D and 3D imaging of elemental distributions. Two directions are currently under development: the SR-based fluorescence tomography in polar scanning geometry, provided by the new generation of X-ray microprobes and the confocal scanning geometry, which can be fielded in both SR and laboratory environments. The new probes bring forth a new age in fluorescence spectrometry: high resolution, high intensity and high sensitivity which allow 3D elemental mapping of volumes. The major task now is the development of these complex tools into fully quantitative probes, reproducible and straightforward for general use. In this work we analyze two X-ray fluorescence microtomography techniques: an apparatus tomography using a confocal collimator for the data collection and a standard first generation Computed Tomography (CT) in the parallel scanning scheme. We calculate the deposited dose (amount of energy deposited and distributed in the sample during the data collection time) and find the conditions for the choice of the tomography scheme.

  6. Quantitative analysis of essential oils of Thymus daenensis using laser-induced fluorescence and Raman spectroscopy.

    Science.gov (United States)

    Khoshroo, H; Khadem, H; Bahreini, M; Tavassoli, S H; Hadian, J

    2015-11-10

    Laser-induced fluorescence and Raman spectroscopy are used for the investigation of different genotypes of Thymus daenensis native to the Ilam province of Iran. Different genotypes of T. daenensis essential oils, labeled T1 through T7, possess slight differences with regard to the composition of the thymol. The gas chromatography-mass spectrometry (GC-MS) method is performed to determine the concentration of each constituent as a reference method. The Raman spectra of different concentrations of pure thymol dissolved in hexane as standard samples are obtained via a laboratory prototype Raman spectroscopy setup for the calculation of the calibration curve. The regression coefficient and limit of detection are calculated. The possibility of the differentiation of different genotypes of T. daenensis is also examined by laser-induced fluorescence spectroscopy, although we do not know the exact amounts of their components. All the fluorescence spectral information is used jointly by cluster analysis to differentiate between 7 genotypes. Our results demonstrate the acceptable precision of Raman spectroscopy with GC-MS and corroborate the capacity of Raman spectroscopy in applications in the quantitative analysis field. Furthermore, the cluster analysis results show that laser-induced fluorescence spectroscopy is an acceptable technique for the rapid classification of different genotypes of T. daenensis without having any previous information of their exact amount of constituents. So, the ability to rapidly and nondestructively differentiate between genotypes makes it possible to efficiently select high-quality herbs from many samples.

  7. Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography.

    Science.gov (United States)

    Maia, Ana Marly Araújo; de Freitas, Anderson Zanardi; de L Campello, Sergio; Gomes, Anderson Stevens Leônidas; Karlsson, Lena

    2016-06-01

    An in vitro study of morphological alterations between sound dental structure and artificially induced w