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Sample records for super-resolution fluorescence microscopy

  1. 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.

  2. 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

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

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    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.

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

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    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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

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    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

  11. 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.

  12. 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.

  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. The 2015 super-resolution microscopy roadmap

    International Nuclear Information System (INIS)

    Hell, Stefan W; Sahl, Steffen J; Bates, Mark; Jakobs, Stefan; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J; Eggeling, Christian; Klenerman, David; Willig, Katrin I

    2015-01-01

    Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough

  15. Developing a New Biophysical Tool to Combine Magneto-Optical Tweezers with Super-Resolution Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Zhaokun Zhou

    2015-06-01

    Full Text Available We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which, in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM, achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing a system which combines tweezing and super-resolution fluorescence imaging. The instrument also features an acousto-optic deflector that temporally divides the laser beam to form multiple traps for high throughput statistics collection. Our motivation for developing the new tool is to enable direct observation of detailed molecular topological transformation and protein binding event localisation in a stretching/twisting mechanical assay that previously could hitherto only be deduced indirectly from the end-to-end length variation of DNA. Our approach is simple and robust enough for reproduction in the lab without the requirement of precise hardware engineering, yet is capable of unveiling the elastic and dynamic properties of filamentous molecules that have been hidden using traditional tools.

  16. 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)

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

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    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.

  18. 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.

  19. Application of super-resolution optical microscopy in biology

    International Nuclear Information System (INIS)

    Mao Xiuhai; Du Jiancong; Huang Qing; Fan Chunhai; Deng Suhui

    2013-01-01

    Background: A noninvasive, real-time far-field optical microscopy is needed to study the dynamic function inside cells and proteins. However, the resolution limit of traditional optical microscope is about 200 nm due to the diffraction limit of light. So, it's hard to directly observe the subcellular structures. Over the past several years of microscopy development, the diffraction limit of fluorescence microscopy has been overcome and its resolution limit is about tens of nanometers. Methods: To overcome the diffraction limit of light, many super-resolution fluoresce microscopes, including stimulated emission of depletion microscopy (STED), photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), have been developed. Conclusions: These methods have been applied in cell biology, microbiology and neurobiology, and the technology of super-resolution provides a new insight into the life science. (authors)

  20. Aberrations and adaptive optics in super-resolution microscopy

    Science.gov (United States)

    Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

    2015-01-01

    As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

  1. Shedding light on endocytosis with optimized super-resolution microscopy

    NARCIS (Netherlands)

    Leyton Puig, D.M.

    2017-01-01

    Super-resolution microscopy is a relatively new microscopy technique that is still under optimization. In this thesis we focus on the improvement of the quality of super-resolution images, to apply them to the study of the processes of cell signaling and endocytosis. First, we show that the use of a

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

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    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.

  3. Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

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    Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

    2017-06-14

    Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

  4. Super-resolution Microscopy in Plant Cell Imaging.

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    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Application of spectroscopy and super-resolution microscopy: Excited state

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Ujjal [Iowa State Univ., Ames, IA (United States)

    2016-02-19

    Photophysics of inorganic materials and organic molecules in complex systems have been extensively studied with absorption and emission spectroscopy.1-4 Steady-state and time-resolved fluorescence studies are commonly carried out to characterize excited-state properties of fluorophores. Although steady-state fluorescence measurements are widely used for analytical applications, time-resolved fluorescence measurements provide more detailed information about excited-state properties and the environment in the vicinity of the fluorophore. Many photophysical processes, such as photoinduced electron transfer (PET), rotational reorientation, solvent relaxation, and energy transfer, occur on a nanosecond (10-9 s) timescale, thus affecting the lifetime of the fluorophores. Moreover, time-resolved microscopy methods, such as lifetimeimaging, combine the benefits of the microscopic measurement and information-rich, timeresolved data. Thus, time-resolved fluorescence spectroscopy combined with microscopy can be used to quantify these processes and to obtain a deeper understanding of the chemical surroundings of the fluorophore in a small area under investigation. This thesis discusses various photophysical and super-resolution microscopic studies of organic and inorganic materials, which have been outlined below.

  6. Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

    Science.gov (United States)

    Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

    2015-06-11

    The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

  7. Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

    Science.gov (United States)

    Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

    2016-01-01

    Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

  8. Super-resolution fluorescence imaging of membrane nanoscale architectures of hematopoietic stem cell homing and migration molecules

    KAUST Repository

    AbuZineh, Karmen

    2017-01-01

    Recent development of super-resolution (SR) fluorescence microscopy techniques has provided a new tool for direct visualization of subcellular structures and their dynamics in cells. The homing of Hematopoietic stem/progenitor cells (HSPCs) to bone

  9. Microsphere-aided optical microscopy and its applications for super-resolution imaging

    Science.gov (United States)

    Upputuri, Paul Kumar; Pramanik, Manojit

    2017-12-01

    The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

  10. An introduction to optical super-resolution microscopy for the adventurous biologist

    Science.gov (United States)

    Vangindertael, J.; Camacho, R.; Sempels, W.; Mizuno, H.; Dedecker, P.; Janssen, K. P. F.

    2018-04-01

    Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist’s toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

  11. Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

    Science.gov (United States)

    Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

    2011-12-01

    Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

  12. 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.

  13. Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

    Science.gov (United States)

    Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

    2018-03-01

    Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

  14. Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI)

    Science.gov (United States)

    Hainsworth, A. H.; Lee, S.; Patel, A.; Poon, W. W.; Knight, A. E.

    2018-01-01

    Aims The spatial resolution of light microscopy is limited by the wavelength of visible light (the ‘diffraction limit’, approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Methods Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8–32 nm) and for SOFI (effective pixel size 80 nm). Results In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Conclusions Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. PMID:28696566

  15. Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI).

    Science.gov (United States)

    Hainsworth, A H; Lee, S; Foot, P; Patel, A; Poon, W W; Knight, A E

    2017-07-11

    The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. © 2017 British Neuropathological Society.

  16. DMD-based LED-illumination super-resolution and optical sectioning microscopy.

    Science.gov (United States)

    Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

    2013-01-01

    Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×10(7) pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

  17. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

    Science.gov (United States)

    Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  18. Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

    Science.gov (United States)

    Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

    2016-06-06

    Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

  19. Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy.

    Science.gov (United States)

    Grimm, Jonathan B; Klein, Teresa; Kopek, Benjamin G; Shtengel, Gleb; Hess, Harald F; Sauer, Markus; Lavis, Luke D

    2016-01-26

    The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  20. Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

    OpenAIRE

    Traenkle, Bjoern; Rothbauer, Ulrich

    2017-01-01

    Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies)...

  1. Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy

    Science.gov (United States)

    German, Christopher L.; Gudheti, Manasa V.; Fleckenstein, Annette E.; Jorgensen, Erik M.

    2018-01-01

    Localization microscopy techniques – such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM) – provide the highest precision for single molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single molecule localizations of synaptic vesicle and active zone proteins in three-dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging. PMID:28924666

  2. Managing the Introduction of Super-Resolution Microscopy into a Core Facility.

    Science.gov (United States)

    Kamykowski, Jeffrey A; Storrie, Brian

    2017-01-01

    Super resolution techniques place the resolution of fluorescence microscopy closer to the size of the underlying cell structure or molecular machine being studied. Structured illumination techniques will give users a set of tools that are close to their past experience and relatively simple and quick to learn. The present dyes can be used. Resolution approaching 100 nm XY can be achieved. In contrast, stochastic methods such as PALM/STORM typically require the choice of new dyes and a much greater learning curve to master the technology and calculations. However, a further fivefold resolution improvement is possible. Stimulated depletion techniques such as STED offer a third set of approaches that will again require the use of new dyes. All these approaches require substantial investment in new equipment and in user training. There is no free lunch in the search for better resolution.

  3. 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.

    Directory of Open Access Journals (Sweden)

    David Baddeley

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

  4. Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

    Directory of Open Access Journals (Sweden)

    Isuru D. Jayasinghe

    2014-12-01

    Full Text Available The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM, has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

  5. A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

    Science.gov (United States)

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

  6. Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

    Science.gov (United States)

    Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

    2017-06-14

    Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

  7. Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

    Directory of Open Access Journals (Sweden)

    Bjoern Traenkle

    2017-08-01

    Full Text Available Single-domain antibodies (sdAbs have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

  8. Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

    Science.gov (United States)

    Traenkle, Bjoern; Rothbauer, Ulrich

    2017-01-01

    Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

  9. Super-resolution microscopy in studying neuroendocrine cell function

    Directory of Open Access Journals (Sweden)

    Anneka eBost

    2013-11-01

    Full Text Available The last two decades have seen a tremendous development in high resolution microscopy techniques giving rise to acronyms such as TIRFM, SIM, PALM, STORM, and STED. The goal of all these techniques is to overcome the physical resolution barrier of light microscopy in order to resolve precise protein localization and possibly their interaction in cells. Neuroendocrine cell function is to secrete hormones and peptides on demand. This fine-tuned multi-step process is mediated by a large array of proteins. Here, we review the new microscopy techniques used to obtain high resolution and how they have been applied to increase our knowledge of the molecular mechanisms involved in neuroendocrine cell secretion. Further the limitations of these methods are discussed and insights in possible new applications are provided.

  10. Super-resolution optical microscopy resolves network morphology of smart colloidal microgels.

    Science.gov (United States)

    Bergmann, Stephan; Wrede, Oliver; Huser, Thomas; Hellweg, Thomas

    2018-02-14

    We present a new method to resolve the network morphology of colloidal particles in an aqueous environment via super-resolution microscopy. By localization of freely diffusing fluorophores inside the particle network we can resolve the three dimensional structure of one species of colloidal particles (thermoresponsive microgels) without altering their chemical composition through copolymerization with fluorescent monomers. Our approach utilizes the interaction of the fluorescent dye rhodamine 6G with the polymer network to achieve an indirect labeling. We calculate the 3D structure from the 2D images and compare the structure to previously published models for the microgel morphology, e.g. the fuzzy sphere model. To describe the differences in the data an extension of this model is suggested. Our method enables the tailor-made fabrication of colloidal particles which are used in various applications, such as paints or cosmetics, and are promising candidates for drug delivery, smart surface coatings, and nanocatalysis. With the precise knowledge of the particle morphology an understanding of the underlying structure-property relationships for various colloidal systems is possible.

  11. Painting Supramolecular Polymers in Organic Solvents by Super-resolution Microscopy

    Science.gov (United States)

    2018-01-01

    Despite the rapid development of complex functional supramolecular systems, visualization of these architectures under native conditions at high resolution has remained a challenging endeavor. Super-resolution microscopy was recently proposed as an effective tool to unveil one-dimensional nanoscale structures in aqueous media upon chemical functionalization with suitable fluorescent probes. Building upon our previous work, which enabled photoactivation localization microscopy in organic solvents, herein, we present the imaging of one-dimensional supramolecular polymers in their native environment by interface point accumulation for imaging in nanoscale topography (iPAINT). The noncovalent staining, typical of iPAINT, allows the investigation of supramolecular polymers’ structure in situ without any chemical modification. The quasi-permanent adsorption of the dye to the polymer is exploited to identify block-like arrangements within supramolecular fibers, which were obtained upon mixing homopolymers that were prestained with different colors. The staining of the blocks, maintained by the lack of exchange of the dyes, permits the imaging of complex structures for multiple days. This study showcases the potential of PAINT-like strategies such as iPAINT to visualize multicomponent dynamic systems in their native environment with an easy, synthesis-free approach and high spatial resolution. PMID:29697958

  12. The tetraspanin web revisited by super-resolution microscopy.

    Science.gov (United States)

    Zuidscherwoude, Malou; Göttfert, Fabian; Dunlock, Vera Marie E; Figdor, Carl G; van den Bogaart, Geert; van Spriel, Annemiek B

    2015-07-17

    The spatial organization of membrane proteins in the plasma membrane is critical for signal transduction, cell communication and membrane trafficking. Tetraspanins organize functional higher-order protein complexes called 'tetraspanin-enriched microdomains (TEMs)' via interactions with partner molecules and other tetraspanins. Still, the nanoscale organization of TEMs in native plasma membranes has not been resolved. Here, we elucidated the size, density and distribution of TEMs in the plasma membrane of human B cells and dendritic cells using dual color stimulated emission depletion (STED) microscopy. We demonstrate that tetraspanins form individual nanoclusters smaller than 120 nm and quantified that a single tetraspanin CD53 cluster contains less than ten CD53 molecules. CD53 and CD37 domains were adjacent to and displayed only minor overlap with clusters containing tetraspanins CD81 or CD82. Moreover, CD53 and CD81 were found in closer proximity to their partners MHC class II and CD19 than to other tetraspanins. Although these results indicate that tetraspanin domains are adjacently positioned in the plasma membrane, they challenge the current view of the tetraspanin web of multiple tetraspanin species organized into a single domain. This study increases the molecular understanding of TEMs at the nanoscale level which is essential for comprehending tetraspanin function in cell biology.

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

    KAUST Repository

    Yabiku, Y.; Kubo, S.; Nakagawa, M.; Vacha, M.; Habuchi, Satoshi

    2013-01-01

    We applied a super-resolution fluorescence imaging based on selective adsorption and redox switching of the fluorescent dye molecules for studying polymer nanostructures. We demonstrate that nano-scale structures of polymer thin films can

  14. Super-resolution optical microscopy for studying membrane structure and dynamics.

    Science.gov (United States)

    Sezgin, Erdinc

    2017-07-12

    Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

  15. 3D single-molecule super-resolution microscopy with a tilted light sheet.

    Science.gov (United States)

    Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

    2018-01-09

    Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

  16. Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

    Science.gov (United States)

    Yan, Rui; Moon, Seonah; Kenny, Samuel J; Xu, Ke

    2018-03-20

    As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with

  17. Super-Resolution Localization Microscopy of γ-H2AX and Heterochromatin after Folate Deficiency.

    Science.gov (United States)

    Bach, Margund; Savini, Claudia; Krufczik, Matthias; Cremer, Christoph; Rösl, Frank; Hausmann, Michael

    2017-08-08

    Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with "high-risk" human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access

  18. Super-resolution and super-localization microscopy: A novel tool for imaging chemical and biological processes

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Bin [Iowa State Univ., Ames, IA (United States)

    2015-01-01

    Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the single molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).

  19. Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation.

    Science.gov (United States)

    Weatherly, Lisa M; Nelson, Andrew J; Shim, Juyoung; Riitano, Abigail M; Gerson, Erik D; Hart, Andrew J; de Juan-Sanz, Jaime; Ryan, Timothy A; Sher, Roger; Hess, Samuel T; Gosse, Julie A

    2018-06-15

    The antimicrobial agent triclosan (TCS) is used in products such as toothpaste and surgical soaps and is readily absorbed into oral mucosa and human skin. These and many other tissues contain mast cells, which are involved in numerous physiologies and diseases. Mast cells release chemical mediators through a process termed degranulation, which is inhibited by TCS. Investigation into the underlying mechanisms led to the finding that TCS is a mitochondrial uncoupler at non-cytotoxic, low-micromolar doses in several cell types and live zebrafish. Our aim was to determine the mechanisms underlying TCS disruption of mitochondrial function and of mast cell signaling. We combined super-resolution (fluorescence photoactivation localization) microscopy and multiple fluorescence-based assays to detail triclosan's effects in living mast cells, fibroblasts, and primary human keratinocytes. TCS disrupts mitochondrial nanostructure, causing mitochondria to undergo fission and to form a toroidal, "donut" shape. TCS increases reactive oxygen species production, decreases mitochondrial membrane potential, and disrupts ER and mitochondrial Ca 2+ levels, processes that cause mitochondrial fission. TCS is 60 × more potent than the banned uncoupler 2,4-dinitrophenol. TCS inhibits mast cell degranulation by decreasing mitochondrial membrane potential, disrupting microtubule polymerization, and inhibiting mitochondrial translocation, which reduces Ca 2+ influx into the cell. Our findings provide mechanisms for both triclosan's inhibition of mast cell signaling and its universal disruption of mitochondria. These mechanisms provide partial explanations for triclosan's adverse effects on human reproduction, immunology, and development. This study is the first to utilize super-resolution microscopy in the field of toxicology. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders.

    Science.gov (United States)

    Westmoreland, D; Shaw, M; Grimes, W; Metcalf, D J; Burden, J J; Gomez, K; Knight, A E; Cutler, D F

    2016-04-01

    Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

  1. Measurement of replication structures at the nanometer scale using super-resolution light microscopy.

    Science.gov (United States)

    Baddeley, D; Chagin, V O; Schermelleh, L; Martin, S; Pombo, A; Carlton, P M; Gahl, A; Domaing, P; Birk, U; Leonhardt, H; Cremer, C; Cardoso, M C

    2010-01-01

    DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.

  2. Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

    Science.gov (United States)

    Lai, Ken; Wu, Xiaojuan; Yong, Jim L. C.; Lee, C. Soon

    2012-01-01

    Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy. PMID:22899862

  3. Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

    Science.gov (United States)

    Killingsworth, Murray C; Lai, Ken; Wu, Xiaojuan; Yong, Jim L C; Lee, C Soon

    2012-11-01

    Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

  4. Super-Resolution Microscopy Reveals the Native Ultrastructure of the Erythrocyte Cytoskeleton

    Directory of Open Access Journals (Sweden)

    Leiting Pan

    2018-01-01

    Full Text Available The erythrocyte cytoskeleton is a textbook prototype for the submembrane cytoskeleton of metazoan cells. While early experiments suggest a triangular network of actin-based junctional complexes connected by ∼200-nm-long spectrin tetramers, later studies indicate much smaller junction-to-junction distances in the range of 25-60 nm. Through super-resolution microscopy, we resolve the native ultrastructure of the cytoskeleton of membrane-preserved erythrocytes for the N and C termini of β-spectrin, F-actin, protein 4.1, tropomodulin, and adducin. This allows us to determine an ∼80-nm junction-to-junction distance, a length consistent with relaxed spectrin tetramers and theories based on spectrin abundance. Through two-color data, we further show that the cytoskeleton meshwork often contains nanoscale voids where the cell membrane remains intact and that actin filaments and capping proteins localize to a subset of, but not all, junctional complexes. Together, our results call for a reassessment of the structure and function of the submembrane cytoskeleton.

  5. Super-resolution fluorescence imaging of membrane nanoscale architectures of hematopoietic stem cell homing and migration molecules

    KAUST Repository

    AbuZineh, Karmen

    2017-12-01

    Recent development of super-resolution (SR) fluorescence microscopy techniques has provided a new tool for direct visualization of subcellular structures and their dynamics in cells. The homing of Hematopoietic stem/progenitor cells (HSPCs) to bone marrow is a multistep process that is initiated by tethering of HSPCs to endothelium and mediated by spatiotemporally organised ligand-receptor interactions of selectins expressed on endothelial cells to their ligands expressed on HSPCs which occurs against the shear stress exerted by blood flow. Although molecules and biological processes involved in this multi-step cellular interaction have been studied extensively, molecular mechanisms of the homing, in particular the nanoscale spatiotemporal behaviour of ligand-receptor interactions and their role in the cellular interaction, remain elusive. Using our new method of microfluidics-based super-resolution fluorescence imaging platform we can now characterize the correlation between both nanoscale ligand-receptor interactions and tethering/rolling of cells under external shear stress. We found that cell rolling on E-selectin caused significant reorganization of the nanoscale clustering behavior of CD44 and CD43, from a patchy clusters of ~ 200 nm in size to an elongated network-like structures where for PSGL-1 the clustering size did not change significantly as it was 85 nm and after cell rolling the PSGL-1 aggregated to one side or even exhibited an increase in the footprint. Furthermore, I have established the use of 3D SR images that indicated that the patchy clusters of CD44 localize to protruding structures of the cell surface. On the other hand, a significant amount of the network-like elongated CD44 clusters observed after the rolling were located in the close proximity to the E-selectin surface. The effect of the nanoscale reorganization of the clusters on the HSPC rolling over selectins is still an open question at this stage. Nevertheless, my results further

  6. Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins.

    Directory of Open Access Journals (Sweden)

    Felix Dempwolff

    2016-06-01

    Full Text Available Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro-and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.

  7. Spatio-temporal image correlation spectroscopy and super-resolution microscopy to quantify molecular dynamics in T cells.

    Science.gov (United States)

    Ashdown, George W; Owen, Dylan M

    2018-02-02

    Many cellular processes are regulated by the spatio-temporal organisation of signalling complexes, cytoskeletal components and membranes. One such example is at the T cell immunological synapse where the retrograde flow of cortical filamentous (F)-actin from the synapse periphery drives signalling protein microclusters towards the synapse centre. The density of this mesh however, makes visualisation and analysis of individual actin fibres difficult due to the resolution limit of conventional microscopy. Recently, super-resolution methods such as structured illumination microscopy (SIM) have surpassed this resolution limit. Here, we apply SIM to better visualise the dense cortical actin meshwork in T cell synapses formed against activating, antibody-coated surfaces and image under total-internal reflection fluorescence (TIRF) illumination. To analyse the observed molecular flows, and the relationship between them, we apply spatio-temporal image correlation spectroscopy (STICS) and its cross-correlation variant (STICCS). We show that the dynamic cortical actin mesh can be visualised with unprecedented detail and that STICS/STICCS can output accurate, quantitative maps of molecular flow velocity and directionality from such data. We find that the actin flow can be disrupted using small molecule inhibitors of actin polymerisation. This combination of imaging and quantitative analysis may provide an important new tool for researchers to investigate the molecular dynamics at cellular length scales. Here we demonstrate the retrograde flow of F-actin which may be important for the clustering and dynamics of key signalling proteins within the plasma membrane, a phenomenon which is vital to correct T cell activation and therefore the mounting of an effective immune response. Copyright © 2018. Published by Elsevier Inc.

  8. Contrast enhancement of microsphere-assisted super-resolution imaging in dark-field microscopy

    Science.gov (United States)

    Zhou, Yi; Tang, Yan; Deng, Qinyuan; Zhao, Lixin; Hu, Song

    2017-08-01

    We report a method of boosting the imaging contrast of microsphere-assisted super-resolution visualization by utilizing dark-field illumination (DFI). We conducted experiments on both 10-µm-diameter silica (SiO2) microspheres with refractive index n ∼ 1.46 under no and partial immersion in ethyl alcohol (n ∼ 1.36) and 20-µm-diameter barium titanate glass (BTG, n ∼ 1.9) microspheres with full immersion to show the super-resolution capability. We experimentally demonstrated that the imaging contrast and uniformity were extraordinarily improved in the DFI mode. The intensity profiles in the visualization also numerically confirm the enhanced sharpness for a better imaging quality when applying DFI.

  9. Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

    Science.gov (United States)

    Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

    2018-01-01

    Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

  10. Measuring true localization accuracy in super resolution microscopy with DNA-origami nanostructures

    International Nuclear Information System (INIS)

    Reuss, Matthias; Blom, Hans; Brismar, Hjalmar; Fördős, Ferenc; Högberg, Björn; Öktem, Ozan

    2017-01-01

    A common method to assess the performance of (super resolution) microscopes is to use the localization precision of emitters as an estimate for the achieved resolution. Naturally, this is widely used in super resolution methods based on single molecule stochastic switching. This concept suffers from the fact that it is hard to calibrate measures against a real sample (a phantom), because true absolute positions of emitters are almost always unknown. For this reason, resolution estimates are potentially biased in an image since one is blind to true position accuracy, i.e. deviation in position measurement from true positions. We have solved this issue by imaging nanorods fabricated with DNA-origami. The nanorods used are designed to have emitters attached at each end in a well-defined and highly conserved distance. These structures are widely used to gauge localization precision. Here, we additionally determined the true achievable localization accuracy and compared this figure of merit to localization precision values for two common super resolution microscope methods STED and STORM. (paper)

  11. Super-resolution microscopy reveals presynaptic localization of the ALS / FTD related protein FUS in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Michael eSchoen

    2016-01-01

    Full Text Available Fused in Sarcoma (FUS is a multifunctional RNA- / DNA-binding protein, which is involved in the pathogenesis of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. A common hallmark of these disorders is the abnormal accumulation of mutated FUS protein in the cytoplasm. Under normal conditions FUS is confined to the nuclear compartment, in neurons however, additional somatodendritic localization can be observed. In this study, we carefully analyzed the subcellular localization of endogenous FUS at synaptic sites of hippocampal neurons which are among the most affected cell types in frontotemporal dementia with FUS pathology. We could confirm a strong nuclear localization of FUS as well as its prominent and widespread neuronal expression throughout the adult and developing rat brain, particularly in the hippocampus, the cerebellum and the outer layers of the cortex. Intriguingly, FUS was also consistently observed at synaptic sites as detected by neuronal subcellular fractionation as well as by immunolabeling. To define a pre- and / or postsynaptic localization of FUS, we employed super-resolution fluorescence localization microscopy. FUS was found to be localized within the axon terminal in close proximity to the presynaptic vesicle protein Synaptophysin1 and adjacent to the active zone protein Bassoon, but well separated from the postsynaptic protein PSD-95. Having shown the presynaptic localization of FUS in the nervous system, a novel extranuclear role of FUS at neuronal contact sites has to be considered. Since there is growing evidence that local presynaptic translation might also be an important mechanism for plasticity, FUS - like the fragile X mental retardation protein FMRP - might act as one of the presynaptic RNA-binding proteins regulating this machinery. Our observation of presynaptic FUS should foster further investigations to determine its role in neurodegenerative diseases such as

  12. Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

    Science.gov (United States)

    Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

    2015-02-11

    Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  13. Localization-based super-resolution imaging of cellular structures.

    Science.gov (United States)

    Kanchanawong, Pakorn; Waterman, Clare M

    2013-01-01

    Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction to ~250 nm, prompting the development of super-resolution microscopy which offers resolution approaching the scale of single proteins, i.e., ~20 nm. Here, we describe protocols for single molecule localization-based super-resolution imaging, using focal adhesion proteins as an example and employing either photoswitchable fluorophores or photoactivatable fluorescent proteins. These protocols should also be easily adaptable to imaging a broad array of macromolecular assemblies in cells whose components can be fluorescently tagged and assemble into high density structures.

  14. Structured Illumination-Based Super-Resolution Optical Microscopy for Hemato- and Cyto-Pathology Applications

    Directory of Open Access Journals (Sweden)

    Tieqiao Zhang

    2013-01-01

    Full Text Available Structured illumination fluorescence microscopy utilizes interfering light and the moiré effect to enhance spatial resolution to about a half of that of conventional light microscopy, i.e. approximately 90 nm. In addition to the enhancement in the x and y directions, it also allows enhancement of resolution in the z- direction by the same factor of two (to approximately 220 nm, making it a powerful tool for 3-D morphology studies of fluorescently labeled cells or thin tissue sections. In this report, we applied this technique to several types of blood cells that are commonly seen in hematopathology. Compared with standard brightfield and ordinary fluorescence microscopy images, the 3-D morphology results clearly reveal the morphological features of different types of normal blood cells. We have also used this technique to evaluate morphologies of abnormal erythrocytes and compare them with those recorded on normal cells. The results give a very intuitive presentation of morphological structures of erythrocytes with great details. This research illustrates the potential of this technique to be used in hematology and cyto-pathology studies aimed at identifying nanometer-sized features that cannot be distinguished otherwise with conventional optical microscopy.

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

    Directory of Open Access Journals (Sweden)

    Yu Yabiku

    2013-10-01

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

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

    KAUST Repository

    Yabiku, Y.

    2013-10-22

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

  17. Parallel detecting super-resolution microscopy using correlation based image restoration

    Science.gov (United States)

    Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

    2017-12-01

    A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

  18. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging

    Science.gov (United States)

    Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

    2015-11-01

    Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents—inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non

  19. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

    Science.gov (United States)

    Shirai, Yuki M.; Tsunoyama, Taka A.; Hiramoto-Yamaki, Nao; Hirosawa, Koichiro M.; Shibata, Akihiro C. E.; Kondo, Kenichi; Tsurumune, Atsushi; Ishidate, Fumiyoshi; Kusumi, Akihiro

    2017-01-01

    Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”. PMID:29190677

  20. Fluorescent Nanodiamond: A Versatile Tool for Long-Term Cell Tracking, Super-Resolution Imaging, and Nanoscale Temperature Sensing.

    Science.gov (United States)

    Hsiao, Wesley Wei-Wen; Hui, Yuen Yung; Tsai, Pei-Chang; Chang, Huan-Cheng

    2016-03-15

    Fluorescent nanodiamond (FND) has recently played a central role in fueling new discoveries in interdisciplinary fields spanning biology, chemistry, physics, and materials sciences. The nanoparticle is unique in that it contains a high density ensemble of negatively charged nitrogen-vacancy (NV(-)) centers as built-in fluorophores. The center possesses a number of outstanding optical and magnetic properties. First, NV(-) has an absorption maximum at ∼550 nm, and when exposed to green-orange light, it emits bright fluorescence at ∼700 nm with a lifetime of longer than 10 ns. These spectroscopic properties are little affected by surface modification but are distinctly different from those of cell autofluorescence and thus enable background-free imaging of FNDs in tissue sections. Such characteristics together with its excellent biocompatibility render FND ideal for long-term cell tracking applications, particularly in stem cell research. Next, as an artificial atom in the solid state, the NV(-) center is perfectly photostable, without photobleaching and blinking. Therefore, the NV-containing FND is suitable as a contrast agent for super-resolution imaging by stimulated emission depletion (STED). An improvement of the spatial resolution by 20-fold is readily achievable by using a high-power STED laser to deplete the NV(-) fluorescence. Such improvement is crucial in revealing the detailed structures of biological complexes and assemblies, including cellular organelles and subcellular compartments. Further enhancement of the resolution for live cell imaging is possible by manipulating the charge states of the NV centers. As the "brightest" member of the nanocarbon family, FND holds great promise and potential for bioimaging with unprecedented resolution and precision. Lastly, the NV(-) center in diamond is an atom-like quantum system with a total electron spin of 1. The ground states of the spins show a crystal field splitting of 2.87 GHz, separating the ms = 0 and

  1. Nanoscale clustering of the neurotrophin receptor TrkB revealed by super-resolution STED microscopy

    Czech Academy of Sciences Publication Activity Database

    Angelov, Borislav; Angelova, A.

    2017-01-01

    Roč. 9, č. 28 (2017), s. 9797-9804 ISSN 2040-3364 R&D Projects: GA MŠk EF15_003/0000447; GA MŠk EF15_008/0000162; GA ČR(CZ) GA17-00973S Grant - others:OP VVV - ELIBIO(XE) CZ.02.1.01/0.0/0.0/15_003/0000447; ELI Beamlines(XE) CZ.02.1.01/0.0/0.0/15_008/0000162 Institutional support: RVO:68378271 Keywords : emission depletion microscopy * higher-order oligomers * neurodegenerative diseases * mechanistic insights Subject RIV: BL - Plasma and Gas Discharge Physics OBOR OECD: Fluids and plasma physics (including surface physics) Impact factor: 7.367, year: 2016

  2. Radial super-resolution in digital holographic microscopy using structured illumination with circular symmetry

    Science.gov (United States)

    Yin, Yujian; Su, Ping; Ma, Jianshe

    2018-01-01

    A method to improve the radial resolution using special structured light is proposed in the field of digital holographic microscopy (DHM). A specimen is illuminated with circular symmetrical structured light that makes the spectrum have radial movement, so that high frequency components of the specimen are moved into the passband of the receiver to overcome the diffraction limit. In the DHM imaging system, Computer Generated Hologram (CGH) technology is used to generate the required structured light grating. Then the grating is loaded into a spatial light modulator (SLM) to obtain specific structured illumination. After recording the hologram, digital reconstruction, for the microstructure of a binary optical element that needs to observe radial distribution, the radial resolution of the specimen is improved experimentally compare it with the result of one-dimensional sinusoidal structured light imaging. And a method of designing structured light is presented.

  3. Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media

    Science.gov (United States)

    Edrei, Eitan; Scarcelli, Giuliano

    2016-09-01

    High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

  4. Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

    Science.gov (United States)

    Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

    2018-02-01

    To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

  5. Super-resolution Phase Tomography

    KAUST Repository

    Depeursinge, Christian; Cotte, Yann; Toy, Fatih; Jourdain, Pascal; Boss, Daiel; Marquet, Pierre; Magistretti, Pierre J.

    2013-01-01

    Digital Holographic Microscopy (DHM) yields reconstructed complex wavefields. It allows synthesizing the aperture of a virtual microscope up to 2π, offering super-resolution phase images. Live images of micro-organisms and neurons with resolution less than 100 nm are presented.

  6. Super-resolution Phase Tomography

    KAUST Repository

    Depeursinge, Christian

    2013-04-21

    Digital Holographic Microscopy (DHM) yields reconstructed complex wavefields. It allows synthesizing the aperture of a virtual microscope up to 2π, offering super-resolution phase images. Live images of micro-organisms and neurons with resolution less than 100 nm are presented.

  7. Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

    Science.gov (United States)

    Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

    2018-02-01

    To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

  8. 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.

  9. Light-Induced Fluorescence Modulation of Quantum Dot-Crystal Violet Conjugates: Stochastic Off-On-Off Cycles for Multicolor Patterning and Super-Resolution.

    Science.gov (United States)

    Jung, Sungwook; Park, Joonhyuck; Bang, Jiwon; Kim, Jae-Yeol; Kim, Cheolhee; Jeon, Yongmoon; Lee, Seung Hwan; Jin, Ho; Choi, Sukyung; Kim, Bomi; Lee, Woo Jin; Pack, Chan-Gi; Lee, Jong-Bong; Lee, Nam Ki; Kim, Sungjee

    2017-06-07

    Photoswitching or modulation of quantum dots (QDs) can be promising for many fields that include display, memory, and super-resolution imaging. However, such modulations have mostly relied on photomodulations of conjugated molecules in QD vicinity, which typically require high power of high energy photons at UV. We report a visible light-induced facile modulation route for QD-dye conjugates. QD crystal violets conjugates (QD-CVs) were prepared and the crystal violet (CV) molecules on QD quenched the fluorescence efficiently. The fluorescence of QD-CVs showed a single cycle of emission burst as they go through three stages of (i) initially quenched "off" to (ii) photoactivated "on" as the result of chemical change of CVs induced by photoelectrons from QD and (iii) back to photodarkened "off" by radical-associated reactions. Multicolor on-demand photopatterning was demonstrated using QD-CV solid films. QD-CVs were introduced into cells, and excitation with visible light yielded photomodulation from "off" to "on" and "off" by nearly ten fold. Individual photoluminescence dynamics of QD-CVs was investigated using fluorescence correlation spectroscopy and single QD emission analysis, which revealed temporally stochastic photoactivations and photodarkenings. Exploiting the stochastic fluorescence burst of QD-CVs, simultaneous multicolor super-resolution localizations were demonstrated.

  10. Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.

    Directory of Open Access Journals (Sweden)

    Joseph Andronic

    Full Text Available Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100-275 mOsm in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200-275 mOsm, P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼ 3 nm/s at 100-125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM. dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200-2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80-800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

  11. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    International Nuclear Information System (INIS)

    Rossier, Olivier; Giannone, Grégory

    2016-01-01

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

  12. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    Energy Technology Data Exchange (ETDEWEB)

    Rossier, Olivier; Giannone, Grégory [Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France); CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France)

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

  13. Super-resolution

    DEFF Research Database (Denmark)

    Nasrollahi, Kamal; Moeslund, Thomas B.

    2014-01-01

    Super-resolution, the process of obtaining one or more high-resolution images from one or more low-resolution observations, has been a very attractive research topic over the last two decades. It has found practical applications in many real world problems in different fields, from satellite...

  14. The super-resolution debate

    Science.gov (United States)

    Won, Rachel

    2018-05-01

    In the quest for nanoscopy with super-resolution, consensus from the imaging community is that super-resolution is not always needed and that scientists should choose an imaging technique based on their specific application.

  15. Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains

    DEFF Research Database (Denmark)

    Rahbek-Clemmensen, Troels; Lycas, Matthew D.; Erlendsson, Simon

    2017-01-01

    is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive...... to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to...

  16. 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.

  17. 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.

  18. 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.

  19. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

    Science.gov (United States)

    Rossier, Olivier; Giannone, Grégory

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

  20. Super-resolution microscopy reveals the insulin-resistance-regulated reorganization of GLUT4 on plasma membranes.

    Science.gov (United States)

    Gao, Lan; Chen, Junling; Gao, Jing; Wang, Hongda; Xiong, Wenyong

    2017-01-15

    GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F 5 QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F 5 QQI (F 5 QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F 5 QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance. © 2017. Published by The Company of Biologists Ltd.

  1. 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.

  2. Diverse Protocols for Correlative Super-Resolution Fluorescence Imaging and Electron Microscopy of Cells and Tissue

    Science.gov (United States)

    2016-05-25

    polymerization36. Finally, we mutated surface residues on mEos2 to remove nucleophilic groups, which are involved in cross-linking with aldehydes and...Sites of Clathrin-Mediated Endocytosis. Current Biology 21, 1167– 1175 (2011). 49. Caplan, J. et al. Correlative Protein Localization in Yeast . (2013

  3. 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...

  4. Super-resolution from single photon emission: toward biological application

    Science.gov (United States)

    Moreva, E.; Traina, P.; Forneris, J.; Ditalia Tchernij, S.; Guarina, L.; Franchino, C.; Picollo, F.; Ruo Berchera, I.; Brida, G.; Degiovanni, I. P.; Carabelli, V.; Olivero, P.; Genovese, M.

    2017-08-01

    Properties of quantum light represent a tool for overcoming limits of classical optics. Several experiments have demonstrated this advantage ranging from quantum enhanced imaging to quantum illumination. In this work, experimental demonstration of quantum-enhanced resolution in confocal fluorescence microscopy will be presented. This is achieved by exploiting the non-classical photon statistics of fluorescence emission of single nitrogen-vacancy (NV) color centers in diamond. By developing a general model of super-resolution based on the direct sampling of the kth-order autocorrelation function of the photoluminescence signal, we show the possibility to resolve, in principle, arbitrarily close emitting centers. Finally, possible applications of NV-based fluorescent nanodiamonds in biosensing and future developments will be presented.

  5. Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci.

    Science.gov (United States)

    Smeets, Daniel; Markaki, Yolanda; Schmid, Volker J; Kraus, Felix; Tattermusch, Anna; Cerase, Andrea; Sterr, Michael; Fiedler, Susanne; Demmerle, Justin; Popken, Jens; Leonhardt, Heinrich; Brockdorff, Neil; Cremer, Thomas; Schermelleh, Lothar; Cremer, Marion

    2014-01-01

    A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an 'autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform

  6. 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.

  7. Adaptive optics improves multiphoton super-resolution imaging

    Science.gov (United States)

    Zheng, Wei; Wu, Yicong; Winter, Peter; Shroff, Hari

    2018-02-01

    Three dimensional (3D) fluorescence microscopy has been essential for biological studies. It allows interrogation of structure and function at spatial scales spanning the macromolecular, cellular, and tissue levels. Critical factors to consider in 3D microscopy include spatial resolution, signal-to-noise (SNR), signal-to-background (SBR), and temporal resolution. Maintaining high quality imaging becomes progressively more difficult at increasing depth (where optical aberrations, induced by inhomogeneities of refractive index in the sample, degrade resolution and SNR), and in thick or densely labeled samples (where out-of-focus background can swamp the valuable, in-focus-signal from each plane). In this report, we introduce our new instrumentation to address these problems. A multiphoton structured illumination microscope was simply modified to integrate an adpative optics system for optical aberrations correction. Firstly, the optical aberrations are determined using direct wavefront sensing with a nonlinear guide star and subsequently corrected using a deformable mirror, restoring super-resolution information. We demonstrate the flexibility of our adaptive optics approach on a variety of semi-transparent samples, including bead phantoms, cultured cells in collagen gels and biological tissues. The performance of our super-resolution microscope is improved in all of these samples, as peak intensity is increased (up to 40-fold) and resolution recovered (up to 176+/-10 nm laterally and 729+/-39 nm axially) at depths up to 250 μm from the coverslip surface.

  8. Focusing super resolution on the cytoskeleton [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Eric A. Shelden

    2016-05-01

    Full Text Available Super resolution imaging is becoming an increasingly important tool in the arsenal of methods available to cell biologists. In recognition of its potential, the Nobel Prize for chemistry was awarded to three investigators involved in the development of super resolution imaging methods in 2014. The availability of commercial instruments for super resolution imaging has further spurred the development of new methods and reagents designed to take advantage of super resolution techniques. Super resolution offers the advantages traditionally associated with light microscopy, including the use of gentle fixation and specimen preparation methods, the ability to visualize multiple elements within a single specimen, and the potential to visualize dynamic changes in living specimens over time. However, imaging of living cells over time is difficult and super resolution imaging is computationally demanding. In this review, we discuss the advantages/disadvantages of different super resolution systems for imaging fixed live specimens, with particular regard to cytoskeleton structures.

  9. Super resolution imaging of genetically labelled synapses in Drosophila brain tissue

    Directory of Open Access Journals (Sweden)

    Isabelle Ayumi Spühler

    2016-05-01

    Full Text Available Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy, dSTORM, we image synaptic proteins in the brain tissue of the fruit fly, Drosophila melanogaster. Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labelled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation

  10. Super Resolution Imaging of Genetically Labeled Synapses in Drosophila Brain Tissue.

    Science.gov (United States)

    Spühler, Isabelle A; Conley, Gaurasundar M; Scheffold, Frank; Sprecher, Simon G

    2016-01-01

    Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy, dSTORM, we image synaptic proteins in the brain tissue of the fruit fly, Drosophila melanogaster. Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labeled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation.

  11. 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

  12. Controlled power delivery for super-resolution imaging of biological samples using digital micromirror device

    Science.gov (United States)

    Valiya Peedikakkal, Liyana; Cadby, Ashley

    2017-02-01

    Localization based super resolution images of a biological sample is generally achieved by using high power laser illumination with long exposure time which unfortunately increases photo-toxicity of a sample, making super resolution microscopy, in general, incompatible with live cell imaging. Furthermore, the limitation of photobleaching reduces the ability to acquire time lapse images of live biological cells using fluorescence microscopy. Digital Light Processing (DLP) technology can deliver light at grey scale levels by flickering digital micromirrors at around 290 Hz enabling highly controlled power delivery to samples. In this work, Digital Micromirror Device (DMD) is implemented in an inverse Schiefspiegler telescope setup to control the power and pattern of illumination for super resolution microscopy. We can achieve spatial and temporal patterning of illumination by controlling the DMD pixel by pixel. The DMD allows us to control the power and spatial extent of the laser illumination. We have used this to show that we can reduce the power delivered to the sample to allow for longer time imaging in one area while achieving sub-diffraction STORM imaging in another using higher power densities.

  13. Fabrication of optical multilayer for two-color phase plate in super-resolution microscope.

    Science.gov (United States)

    Iketaki, Yoshinori; Kitagawa, Katsuichi; Hidaka, Kohjiro; Kato, Naoki; Hirabayashi, Akira; Bokor, Nandor

    2014-07-01

    In super-resolution microscopy based on fluorescence depletion, the two-color phase plate (TPP) is an indispensable optical element, which can independently control the phase shifts for two beams of different color, i.e., the pump and erase beams. By controlling a phase shift of the erase beam through the TPP, the erase beam can be modulated into a doughnut shape, while the pump beam maintains the initial Gaussian shape. To obtain a reliable optical multiplayer (ML) for the TPP, we designed a ML with only two optical layers by performing numerical optimization. The measured phase shifts generated by the fabricated ML using interferometry correspond to the design values. The beam profiles in the focal plane are also consistent with theoretical results. Although the fabricated ML consists of only two optical layers, the ML can provide a suitable phase modulation function for the TPP in a practical super-resolution microscope.

  14. Fabrication of optical multilayer for two-color phase plate in super-resolution microscope

    International Nuclear Information System (INIS)

    Iketaki, Yoshinori; Kitagawa, Katsuichi; Hidaka, Kohjiro; Kato, Naoki; Hirabayashi, Akira; Bokor, Nandor

    2014-01-01

    In super-resolution microscopy based on fluorescence depletion, the two-color phase plate (TPP) is an indispensable optical element, which can independently control the phase shifts for two beams of different color, i.e., the pump and erase beams. By controlling a phase shift of the erase beam through the TPP, the erase beam can be modulated into a doughnut shape, while the pump beam maintains the initial Gaussian shape. To obtain a reliable optical multiplayer (ML) for the TPP, we designed a ML with only two optical layers by performing numerical optimization. The measured phase shifts generated by the fabricated ML using interferometry correspond to the design values. The beam profiles in the focal plane are also consistent with theoretical results. Although the fabricated ML consists of only two optical layers, the ML can provide a suitable phase modulation function for the TPP in a practical super-resolution microscope

  15. 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.

  16. 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.

  17. 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.

  18. Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

    Science.gov (United States)

    Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

    2015-01-01

    Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

  19. Microsphere-based super-resolution scanning optical microscope.

    Science.gov (United States)

    Huszka, Gergely; Yang, Hui; Gijs, Martin A M

    2017-06-26

    High-refractive index dielectric microspheres positioned within the field of view of a microscope objective in a dielectric medium can focus the light into a so-called photonic nanojet. A sample placed in such nanojet can be imaged by the objective with super-resolution, i.e. with a resolution beyond the classical diffraction limit. However, when imaging nanostructures on a substrate, the propagation distance of a light wave in the dielectric medium in between the substrate and the microsphere must be small enough to reveal the sample's nanometric features. Therefore, only the central part of an image obtained through a microsphere shows super-resolution details, which are typically ∼100 nm using white light (peak at λ = 600 nm). We have performed finite element simulations of the role of this critical distance in the super-resolution effect. Super-resolution imaging of a sample placed beneath the microsphere is only possible within a very restricted central area of ∼10 μm 2 , where the separation distance between the substrate and the microsphere surface is very small (∼1 μm). To generate super-resolution images over larger areas of the sample, we have fixed a microsphere on a frame attached to the microscope objective, which is automatically scanned over the sample in a step-by-step fashion. This generates a set of image tiles, which are subsequently stitched into a single super-resolution image (with resolution of λ/4-λ/5) of a sample area of up to ∼10 4 μm 2 . Scanning a standard optical microscope objective with microsphere therefore enables super-resolution microscopy over the complete field-of-view of the objective.

  20. 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...

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

    KAUST Repository

    Habuchi, Satoshi

    2014-06-12

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

  2. Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space

    Science.gov (United States)

    Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

    2014-07-01

    Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

  3. Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space

    Science.gov (United States)

    Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

    2014-01-01

    Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions. PMID:25030837

  4. 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

  5. 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.

  6. 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 ...

  7. Super resolution for astronomical observations

    Science.gov (United States)

    Li, Zhan; Peng, Qingyu; Bhanu, Bir; Zhang, Qingfeng; He, Haifeng

    2018-05-01

    In order to obtain detailed information from multiple telescope observations a general blind super-resolution (SR) reconstruction approach for astronomical images is proposed in this paper. A pixel-reliability-based SR reconstruction algorithm is described and implemented, where the developed process incorporates flat field correction, automatic star searching and centering, iterative star matching, and sub-pixel image registration. Images captured by the 1-m telescope at Yunnan Observatory are used to test the proposed technique. The results of these experiments indicate that, following SR reconstruction, faint stars are more distinct, bright stars have sharper profiles, and the backgrounds have higher details; thus these results benefit from the high-precision star centering and image registration provided by the developed method. Application of the proposed approach not only provides more opportunities for new discoveries from astronomical image sequences, but will also contribute to enhancing the capabilities of most spatial or ground-based telescopes.

  8. 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).

  9. Live-cell super-resolution imaging of intrinsically fast moving flagellates

    International Nuclear Information System (INIS)

    Glogger, M; Subota, I; Spindler, M-C; Engstler, M; Fenz, S F; Stichler, S; Bertlein, S; Teßmar, J; Groll, J

    2017-01-01

    Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μ s. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. (paper)

  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. A novel super-resolution camera model

    Science.gov (United States)

    Shao, Xiaopeng; Wang, Yi; Xu, Jie; Wang, Lin; Liu, Fei; Luo, Qiuhua; Chen, Xiaodong; Bi, Xiangli

    2015-05-01

    Aiming to realize super resolution(SR) to single image and video reconstruction, a super resolution camera model is proposed for the problem that the resolution of the images obtained by traditional cameras behave comparatively low. To achieve this function we put a certain driving device such as piezoelectric ceramics in the camera. By controlling the driving device, a set of continuous low resolution(LR) images can be obtained and stored instantaneity, which reflect the randomness of the displacements and the real-time performance of the storage very well. The low resolution image sequences have different redundant information and some particular priori information, thus it is possible to restore super resolution image factually and effectively. The sample method is used to derive the reconstruction principle of super resolution, which analyzes the possible improvement degree of the resolution in theory. The super resolution algorithm based on learning is used to reconstruct single image and the variational Bayesian algorithm is simulated to reconstruct the low resolution images with random displacements, which models the unknown high resolution image, motion parameters and unknown model parameters in one hierarchical Bayesian framework. Utilizing sub-pixel registration method, a super resolution image of the scene can be reconstructed. The results of 16 images reconstruction show that this camera model can increase the image resolution to 2 times, obtaining images with higher resolution in currently available hardware levels.

  13. Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis.

    Science.gov (United States)

    Lee, Antony; Tsekouras, Konstantinos; Calderon, Christopher; Bustamante, Carlos; Pressé, Steve

    2017-06-14

    Super-resolution microscopy provides direct insight into fundamental biological processes occurring at length scales smaller than light's diffraction limit. The analysis of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data analysis methods starting from an overview of basic statistical techniques underlying the analysis of super-resolution and, more broadly, imaging data. We subsequently break down the analysis of super-resolution data into four problems: the localization problem, the counting problem, the linking problem, and what we've termed the interpretation problem.

  14. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

    Science.gov (United States)

    Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

    2013-08-01

    Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

  15. Learning from errors in super-resolution.

    Science.gov (United States)

    Tang, Yi; Yuan, Yuan

    2014-11-01

    A novel framework of learning-based super-resolution is proposed by employing the process of learning from the estimation errors. The estimation errors generated by different learning-based super-resolution algorithms are statistically shown to be sparse and uncertain. The sparsity of the estimation errors means most of estimation errors are small enough. The uncertainty of the estimation errors means the location of the pixel with larger estimation error is random. Noticing the prior information about the estimation errors, a nonlinear boosting process of learning from these estimation errors is introduced into the general framework of the learning-based super-resolution. Within the novel framework of super-resolution, a low-rank decomposition technique is used to share the information of different super-resolution estimations and to remove the sparse estimation errors from different learning algorithms or training samples. The experimental results show the effectiveness and the efficiency of the proposed framework in enhancing the performance of different learning-based algorithms.

  16. Live-cell super-resolution imaging of intrinsically fast moving flagellates

    Science.gov (United States)

    Glogger, M.; Stichler, S.; Subota, I.; Bertlein, S.; Spindler, M.-C.; Teßmar, J.; Groll, J.; Engstler, M.; Fenz, S. F.

    2017-02-01

    Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μs. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. , which features invited work from the best early-career researchers working within the scope of J Phys D. This project is part of the Journal of Physics series’ 50th anniversary celebrations in 2017. Susanne Fenz was selected by the Editorial Board of J Phys D as an Emerging Talent/Leader.

  17. Single Image Super Resolution via Sparse Reconstruction

    NARCIS (Netherlands)

    Kruithof, M.C.; Eekeren, A.W.M. van; Dijk, J.; Schutte, K.

    2012-01-01

    High resolution sensors are required for recognition purposes. Low resolution sensors, however, are still widely used. Software can be used to increase the resolution of such sensors. One way of increasing the resolution of the images produced is using multi-frame super resolution algorithms.

  18. 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...

  19. Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Ziomkiewicz, Iwona; Schulz, Alexander

    2013-01-01

    of cellulose fibril orientation and growth. The fluorescent dye Pontamine Fast Scarlet 4BS (PFS) was shown to stain cellulose with high specificity and could be used to visualize cellulose bundles in cell walls of Arabidopsis root epidermal cells with confocal microscopy. The resolution limit of confocal...... present the first super-resolution images of cellulose bundles in the plant cell wall produced by direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection fluorescence (TIRF) microscopy. Since TIRF limits observation to the cell surface, we tested...... as alternatives 3D-structured illumination microscopy (3D-SIM) and confocal microscopy, combined with image deconvolution. Both methods offer lower resolution than STORM, but enable 3D imaging. While 3D-SIM produced strong artifacts, deconvolution gave good results. The resolution was improved over conventional...

  20. 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.

  1. 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.

  2. Temporal super resolution using variational methods

    DEFF Research Database (Denmark)

    Keller, Sune Høgild; Lauze, Francois Bernard; Nielsen, Mads

    2010-01-01

    Temporal super resolution (TSR) is the ability to convert video from one frame rate to another and is as such a key functionality in modern video processing systems. A higher frame rate than what is recorded is desired for high frame rate displays, for super slow-motion, and for video/film format...... observed when watching video on large and bright displays where the motion of high contrast edges often seem jerky and unnatural. A novel motion compensated (MC) TSR algorithm using variational methods for both optical flow calculation and the actual new frame interpolation is presented. The flow...

  3. 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.

  4. Simultaneous super-resolution and blind deconvolution

    International Nuclear Information System (INIS)

    Sroubek, F; Flusser, J; Cristobal, G

    2008-01-01

    In many real applications, blur in input low-resolution images is a nuisance, which prevents traditional super-resolution methods from working correctly. This paper presents a unifying approach to the blind deconvolution and superresolution problem of multiple degraded low-resolution frames of the original scene. We introduce a method which assumes no prior information about the shape of degradation blurs and which is properly defined for any rational (fractional) resolution factor. The method minimizes a regularized energy function with respect to the high-resolution image and blurs, where regularization is carried out in both the image and blur domains. The blur regularization is based on a generalized multichannel blind deconvolution constraint. Experiments on real data illustrate robustness and utilization of the method

  5. Super-Resolution Enhancement From Multiple Overlapping Images: A Fractional Area Technique

    Science.gov (United States)

    Michaels, Joshua A.

    With the availability of large quantities of relatively low-resolution data from several decades of space borne imaging, methods of creating an accurate, higher-resolution image from the multiple lower-resolution images (i.e. super-resolution), have been developed almost since such imagery has been around. The fractional-area super-resolution technique developed in this thesis has never before been documented. Satellite orbits, like Landsat, have a quantifiable variation, which means each image is not centered on the exact same spot more than once and the overlapping information from these multiple images may be used for super-resolution enhancement. By splitting a single initial pixel into many smaller, desired pixels, a relationship can be created between them using the ratio of the area within the initial pixel. The ideal goal for this technique is to obtain smaller pixels with exact values and no error, yielding a better potential result than those methods that yield interpolated pixel values with consequential loss of spatial resolution. A Fortran 95 program was developed to perform all calculations associated with the fractional-area super-resolution technique. The fractional areas are calculated using traditional trigonometry and coordinate geometry and Linear Algebra Package (LAPACK; Anderson et al., 1999) is used to solve for the higher-resolution pixel values. In order to demonstrate proof-of-concept, a synthetic dataset was created using the intrinsic Fortran random number generator and Adobe Illustrator CS4 (for geometry). To test the real-life application, digital pictures from a Sony DSC-S600 digital point-and-shoot camera with a tripod were taken of a large US geological map under fluorescent lighting. While the fractional-area super-resolution technique works in perfect synthetic conditions, it did not successfully produce a reasonable or consistent solution in the digital photograph enhancement test. The prohibitive amount of processing time (up to

  6. Super-resolution structure of DNA significantly differs in buccal cells of controls and Alzheimer's patients.

    Science.gov (United States)

    Garcia, Angeles; Huang, David; Righolt, Amanda; Righolt, Christiaan; Kalaw, Maria Carmela; Mathur, Shubha; McAvoy, Elizabeth; Anderson, James; Luedke, Angela; Itorralba, Justine; Mai, Sabine

    2017-09-01

    The advent of super-resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super-resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age- and gender-matched healthy, non-caregiver controls. In this super-resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D-SIM). Quantitation of the super-resolution DNA structure revealed that the nuclear super-resolution DNA structure of individuals with AD significantly differs from that of their controls (p structure of AD significantly differs in mild, moderate, and severe disease with respect to the DNA-containing and DNA-free/poor spaces. We conclude that whole genome remodeling is a feature of buccal cells in AD. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  7. 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.

  8. Super-Resolution for Synthetic Zooming

    Directory of Open Access Journals (Sweden)

    Li Xin

    2006-01-01

    Full Text Available Optical zooming is an important feature of imaging systems. In this paper, we investigate a low-cost signal processing alternative to optical zooming—synthetic zooming by super-resolution (SR techniques. Synthetic zooming is achieved by registering a sequence of low-resolution (LR images acquired at varying focal lengths and reconstructing the SR image at a larger focal length or increased spatial resolution. Under the assumptions of constant scene depth and zooming speed, we argue that the motion trajectories of all physical points are related to each other by a unique vanishing point and present a robust technique for estimating its D coordinate. Such a line-geometry-based registration is the foundation of SR for synthetic zooming. We address the issue of data inconsistency arising from the varying focal length of optical lens during the zooming process. To overcome the difficulty of data inconsistency, we propose a two-stage Delaunay-triangulation-based interpolation for fusing the LR image data. We also present a PDE-based nonlinear deblurring to accommodate the blindness and variation of sensor point spread functions. Simulation results with real-world images have verified the effectiveness of the proposed SR techniques for synthetic zooming.

  9. Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging.

    Directory of Open Access Journals (Sweden)

    Lina Carlini

    Full Text Available Three-dimensional (3D localization-based super-resolution microscopy (SR requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope's pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.

  10. Super-resolution imaging applied to moving object tracking

    Science.gov (United States)

    Swalaganata, Galandaru; Ratna Sulistyaningrum, Dwi; Setiyono, Budi

    2017-10-01

    Moving object tracking in a video is a method used to detect and analyze changes that occur in an object that being observed. Visual quality and the precision of the tracked target are highly wished in modern tracking system. The fact that the tracked object does not always seem clear causes the tracking result less precise. The reasons are low quality video, system noise, small object, and other factors. In order to improve the precision of the tracked object especially for small object, we propose a two step solution that integrates a super-resolution technique into tracking approach. First step is super-resolution imaging applied into frame sequences. This step was done by cropping the frame in several frame or all of frame. Second step is tracking the result of super-resolution images. Super-resolution image is a technique to obtain high-resolution images from low-resolution images. In this research single frame super-resolution technique is proposed for tracking approach. Single frame super-resolution was a kind of super-resolution that it has the advantage of fast computation time. The method used for tracking is Camshift. The advantages of Camshift was simple calculation based on HSV color that use its histogram for some condition and color of the object varies. The computational complexity and large memory requirements required for the implementation of super-resolution and tracking were reduced and the precision of the tracked target was good. Experiment showed that integrate a super-resolution imaging into tracking technique can track the object precisely with various background, shape changes of the object, and in a good light conditions.

  11. Photobleaching correction in fluorescence microscopy images

    International Nuclear Information System (INIS)

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

    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

  12. Confocal pore size measurement based on super-resolution image restoration.

    Science.gov (United States)

    Liu, Dali; Wang, Yun; Qiu, Lirong; Mao, Xinyue; Zhao, Weiqian

    2014-09-01

    A confocal pore size measurement based on super-resolution image restoration is proposed to obtain a fast and accurate measurement for submicrometer pore size of nuclear track-etched membranes (NTEMs). This method facilitates the online inspection of the pore size evolution during etching. Combining confocal microscopy with super-resolution image restoration significantly improves the lateral resolution of the NTEM image, yields a reasonable circle edge-setting criterion of 0.2408, and achieves precise pore edge detection. Theoretical analysis shows that the minimum measuring diameter can reach 0.19 μm, and the root mean square of the residuals is only 1.4 nm. Edge response simulation and experiment reveal that the edge response of the proposed method is better than 80 nm. The NTEM pore size measurement results obtained by the proposed method agree well with that obtained by scanning electron microscopy.

  13. Localization-based super-resolution imaging meets high-content screening.

    Science.gov (United States)

    Beghin, Anne; Kechkar, Adel; Butler, Corey; Levet, Florian; Cabillic, Marine; Rossier, Olivier; Giannone, Gregory; Galland, Rémi; Choquet, Daniel; Sibarita, Jean-Baptiste

    2017-12-01

    Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

  14. 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.

  15. Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

    Science.gov (United States)

    Ni, Yanxiang; Cao, Bo; Ma, Tszshan; Niu, Gang; Huo, Yingdong; Huang, Jiandong; Chen, Danni; Liu, Yi; Yu, Bin; Zhang, Michael Q; Niu, Hanben

    2017-01-01

    High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution. DOI: http://dx.doi.org/10.7554/eLife.21660.001 PMID:28485713

  16. A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

    Science.gov (United States)

    D'Incecco, P; Ong, L; Gras, S; Pellegrino, L

    2018-04-18

    Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Dynamic placement of plasmonic hotspots for super-resolution surface-enhanced Raman scattering.

    Science.gov (United States)

    Ertsgaard, Christopher T; McKoskey, Rachel M; Rich, Isabel S; Lindquist, Nathan C

    2014-10-28

    In this paper, we demonstrate dynamic placement of locally enhanced plasmonic fields using holographic laser illumination of a silver nanohole array. To visualize these focused "hotspots", the silver surface was coated with various biological samples for surface-enhanced Raman spectroscopy (SERS) imaging. Due to the large field enhancements, blinking behavior of the SERS hotspots was observed and processed using a stochastic optical reconstruction microscopy algorithm enabling super-resolution localization of the hotspots to within 10 nm. These hotspots were then shifted across the surface in subwavelength (hotspots. Using this technique, we also show that such subwavelength shifting and localization of plasmonic hotspots has potential for imaging applications. Interestingly, illuminating the surface with randomly shifting SERS hotspots was sufficient to completely fill in a wide field of view for super-resolution chemical imaging.

  18. 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

  19. Oblique reconstructions in tomosynthesis. II. Super-resolution

    International Nuclear Information System (INIS)

    Acciavatti, Raymond J.; Maidment, Andrew D. A.

    2013-01-01

    Purpose: In tomosynthesis, super-resolution has been demonstrated using reconstruction planes parallel to the detector. Super-resolution allows for subpixel resolution relative to the detector. The purpose of this work is to develop an analytical model that generalizes super-resolution to oblique reconstruction planes.Methods: In a digital tomosynthesis system, a sinusoidal test object is modeled along oblique angles (i.e., “pitches”) relative to the plane of the detector in a 3D divergent-beam acquisition geometry. To investigate the potential for super-resolution, the input frequency is specified to be greater than the alias frequency of the detector. Reconstructions are evaluated in an oblique plane along the extent of the object using simple backprojection (SBP) and filtered backprojection (FBP). By comparing the amplitude of the reconstruction against the attenuation coefficient of the object at various frequencies, the modulation transfer function (MTF) is calculated to determine whether modulation is within detectable limits for super-resolution. For experimental validation of super-resolution, a goniometry stand was used to orient a bar pattern phantom along various pitches relative to the breast support in a commercial digital breast tomosynthesis system.Results: Using theoretical modeling, it is shown that a single projection image cannot resolve a sine input whose frequency exceeds the detector alias frequency. The high frequency input is correctly visualized in SBP or FBP reconstruction using a slice along the pitch of the object. The Fourier transform of this reconstructed slice is maximized at the input frequency as proof that the object is resolved. Consistent with the theoretical results, experimental images of a bar pattern phantom showed super-resolution in oblique reconstructions. At various pitches, the highest frequency with detectable modulation was determined by visual inspection of the bar patterns. The dependency of the highest

  20. Oblique reconstructions in tomosynthesis. II. Super-resolution

    Science.gov (United States)

    Acciavatti, Raymond J.; Maidment, Andrew D. A.

    2013-01-01

    Purpose: In tomosynthesis, super-resolution has been demonstrated using reconstruction planes parallel to the detector. Super-resolution allows for subpixel resolution relative to the detector. The purpose of this work is to develop an analytical model that generalizes super-resolution to oblique reconstruction planes. Methods: In a digital tomosynthesis system, a sinusoidal test object is modeled along oblique angles (i.e., “pitches”) relative to the plane of the detector in a 3D divergent-beam acquisition geometry. To investigate the potential for super-resolution, the input frequency is specified to be greater than the alias frequency of the detector. Reconstructions are evaluated in an oblique plane along the extent of the object using simple backprojection (SBP) and filtered backprojection (FBP). By comparing the amplitude of the reconstruction against the attenuation coefficient of the object at various frequencies, the modulation transfer function (MTF) is calculated to determine whether modulation is within detectable limits for super-resolution. For experimental validation of super-resolution, a goniometry stand was used to orient a bar pattern phantom along various pitches relative to the breast support in a commercial digital breast tomosynthesis system. Results: Using theoretical modeling, it is shown that a single projection image cannot resolve a sine input whose frequency exceeds the detector alias frequency. The high frequency input is correctly visualized in SBP or FBP reconstruction using a slice along the pitch of the object. The Fourier transform of this reconstructed slice is maximized at the input frequency as proof that the object is resolved. Consistent with the theoretical results, experimental images of a bar pattern phantom showed super-resolution in oblique reconstructions. At various pitches, the highest frequency with detectable modulation was determined by visual inspection of the bar patterns. The dependency of the highest

  1. Extreme super-resolution using the spherical geodesic waveguide

    Science.gov (United States)

    Miñano, Juan Carlos; González, Juan Carlos; Benítez, Pablo; Grabovičkić, Dejan

    2012-06-01

    Leonhardt demonstrated (2009) that the 2D Maxwell Fish Eye lens (MFE) can focus perfectly 2D Helmholtz waves of arbitrary frequency, i.e., it can transport perfectly an outward (monopole) 2D Helmholtz wave field, generated by a point source, towards a "perfect point drain" located at the corresponding image point. Moreover, a prototype with λ/5 super-resolution (SR) property for one microwave frequency has been manufactured and tested (Ma et al, 2010). Although this prototype has been loaded with an impedance different from the "perfect point drain", it has shown super-resolution property. However, neither software simulations nor experimental measurements for a broad band of frequencies have yet been reported. Here we present steady state simulations for two cases, using perfect drain as suggested by Leonhardt and without perfect drain as in the prototype. All the simulations have been done using a device equivalent to the MFE, called the Spherical Geodesic Waveguide (SGW). The results show the super-resolution up to λ/3000, for the system loaded with the perfect drain, and up to λ /500 for a not perfect load. In both cases super-resolution only happens for discrete number of frequencies. Out of these frequencies, the SGW does not show super-resolution in the analysis carried out.

  2. Nonlinear super-resolution nano-optics and applications

    CERN Document Server

    Wei, Jingsong

    2015-01-01

    This book covers many advances in the subjects of nano-optics and nano photonics. The author describes the principle and technical schematics of common methods for breaking through the optical diffraction limit and focuses on realizing optical super-resolution with nonlinear effects of thin film materials. The applications of nonlinear optical super-resolution effects in nano-data storage, nanolithography, and nano-imaging are also presented. This book is useful to graduate students majoring in optics and nano science and also serves as a reference book for academic researchers, engineers, technical professionals in the fields of super-resolution optics and laser techniques, nano-optics and nano photonics, nano-data storage, nano imaging, micro/nanofabrication and nanolithography and nonlinear optics.

  3. Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

    Science.gov (United States)

    Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

    2017-09-05

    Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

  4. The tetraspanin web revisited by super-resolution microscopy

    NARCIS (Netherlands)

    Zuidscherwoude, M.C.M.; Gottfert, F.; Dunlock, V.M.; Figdor, C.G.; Bogaart, G. van den; Spriel, A.B. van

    2015-01-01

    The spatial organization of membrane proteins in the plasma membrane is critical for signal transduction, cell communication and membrane trafficking. Tetraspanins organize functional higher-order protein complexes called 'tetraspanin-enriched microdomains (TEMs)' via interactions with partner

  5. Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

    Science.gov (United States)

    Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

    2017-04-01

    Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

  6. Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria

    Directory of Open Access Journals (Sweden)

    Sachith D. Gunasinghe

    2017-05-01

    Full Text Available Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.

  7. Deep Learning based Super-Resolution for Improved Action Recognition

    DEFF Research Database (Denmark)

    Nasrollahi, Kamal; Guerrero, Sergio Escalera; Rasti, Pejman

    2015-01-01

    with results of a state-of- the-art deep learning-based super-resolution algorithm, through an alpha-blending approach. The experimental results obtained on down-sampled version of a large subset of Hoolywood2 benchmark database show the importance of the proposed system in increasing the recognition rate...

  8. Video super-resolution using simultaneous motion and intensity calculations

    DEFF Research Database (Denmark)

    Keller, Sune Høgild; Lauze, Francois Bernard; Nielsen, Mads

    2011-01-01

    for the joint estimation of a super-resolution sequence and its flow field. Via the calculus of variations, this leads to a coupled system of partial differential equations for image sequence and motion estimation. We solve a simplified form of this system and as a by-product we indeed provide a motion field...

  9. Generation of super-resolution stills from video

    CSIR Research Space (South Africa)

    Duvenhage, B

    2014-11-01

    Full Text Available plane. If one accurately registers the image of the target on the focal plane to some reference then one can increase the effective sensor pixel density by stacking or appropriately combining the registered images. The super-resolution technique operates...

  10. Improved Interpolation Kernels for Super-resolution Algorithms

    DEFF Research Database (Denmark)

    Rasti, Pejman; Orlova, Olga; Tamberg, Gert

    2016-01-01

    Super resolution (SR) algorithms are widely used in forensics investigations to enhance the resolution of images captured by surveillance cameras. Such algorithms usually use a common interpolation algorithm to generate an initial guess for the desired high resolution (HR) image. This initial guess...... when their original interpolation kernel is replaced by the ones introduced in this work....

  11. 3D high- and super-resolution imaging using single-objective SPIM.

    Science.gov (United States)

    Galland, Remi; Grenci, Gianluca; Aravind, Ajay; Viasnoff, Virgile; Studer, Vincent; Sibarita, Jean-Baptiste

    2015-07-01

    Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam-steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single-molecule-based super-resolution imaging of whole cells or cell aggregates. Using larger mirrors enabled us to broaden the capabilities of our system to image Drosophila embryos.

  12. 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.

  13. 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

  14. Super-resolution imaging based on the temperature-dependent electron-phonon collision frequency effect of metal thin films

    Science.gov (United States)

    Ding, Chenliang; Wei, Jingsong; Xiao, Mufei

    2018-05-01

    We herein propose a far-field super-resolution imaging with metal thin films based on the temperature-dependent electron-phonon collision frequency effect. In the proposed method, neither fluorescence labeling nor any special properties are required for the samples. The 100 nm lands and 200 nm grooves on the Blu-ray disk substrates were clearly resolved and imaged through a laser scanning microscope of wavelength 405 nm. The spot size was approximately 0.80 μm , and the imaging resolution of 1/8 of the laser spot size was experimentally obtained. This work can be applied to the far-field super-resolution imaging of samples with neither fluorescence labeling nor any special properties.

  15. 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...

  16. 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...

  17. Robust microbubble tracking for super resolution imaging in ultrasound

    DEFF Research Database (Denmark)

    Hansen, Kristoffer B.; Villagómez Hoyos, Carlos Armando; Brasen, Jens Christian

    2016-01-01

    Currently ultrasound resolution is limited by diffraction to approximately half the wavelength of the sound wave employed. In recent years, super resolution imaging techniques have overcome the diffraction limit through the localization and tracking of a sparse set of microbubbles through...... the vasculature. However, this has only been performed on fixated tissue, limiting its clinical application. This paper proposes a technique for making super resolution images on non-fixated tissue by first compensating for tissue movement and then tracking the individual microbubbles. The experiment is performed...... on the kidney of a anesthetized Sprage-Dawley rat by infusing SonoVue at 0.1× original concentration. The algorithm demonstrated in vivo that the motion compensation was capable of removing the movement caused by the mechanical ventilator. The results shows that microbubbles were localized with a higher...

  18. Underwater video enhancement using multi-camera super-resolution

    Science.gov (United States)

    Quevedo, E.; Delory, E.; Callicó, G. M.; Tobajas, F.; Sarmiento, R.

    2017-12-01

    Image spatial resolution is critical in several fields such as medicine, communications or satellite, and underwater applications. While a large variety of techniques for image restoration and enhancement has been proposed in the literature, this paper focuses on a novel Super-Resolution fusion algorithm based on a Multi-Camera environment that permits to enhance the quality of underwater video sequences without significantly increasing computation. In order to compare the quality enhancement, two objective quality metrics have been used: PSNR (Peak Signal-to-Noise Ratio) and the SSIM (Structural SIMilarity) index. Results have shown that the proposed method enhances the objective quality of several underwater sequences, avoiding the appearance of undesirable artifacts, with respect to basic fusion Super-Resolution algorithms.

  19. Application of Super-Resolution Image Reconstruction to Digital Holography

    Directory of Open Access Journals (Sweden)

    Zhang Shuqun

    2006-01-01

    Full Text Available We describe a new application of super-resolution image reconstruction to digital holography which is a technique for three-dimensional information recording and reconstruction. Digital holography has suffered from the low resolution of CCD sensors, which significantly limits the size of objects that can be recorded. The existing solution to this problem is to use optics to bandlimit the object to be recorded, which can cause the loss of details. Here super-resolution image reconstruction is proposed to be applied in enhancing the spatial resolution of digital holograms. By introducing a global camera translation before sampling, a high-resolution hologram can be reconstructed from a set of undersampled hologram images. This permits the recording of larger objects and reduces the distance between the object and the hologram. Practical results from real and simulated holograms are presented to demonstrate the feasibility of the proposed technique.

  20. Super-resolution for scanning light stimulation systems

    Energy Technology Data Exchange (ETDEWEB)

    Bitzer, L. A.; Neumann, K.; Benson, N., E-mail: niels.benson@uni-due.de; Schmechel, R. [Faculty of Engineering, NST and CENIDE, University of Duisburg-Essen, Bismarckstr. 81, 47057 Duisburg (Germany)

    2016-09-15

    Super-resolution (SR) is a technique used in digital image processing to overcome the resolution limitation of imaging systems. In this process, a single high resolution image is reconstructed from multiple low resolution images. SR is commonly used for CCD and CMOS (Complementary Metal-Oxide-Semiconductor) sensor images, as well as for medical applications, e.g., magnetic resonance imaging. Here, we demonstrate that super-resolution can be applied with scanning light stimulation (LS) systems, which are common to obtain space-resolved electro-optical parameters of a sample. For our purposes, the Projection Onto Convex Sets (POCS) was chosen and modified to suit the needs of LS systems. To demonstrate the SR adaption, an Optical Beam Induced Current (OBIC) LS system was used. The POCS algorithm was optimized by means of OBIC short circuit current measurements on a multicrystalline solar cell, resulting in a mean square error reduction of up to 61% and improved image quality.

  1. SINGLE FRAME SUPER RESOLUTION OF NONCOOPERATIVE IRIS IMAGES

    Directory of Open Access Journals (Sweden)

    Anand Deshpande

    2016-11-01

    Full Text Available Image super-resolution, a process to enhance image resolution, has important applications in biometrics, satellite imaging, high definition television, medical imaging, etc. The long range captured iris identification systems often suffer from low resolution and meager focus of the captured iris images. These degrade the iris recognition performance. This paper proposes enhanced iterated back projection (EIBP method to super resolute the long range captured iris polar images. The performance of proposed method is tested and analyzed on CASIA long range iris database by comparing peak signal to noise ratio (PSNR and structural similarity index (SSIM with state-of-the-art super resolution (SR algorithms. It is further analyzed by increasing the up-sampling factor. Performance analysis shows that the proposed method is superior to state-of-the-art algorithms, the peak signal-to-noise ratio improved about 0.1-1.5 dB. The results demonstrate that the proposed method is well suited to super resolve the iris polar images captured at a long distance

  2. 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

  3. 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...

  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. 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.

  7. 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 ...

  8. 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.

  9. 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.

  10. Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites.

    Directory of Open Access Journals (Sweden)

    Jens Prescher

    2015-02-01

    Full Text Available The cellular endosomal sorting complex required for transport (ESCRT machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%. All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum between 45 and 60 nm or a diameter (determined using a Ripley's L-function analysis of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices

  11. 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,.

  12. Finite detector based projection model for super resolution CT

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hengyong; Wang, Ge [Wake Forest Univ. Health Sciences, Winston-Salem, NC (United States). Dept. of Radiology; Virgina Tech, Blacksburg, VA (United States). Biomedical Imaging Div.

    2011-07-01

    For finite detector and focal spot sizes, here we propose a projection model for super resolution CT. First, for a given X-ray source point, a projection datum is modeled as an area integral over a narrow fan-beam connecting the detector elemental borders and the X-ray source point. Then, the final projection value is expressed as the integral obtained in the first step over the whole focal spot support. An ordered-subset simultaneous algebraic reconstruction technique (OS-SART) is developed using the proposed projection model. In the numerical simulation, our method produces super spatial resolution and suppresses high-frequency artifacts. (orig.)

  13. Far-field super-resolution imaging of resonant multiples

    KAUST Repository

    Guo, Bowen

    2016-05-20

    We demonstrate for the first time that seismic resonant multiples, usually considered as noise, can be used for super-resolution imaging in the far-field region of sources and receivers. Tests with both synthetic data and field data show that resonant multiples can image reflector boundaries with resolutions more than twice the classical resolution limit. Resolution increases with the order of the resonant multiples. This procedure has important applications in earthquake and exploration seismology, radar, sonar, LIDAR (light detection and ranging), and ultrasound imaging, where the multiples can be used to make high-resolution images.

  14. Super-Resolution Algorithm in Cumulative Virtual Blanking

    Science.gov (United States)

    Montillet, J. P.; Meng, X.; Roberts, G. W.; Woolfson, M. S.

    2008-11-01

    The proliferation of mobile devices and the emergence of wireless location-based services have generated consumer demand for precise location. In this paper, the MUSIC super-resolution algorithm is applied to time delay estimation for positioning purposes in cellular networks. The goal is to position a Mobile Station with UMTS technology. The problem of Base-Stations herability is solved using Cumulative Virtual Blanking. A simple simulator is presented using DS-SS signal. The results show that MUSIC algorithm improves the time delay estimation in both the cases whether or not Cumulative Virtual Blanking was carried out.

  15. 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 ...

  16. Super-resolution in plenoptic cameras using FPGAs.

    Science.gov (United States)

    Pérez, Joel; Magdaleno, Eduardo; Pérez, Fernando; Rodríguez, Manuel; Hernández, David; Corrales, Jaime

    2014-05-16

    Plenoptic cameras are a new type of sensor that extend the possibilities of current commercial cameras allowing 3D refocusing or the capture of 3D depths. One of the limitations of plenoptic cameras is their limited spatial resolution. In this paper we describe a fast, specialized hardware implementation of a super-resolution algorithm for plenoptic cameras. The algorithm has been designed for field programmable graphic array (FPGA) devices using VHDL (very high speed integrated circuit (VHSIC) hardware description language). With this technology, we obtain an acceleration of several orders of magnitude using its extremely high-performance signal processing capability through parallelism and pipeline architecture. The system has been developed using generics of the VHDL language. This allows a very versatile and parameterizable system. The system user can easily modify parameters such as data width, number of microlenses of the plenoptic camera, their size and shape, and the super-resolution factor. The speed of the algorithm in FPGA has been successfully compared with the execution using a conventional computer for several image sizes and different 3D refocusing planes.

  17. Super-Resolution in Plenoptic Cameras Using FPGAs

    Directory of Open Access Journals (Sweden)

    Joel Pérez

    2014-05-01

    Full Text Available Plenoptic cameras are a new type of sensor that extend the possibilities of current commercial cameras allowing 3D refocusing or the capture of 3D depths. One of the limitations of plenoptic cameras is their limited spatial resolution. In this paper we describe a fast, specialized hardware implementation of a super-resolution algorithm for plenoptic cameras. The algorithm has been designed for field programmable graphic array (FPGA devices using VHDL (very high speed integrated circuit (VHSIC hardware description language. With this technology, we obtain an acceleration of several orders of magnitude using its extremely high-performance signal processing capability through parallelism and pipeline architecture. The system has been developed using generics of the VHDL language. This allows a very versatile and parameterizable system. The system user can easily modify parameters such as data width, number of microlenses of the plenoptic camera, their size and shape, and the super-resolution factor. The speed of the algorithm in FPGA has been successfully compared with the execution using a conventional computer for several image sizes and different 3D refocusing planes.

  18. Super-resolution thermographic imaging using blind structured illumination

    Science.gov (United States)

    Burgholzer, Peter; Berer, Thomas; Gruber, Jürgen; Mayr, Günther

    2017-07-01

    Using an infrared camera for thermographic imaging allows the contactless temperature measurement of many surface pixels simultaneously. From the measured surface data, the structure below the surface, embedded inside a sample or tissue, can be reconstructed and imaged, if heated by an excitation light pulse. The main drawback in active thermographic imaging is the degradation of the spatial resolution with the imaging depth, which results in blurred images for deeper lying structures. We circumvent this degradation by using blind structured illumination combined with a non-linear joint sparsity reconstruction algorithm. We demonstrate imaging of a line pattern and a star-shaped structure through a 3 mm thick steel sheet with a resolution four times better than the width of the thermal point-spread-function. The structured illumination is realized by parallel slits cut in an aluminum foil, where the excitation coming from a flashlight can penetrate. This realization of super-resolution thermographic imaging demonstrates that blind structured illumination allows thermographic imaging without high degradation of the spatial resolution for deeper lying structures. The groundbreaking concept of super-resolution can be transferred from optics to diffusive imaging by defining a thermal point-spread-function, which gives the principle resolution limit for a certain signal-to-noise ratio, similar to the Abbe limit for a certain optical wavelength. In future work, the unknown illumination pattern could be the speckle pattern generated by a short laser pulse inside a light scattering sample or tissue.

  19. Field-portable pixel super-resolution colour microscope.

    Directory of Open Access Journals (Sweden)

    Alon Greenbaum

    Full Text Available Based on partially-coherent digital in-line holography, we report a field-portable microscope that can render lensfree colour images over a wide field-of-view of e.g., >20 mm(2. This computational holographic microscope weighs less than 145 grams with dimensions smaller than 17×6×5 cm, making it especially suitable for field settings and point-of-care use. In this lensfree imaging design, we merged a colorization algorithm with a source shifting based multi-height pixel super-resolution technique to mitigate 'rainbow' like colour artefacts that are typical in holographic imaging. This image processing scheme is based on transforming the colour components of an RGB image into YUV colour space, which separates colour information from brightness component of an image. The resolution of our super-resolution colour microscope was characterized using a USAF test chart to confirm sub-micron spatial resolution, even for reconstructions that employ multi-height phase recovery to handle dense and connected objects. To further demonstrate the performance of this colour microscope Papanicolaou (Pap smears were also successfully imaged. This field-portable and wide-field computational colour microscope could be useful for tele-medicine applications in resource poor settings.

  20. 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.

  1. 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

  2. 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...

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Wavelet Filter Banks for Super-Resolution SAR Imaging

    Science.gov (United States)

    Sheybani, Ehsan O.; Deshpande, Manohar; Memarsadeghi, Nargess

    2011-01-01

    This paper discusses Innovative wavelet-based filter banks designed to enhance the analysis of super resolution Synthetic Aperture Radar (SAR) images using parametric spectral methods and signal classification algorithms, SAR finds applications In many of NASA's earth science fields such as deformation, ecosystem structure, and dynamics of Ice, snow and cold land processes, and surface water and ocean topography. Traditionally, standard methods such as Fast-Fourier Transform (FFT) and Inverse Fast-Fourier Transform (IFFT) have been used to extract Images from SAR radar data, Due to non-parametric features of these methods and their resolution limitations and observation time dependence, use of spectral estimation and signal pre- and post-processing techniques based on wavelets to process SAR radar data has been proposed. Multi-resolution wavelet transforms and advanced spectral estimation techniques have proven to offer efficient solutions to this problem.

  8. Single image super-resolution based on convolutional neural networks

    Science.gov (United States)

    Zou, Lamei; Luo, Ming; Yang, Weidong; Li, Peng; Jin, Liujia

    2018-03-01

    We present a deep learning method for single image super-resolution (SISR). The proposed approach learns end-to-end mapping between low-resolution (LR) images and high-resolution (HR) images. The mapping is represented as a deep convolutional neural network which inputs the LR image and outputs the HR image. Our network uses 5 convolution layers, which kernels size include 5×5, 3×3 and 1×1. In our proposed network, we use residual-learning and combine different sizes of convolution kernels at the same layer. The experiment results show that our proposed method performs better than the existing methods in reconstructing quality index and human visual effects on benchmarked images.

  9. Color image guided depth image super resolution using fusion filter

    Science.gov (United States)

    He, Jin; Liang, Bin; He, Ying; Yang, Jun

    2018-04-01

    Depth cameras are currently playing an important role in many areas. However, most of them can only obtain lowresolution (LR) depth images. Color cameras can easily provide high-resolution (HR) color images. Using color image as a guide image is an efficient way to get a HR depth image. In this paper, we propose a depth image super resolution (SR) algorithm, which uses a HR color image as a guide image and a LR depth image as input. We use the fusion filter of guided filter and edge based joint bilateral filter to get HR depth image. Our experimental results on Middlebury 2005 datasets show that our method can provide better quality in HR depth images both numerically and visually.

  10. Optimized multiple linear mappings for single image super-resolution

    Science.gov (United States)

    Zhang, Kaibing; Li, Jie; Xiong, Zenggang; Liu, Xiuping; Gao, Xinbo

    2017-12-01

    Learning piecewise linear regression has been recognized as an effective way for example learning-based single image super-resolution (SR) in literature. In this paper, we employ an expectation-maximization (EM) algorithm to further improve the SR performance of our previous multiple linear mappings (MLM) based SR method. In the training stage, the proposed method starts with a set of linear regressors obtained by the MLM-based method, and then jointly optimizes the clustering results and the low- and high-resolution subdictionary pairs for regression functions by using the metric of the reconstruction errors. In the test stage, we select the optimal regressor for SR reconstruction by accumulating the reconstruction errors of m-nearest neighbors in the training set. Thorough experimental results carried on six publicly available datasets demonstrate that the proposed SR method can yield high-quality images with finer details and sharper edges in terms of both quantitative and perceptual image quality assessments.

  11. 3-D Cellular Ultrastructure Can Be Resolved by X-ray Microscopy | Center for Cancer Research

    Science.gov (United States)

    X-ray microscopy (XRM) is more rapid than cryoelectron tomography or super-resolution fluorescence microscopy and could fill an important gap in current technologies used to investigate in situ three-dimensional structure of cells. New XRM methods developed by first author Gerd Schneider, Ph.D., working with James McNally. Ph.D., and a team of colleagues, is capable of revealing full cellular ultrastructure without requiring fixation, staining, or sectioning.

  12. 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

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

    KAUST Repository

    Habuchi, Satoshi

    2014-01-01

    fluorescence microscopy techniques along with the latest developments of fluorophores and labeling for the SR microscopy. I discuss the applications of SR microscopy in the fields of life science and materials science with a special emphasis on quantitative

  14. Concepts in Light Microscopy of Viruses

    Science.gov (United States)

    Witte, Robert; Georgi, Fanny

    2018-01-01

    Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Performance Evaluations for Super-Resolution Mosaicing on UAS Surveillance Videos

    Directory of Open Access Journals (Sweden)

    Aldo Camargo

    2013-05-01

    Full Text Available Abstract Unmanned Aircraft Systems (UAS have been widely applied for reconnaissance and surveillance by exploiting information collected from the digital imaging payload. The super-resolution (SR mosaicing of low-resolution (LR UAS surveillance video frames has become a critical requirement for UAS video processing and is important for further effective image understanding. In this paper we develop a novel super-resolution framework, which does not require the construction of sparse matrices. The proposed method implements image operations in the spatial domain and applies an iterated back-projection to construct super-resolution mosaics from the overlapping UAS surveillance video frames. The Steepest Descent method, the Conjugate Gradient method and the Levenberg-Marquardt algorithm are used to numerically solve the nonlinear optimization problem for estimating a super-resolution mosaic. A quantitative performance comparison in terms of computation time and visual quality of the super-resolution mosaics through the three numerical techniques is presented.

  20. Adaptive Markov Random Fields for Example-Based Super-resolution of Faces

    Science.gov (United States)

    Stephenson, Todd A.; Chen, Tsuhan

    2006-12-01

    Image enhancement of low-resolution images can be done through methods such as interpolation, super-resolution using multiple video frames, and example-based super-resolution. Example-based super-resolution, in particular, is suited to images that have a strong prior (for those frameworks that work on only a single image, it is more like image restoration than traditional, multiframe super-resolution). For example, hallucination and Markov random field (MRF) methods use examples drawn from the same domain as the image being enhanced to determine what the missing high-frequency information is likely to be. We propose to use even stronger prior information by extending MRF-based super-resolution to use adaptive observation and transition functions, that is, to make these functions region-dependent. We show with face images how we can adapt the modeling for each image patch so as to improve the resolution.

  1. Adaptive Markov Random Fields for Example-Based Super-resolution of Faces

    Directory of Open Access Journals (Sweden)

    Stephenson Todd A

    2006-01-01

    Full Text Available Image enhancement of low-resolution images can be done through methods such as interpolation, super-resolution using multiple video frames, and example-based super-resolution. Example-based super-resolution, in particular, is suited to images that have a strong prior (for those frameworks that work on only a single image, it is more like image restoration than traditional, multiframe super-resolution. For example, hallucination and Markov random field (MRF methods use examples drawn from the same domain as the image being enhanced to determine what the missing high-frequency information is likely to be. We propose to use even stronger prior information by extending MRF-based super-resolution to use adaptive observation and transition functions, that is, to make these functions region-dependent. We show with face images how we can adapt the modeling for each image patch so as to improve the resolution.

  2. 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.

  3. 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.

  4. 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".

  5. 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.

  6. 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...

  7. 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.

  8. 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 ...

  9. 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.

  10. A Super-resolution Reconstruction Algorithm for Surveillance Video

    Directory of Open Access Journals (Sweden)

    Jian Shao

    2017-01-01

    Full Text Available Recent technological developments have resulted in surveillance video becoming a primary method of preserving public security. Many city crimes are observed in surveillance video. The most abundant evidence collected by the police is also acquired through surveillance video sources. Surveillance video footage offers very strong support for solving criminal cases, therefore, creating an effective policy, and applying useful methods to the retrieval of additional evidence is becoming increasingly important. However, surveillance video has had its failings, namely, video footage being captured in low resolution (LR and bad visual quality. In this paper, we discuss the characteristics of surveillance video and describe the manual feature registration – maximum a posteriori – projection onto convex sets to develop a super-resolution reconstruction method, which improves the quality of surveillance video. From this method, we can make optimal use of information contained in the LR video image, but we can also control the image edge clearly as well as the convergence of the algorithm. Finally, we make a suggestion on how to adjust the algorithm adaptability by analyzing the prior information of target image.

  11. Learning-based compressed sensing for infrared image super resolution

    Science.gov (United States)

    Zhao, Yao; Sui, Xiubao; Chen, Qian; Wu, Shaochi

    2016-05-01

    This paper presents an infrared image super-resolution method based on compressed sensing (CS). First, the reconstruction model under the CS framework is established and a Toeplitz matrix is selected as the sensing matrix. Compared with traditional learning-based methods, the proposed method uses a set of sub-dictionaries instead of two coupled dictionaries to recover high resolution (HR) images. And Toeplitz sensing matrix allows the proposed method time-efficient. Second, all training samples are divided into several feature spaces by using the proposed adaptive k-means classification method, which is more accurate than the standard k-means method. On the basis of this approach, a complex nonlinear mapping from the HR space to low resolution (LR) space can be converted into several compact linear mappings. Finally, the relationships between HR and LR image patches can be obtained by multi-sub-dictionaries and HR infrared images are reconstructed by the input LR images and multi-sub-dictionaries. The experimental results show that the proposed method is quantitatively and qualitatively more effective than other state-of-the-art methods.

  12. Super-resolution for a point source using positive refraction

    Science.gov (United States)

    Miñano, Juan C.; Benítez, Pablo; González, Juan C.; Grabovičkić, Dejan; Ahmadpanahi, Hamed

    Leonhardt demonstrated (2009) that the 2D Maxwell Fish Eye lens (MFE) can focus perfectly 2D Helmholtz waves of arbitrary frequency, i.e., it can transport perfectly an outward (monopole) 2D Helmholtz wave field, generated by a point source, towards a receptor called "perfect drain" (PD) located at the corresponding MFE image point. The PD has the property of absorbing the complete radiation without radiation or scattering and it has been claimed as necessary to obtain super-resolution (SR) in the MFE. However, a prototype using a "drain" different from the PD has shown λ/5 resolution for microwave frequencies (Ma et al, 2010). Recently, the SR properties of a device equivalent to the MFE, called the Spherical Geodesic Waveguide (SGW) (Miñano et al, 2012) have been analyzed. The reported results show resolution up to λ /3000, for the SGW loaded with the perfect drain, and up to λ /500 for the SGW without perfect drain. The perfect drain was realized as a coaxial probe loaded with properly calculated impedance. The SGW provides SR only in a narrow band of frequencies close to the resonance Schumann frequencies. Here we analyze the SGW loaded with a small "perfect drain region" (González et al, 2011). This drain is designed as a region made of a material with complex permittivity. The comparative results show that there is no significant difference in the SR properties for both perfect drain designs.

  13. Super-resolution for imagery from integrated microgrid polarimeters.

    Science.gov (United States)

    Hardie, Russell C; LeMaster, Daniel A; Ratliff, Bradley M

    2011-07-04

    Imagery from microgrid polarimeters is obtained by using a mosaic of pixel-wise micropolarizers on a focal plane array (FPA). Each distinct polarization image is obtained by subsampling the full FPA image. Thus, the effective pixel pitch for each polarization channel is increased and the sampling frequency is decreased. As a result, aliasing artifacts from such undersampling can corrupt the true polarization content of the scene. Here we present the first multi-channel multi-frame super-resolution (SR) algorithms designed specifically for the problem of image restoration in microgrid polarization imagers. These SR algorithms can be used to address aliasing and other degradations, without sacrificing field of view or compromising optical resolution with an anti-aliasing filter. The new SR methods are designed to exploit correlation between the polarimetric channels. One of the new SR algorithms uses a form of regularized least squares and has an iterative solution. The other is based on the faster adaptive Wiener filter SR method. We demonstrate that the new multi-channel SR algorithms are capable of providing significant enhancement of polarimetric imagery and that they outperform their independent channel counterparts.

  14. Video Super-Resolution via Bidirectional Recurrent Convolutional Networks.

    Science.gov (United States)

    Huang, Yan; Wang, Wei; Wang, Liang

    2018-04-01

    Super resolving a low-resolution video, namely video super-resolution (SR), is usually handled by either single-image SR or multi-frame SR. Single-Image SR deals with each video frame independently, and ignores intrinsic temporal dependency of video frames which actually plays a very important role in video SR. Multi-Frame SR generally extracts motion information, e.g., optical flow, to model the temporal dependency, but often shows high computational cost. Considering that recurrent neural networks (RNNs) can model long-term temporal dependency of video sequences well, we propose a fully convolutional RNN named bidirectional recurrent convolutional network for efficient multi-frame SR. Different from vanilla RNNs, 1) the commonly-used full feedforward and recurrent connections are replaced with weight-sharing convolutional connections. So they can greatly reduce the large number of network parameters and well model the temporal dependency in a finer level, i.e., patch-based rather than frame-based, and 2) connections from input layers at previous timesteps to the current hidden layer are added by 3D feedforward convolutions, which aim to capture discriminate spatio-temporal patterns for short-term fast-varying motions in local adjacent frames. Due to the cheap convolutional operations, our model has a low computational complexity and runs orders of magnitude faster than other multi-frame SR methods. With the powerful temporal dependency modeling, our model can super resolve videos with complex motions and achieve well performance.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Image quality assessment for selfies with and without super resolution

    Science.gov (United States)

    Kubota, Aya; Gohshi, Seiichi

    2018-04-01

    With the advent of cellphone cameras, in particular, on smartphones, many people now take photos of themselves alone and with others in the frame; such photos are popularly known as "selfies". Most smartphones are equipped with two cameras: the front-facing and rear cameras. The camera located on the back of the smartphone is referred to as the "out-camera," whereas the one located on the front of the smartphone is called the "in-camera." In-cameras are mainly used for selfies. Some smartphones feature high-resolution cameras. However, the original image quality cannot be obtained because smartphone cameras often have low-performance lenses. Super resolution (SR) is one of the recent technological advancements that has increased image resolution. We developed a new SR technology that can be processed on smartphones. Smartphones with new SR technology are currently available in the market have already registered sales. However, the effective use of new SR technology has not yet been verified. Comparing the image quality with and without SR on smartphone display is necessary to confirm the usefulness of this new technology. Methods that are based on objective and subjective assessments are required to quantitatively measure image quality. It is known that the typical object assessment value, such as Peak Signal to Noise Ratio (PSNR), does not go together with how we feel when we assess image/video. When digital broadcast started, the standard was determined using subjective assessment. Although subjective assessment usually comes at high cost because of personnel expenses for observers, the results are highly reproducible when they are conducted under right conditions and statistical analysis. In this study, the subjective assessment results for selfie images are reported.

  20. Sparsity-Based Super Resolution for SEM Images.

    Science.gov (United States)

    Tsiper, Shahar; Dicker, Or; Kaizerman, Idan; Zohar, Zeev; Segev, Mordechai; Eldar, Yonina C

    2017-09-13

    The scanning electron microscope (SEM) is an electron microscope that produces an image of a sample by scanning it with a focused beam of electrons. The electrons interact with the atoms in the sample, which emit secondary electrons that contain information about the surface topography and composition. The sample is scanned by the electron beam point by point, until an image of the surface is formed. Since its invention in 1942, the capabilities of SEMs have become paramount in the discovery and understanding of the nanometer world, and today it is extensively used for both research and in industry. In principle, SEMs can achieve resolution better than one nanometer. However, for many applications, working at subnanometer resolution implies an exceedingly large number of scanning points. For exactly this reason, the SEM diagnostics of microelectronic chips is performed either at high resolution (HR) over a small area or at low resolution (LR) while capturing a larger portion of the chip. Here, we employ sparse coding and dictionary learning to algorithmically enhance low-resolution SEM images of microelectronic chips-up to the level of the HR images acquired by slow SEM scans, while considerably reducing the noise. Our methodology consists of two steps: an offline stage of learning a joint dictionary from a sequence of LR and HR images of the same region in the chip, followed by a fast-online super-resolution step where the resolution of a new LR image is enhanced. We provide several examples with typical chips used in the microelectronics industry, as well as a statistical study on arbitrary images with characteristic structural features. Conceptually, our method works well when the images have similar characteristics, as microelectronics chips do. This work demonstrates that employing sparsity concepts can greatly improve the performance of SEM, thereby considerably increasing the scanning throughput without compromising on analysis quality and resolution.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Overcoming Registration Uncertainty in Image Super-Resolution: Maximize or Marginalize?

    Directory of Open Access Journals (Sweden)

    Andrew Zisserman

    2007-01-01

    Full Text Available In multiple-image super-resolution, a high-resolution image is estimated from a number of lower-resolution images. This usually involves computing the parameters of a generative imaging model (such as geometric and photometric registration, and blur and obtaining a MAP estimate by minimizing a cost function including an appropriate prior. Two alternative approaches are examined. First, both registrations and the super-resolution image are found simultaneously using a joint MAP optimization. Second, we perform Bayesian integration over the unknown image registration parameters, deriving a cost function whose only variables of interest are the pixel values of the super-resolution image. We also introduce a scheme to learn the parameters of the image prior as part of the super-resolution algorithm. We show examples on a number of real sequences including multiple stills, digital video, and DVDs of movies.

  6. Correlative Light and Electron Microscopy (CLEM) and its applications in infectious disease

    Science.gov (United States)

    2016-05-20

    has been shown to handle OsO4 fixation by withstanding standard EM processing concentrations of 1% [29]. A great utility of FPs is the endogenous...Development of imaging techniques to study the pathogenesis of biosafety level 2/3 infectious agents. Pathog Dis, 2014. 72(3): p. 167-73. 3. Sridhar...3): p. 397-406. 32. Johnson, E., et al., Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Sci Rep

  7. Super resolution PLIF demonstrated in turbulent jet flows seeded with I2

    Science.gov (United States)

    Xu, Wenjiang; Liu, Ning; Ma, Lin

    2018-05-01

    Planar laser induced fluorescence (PLIF) represents an indispensable tool for flow and flame imaging. However, the PLIF technique suffers from limited spatial resolution or blurring in many situations, which restricts its applicability and capability. This work describes a new method, named SR-PLIF (super-resolution PLIF), to overcome these limitations and enhance the capability of PLIF. The method uses PLIF images captured simultaneously from two (or more) orientations to reconstruct a final PLIF image with resolution enhanced or blurring removed. This paper reports the development of the reconstruction algorithm, and the experimental demonstration of the SR-PLIF method both with controlled samples and with turbulent flows seeded with iodine vapor. Using controlled samples with two cameras, the spatial resolution in the best case was improved from 0.06 mm in the projections to 0.03 mm in the SR image, in terms of the spreading width of a sharp edge. With turbulent flows, an image sharpness measure was developed to quantify the spatial resolution, and SR reconstruction with two cameras can effectively improve the spatial resolution compared to the projections in terms of the sharpness measure.

  8. 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

  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. Super-resolution with an optically-addressable liquid crystal spatial light modulator

    International Nuclear Information System (INIS)

    McOrist, J.; Sharma, M.D.; Sheppard, C.J.R.

    2002-01-01

    Full text: An optically-addressable liquid crystal spatial light modulator has been used to generate super-resolving masks. This approach avoids problems of low efficiency and coupling between amplitude and phase modulation, that occur when using conventional liquid crystal modulators. When addressed by a programmed light intensity distribution, it allows filters to be changed rapidly to modify the response of a system or permit the investigation of different filter designs. The device used is not pixellated, with a spatial resolution of 30 line pairs/mm over an area 18mm X 18mm, and can achieve continuously-variable phase modulation up to 1.5 wavelengths. The system consists of a write-beam that is collimated from a white-light source. An input mask was used in our experiments determines the modulation pattern of the read-beam. The read-beam from a HeNe laser reflects from the modulator and is focused by a microscope objective. The value of the phase change induced by the transparent regions of the mask can be altered continuously by adjusting the brightness of the write-beam. We have used this system to attain super-resolution by simple Toraldo filters, consisting of arrays of rings. Copyright (2002) Australian Society for Electron Microscopy Inc

  11. Structural analysis of herpes simplex virus by optical super-resolution imaging

    Science.gov (United States)

    Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

    2015-01-01

    Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

  12. Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.

    Science.gov (United States)

    Ma, Jiong; Kelich, Joseph M; Junod, Samuel L; Yang, Weidong

    2017-04-01

    The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. © 2017. Published by The Company of Biologists Ltd.

  13. Super-resolution for asymmetric resolution of FIB-SEM 3D imaging using AI with deep learning.

    Science.gov (United States)

    Hagita, Katsumi; Higuchi, Takeshi; Jinnai, Hiroshi

    2018-04-12

    Scanning electron microscopy equipped with a focused ion beam (FIB-SEM) is a promising three-dimensional (3D) imaging technique for nano- and meso-scale morphologies. In FIB-SEM, the specimen surface is stripped by an ion beam and imaged by an SEM installed orthogonally to the FIB. The lateral resolution is governed by the SEM, while the depth resolution, i.e., the FIB milling direction, is determined by the thickness of the stripped thin layer. In most cases, the lateral resolution is superior to the depth resolution; hence, asymmetric resolution is generated in the 3D image. Here, we propose a new approach based on an image-processing or deep-learning-based method for super-resolution of 3D images with such asymmetric resolution, so as to restore the depth resolution to achieve symmetric resolution. The deep-learning-based method learns from high-resolution sub-images obtained via SEM and recovers low-resolution sub-images parallel to the FIB milling direction. The 3D morphologies of polymeric nano-composites are used as test images, which are subjected to the deep-learning-based method as well as conventional methods. We find that the former yields superior restoration, particularly as the asymmetric resolution is increased. Our super-resolution approach for images having asymmetric resolution enables observation time reduction.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

    Directory of Open Access Journals (Sweden)

    Fabiana A Caetano

    2015-12-01

    Full Text Available Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

  19. 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

  20. 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

  1. 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

  2. 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...

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.)

  8. 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.

  9. PALM and STORM: unlocking live-cell super-resolution

    CSIR Research Space (South Africa)

    Henriques, R

    2011-05-01

    Full Text Available microscopy methods such as PALM and STORM to achieve this important goal. ? 2011 Wiley Periodicals, Inc. Biopolymers 95: 322?331, 2011. http://onlinelibrary.wiley.com/doi/10.1002/bip.21586/abstract ...

  10. Introduction to the virtual special issue on super-resolution imaging techniques

    Science.gov (United States)

    Cao, Liangcai; Liu, Zhengjun

    2017-12-01

    Until quite recently, the resolution of optical imaging instruments, including telescopes, cameras and microscopes, was considered to be limited by the diffraction of light and by image sensors. In the past few years, many exciting super-resolution approaches have emerged that demonstrate intriguing ways to bypass the classical limit in optics and detectors. More and more research groups are engaged in the study of advanced super-resolution schemes, devices, algorithms, systems, and applications [1-6]. Super-resolution techniques involve new methods in science and engineering of optics [7,8], measurements [9,10], chemistry [11,12] and information [13,14]. Promising applications, particularly in biomedical research and semiconductor industry, have been successfully demonstrated.

  11. Signal Characteristics of Super-Resolution Near-Field Structure Disks with 100 GB Capacity

    Science.gov (United States)

    Kim, Jooho; Hwang, Inoh; Kim, Hyunki; Park, Insik; Tominaga, Junji

    2005-05-01

    We report the basic characteristics of super resolution near-field structure (Super-RENS) media at a blue laser optical system (laser wavelength 405 nm, numerical aperture 0.85). Using a novel write once read many (WORM) structure for a blue laser system, we obtained a carrier-to-noise ratio (CNR) above 33 dB from the signal of the 37.5 nm mark length, which is equivalent to a 100 GB capacity with a 0.32 micrometer track pitch, and an eye pattern for 50 GB (2T: 75 nm) capacity using a patterned signal. Using a novel super-resolution material (tellurium, Te) with low super-resolution readout power, we also improved the read stability.

  12. Magnetic Resonance Super-resolution Imaging Measurement with Dictionary-optimized Sparse Learning

    Directory of Open Access Journals (Sweden)

    Li Jun-Bao

    2017-06-01

    Full Text Available Magnetic Resonance Super-resolution Imaging Measurement (MRIM is an effective way of measuring materials. MRIM has wide applications in physics, chemistry, biology, geology, medical and material science, especially in medical diagnosis. It is feasible to improve the resolution of MR imaging through increasing radiation intensity, but the high radiation intensity and the longtime of magnetic field harm the human body. Thus, in the practical applications the resolution of hardware imaging reaches the limitation of resolution. Software-based super-resolution technology is effective to improve the resolution of image. This work proposes a framework of dictionary-optimized sparse learning based MR super-resolution method. The framework is to solve the problem of sample selection for dictionary learning of sparse reconstruction. The textural complexity-based image quality representation is proposed to choose the optimal samples for dictionary learning. Comprehensive experiments show that the dictionary-optimized sparse learning improves the performance of sparse representation.

  13. Single Image Super-Resolution Based on Multi-Scale Competitive Convolutional Neural Network.

    Science.gov (United States)

    Du, Xiaofeng; Qu, Xiaobo; He, Yifan; Guo, Di

    2018-03-06

    Deep convolutional neural networks (CNNs) are successful in single-image super-resolution. Traditional CNNs are limited to exploit multi-scale contextual information for image reconstruction due to the fixed convolutional kernel in their building modules. To restore various scales of image details, we enhance the multi-scale inference capability of CNNs by introducing competition among multi-scale convolutional filters, and build up a shallow network under limited computational resources. The proposed network has the following two advantages: (1) the multi-scale convolutional kernel provides the multi-context for image super-resolution, and (2) the maximum competitive strategy adaptively chooses the optimal scale of information for image reconstruction. Our experimental results on image super-resolution show that the performance of the proposed network outperforms the state-of-the-art methods.

  14. Multiband super-resolution imaging of graded-index photonic crystal flat lens

    Science.gov (United States)

    Xie, Jianlan; Wang, Junzhong; Ge, Rui; Yan, Bei; Liu, Exian; Tan, Wei; Liu, Jianjun

    2018-05-01

    Multiband super-resolution imaging of point source is achieved by a graded-index photonic crystal flat lens. With the calculations of six bands in common photonic crystal (CPC) constructed with scatterers of different refractive indices, it can be found that the super-resolution imaging of point source can be realized by different physical mechanisms in three different bands. In the first band, the imaging of point source is based on far-field condition of spherical wave while in the second band, it is based on the negative effective refractive index and exhibiting higher imaging quality than that of the CPC. However, in the fifth band, the imaging of point source is mainly based on negative refraction of anisotropic equi-frequency surfaces. The novel method of employing different physical mechanisms to achieve multiband super-resolution imaging of point source is highly meaningful for the field of imaging.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  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. Comparison of super-resolution benefits for downsampled iages and real low-resolution data

    NARCIS (Netherlands)

    Peng, Y.; Spreeuwers, Lieuwe Jan; Gökberk, B.; Veldhuis, Raymond N.J.

    2013-01-01

    Recently, more and more researchers are exploring the benefits of super-resolution methods on low-resolution face recognition. However, often results presented are obtained on downsampled high-resolution face images. Because downsampled images are different from real images taken at low resolution,

  3. Texton-based super-resolution for achieving high spatiotemporal resolution in hybrid camera system

    Science.gov (United States)

    Kamimura, Kenji; Tsumura, Norimichi; Nakaguchi, Toshiya; Miyake, Yoichi

    2010-05-01

    Many super-resolution methods have been proposed to enhance the spatial resolution of images by using iteration and multiple input images. In a previous paper, we proposed the example-based super-resolution method to enhance an image through pixel-based texton substitution to reduce the computational cost. In this method, however, we only considered the enhancement of a texture image. In this study, we modified this texton substitution method for a hybrid camera to reduce the required bandwidth of a high-resolution video camera. We applied our algorithm to pairs of high- and low-spatiotemporal-resolution videos, which were synthesized to simulate a hybrid camera. The result showed that the fine detail of the low-resolution video can be reproduced compared with bicubic interpolation and the required bandwidth could be reduced to about 1/5 in a video camera. It was also shown that the peak signal-to-noise ratios (PSNRs) of the images improved by about 6 dB in a trained frame and by 1.0-1.5 dB in a test frame, as determined by comparison with the processed image using bicubic interpolation, and the average PSNRs were higher than those obtained by the well-known Freeman’s patch-based super-resolution method. Compared with that of the Freeman’s patch-based super-resolution method, the computational time of our method was reduced to almost 1/10.

  4. Super-resolution links vinculin localization to function in focal adhesions.

    Science.gov (United States)

    Giannone, Grégory

    2015-07-01

    Integrin-based focal adhesions integrate biochemical and biomechanical signals from the extracellular matrix and the actin cytoskeleton. The combination of three-dimensional super-resolution imaging and loss- or gain-of-function protein mutants now links the nanoscale dynamic localization of proteins to their activation and function within focal adhesions.

  5. Deterministic phase measurements exhibiting super-sensitivity and super-resolution

    DEFF Research Database (Denmark)

    Schäfermeier, Clemens; Ježek, Miroslav; Madsen, Lars S.

    2018-01-01

    Phase super-sensitivity is obtained when the sensitivity in a phase measurement goes beyond the quantum shot noise limit, whereas super-resolution is obtained when the interference fringes in an interferometer are narrower than half the input wavelength. Here we show experimentally that these two...

  6. Low-Power Super-resolution Readout with Antimony Bismuth Alloy Film as Mask layer

    International Nuclear Information System (INIS)

    Lai-Xin, Jiang; Yi-Qun, Wu; Yang, Wang; Jing-Song, Wei; Fu-Xi, Gan

    2009-01-01

    Sb–Bi alloy films are proposed as a new kind of super-resolution mask layer with low readout threshold power. Using the Sb–Bi alloy film as a mask layer and SiN as a protective layer in a read-only memory disc, the super-resolution pits with diameters of 380 nm are read out by a dynamic setup, the laser wavelength is 780 nm and the numerical aperture of pickup lens is 0.45. The effects of the Sb–Bi thin film thickness, laser readout power and disc rotating velocity on the readout signal are investigated. The results show that the threshold laser power of super-resolution readout of the Sb–Bi mask layer is about 0.5 mW, and the corresponding carrier-to-noise ratio is about 20 dB at the film thickness of 50 nm. The super-resolution mechanism of the Sb–Bi alloy mask layer is discussed based on its temperature dependence of reflection

  7. Low-Cost Super-Resolution Algorithms Implementation Over a HW/SW Video Compression Platform

    Directory of Open Access Journals (Sweden)

    Llopis Rafael Peset

    2006-01-01

    Full Text Available Two approaches are presented in this paper to improve the quality of digital images over the sensor resolution using super-resolution techniques: iterative super-resolution (ISR and noniterative super-resolution (NISR algorithms. The results show important improvements in the image quality, assuming that sufficient sample data and a reasonable amount of aliasing are available at the input images. These super-resolution algorithms have been implemented over a codesign video compression platform developed by Philips Research, performing minimal changes on the overall hardware architecture. In this way, a novel and feasible low-cost implementation has been obtained by using the resources encountered in a generic hybrid video encoder. Although a specific video codec platform has been used, the methodology presented in this paper is easily extendable to any other video encoder architectures. Finally a comparison in terms of memory, computational load, and image quality for both algorithms, as well as some general statements about the final impact of the sampling process on the quality of the super-resolved (SR image, are also presented.

  8. Evaluation of deep neural networks for single image super-resolution in a maritime context

    NARCIS (Netherlands)

    Nieuwenhuizen, R.P.J.; Kruithof, M.; Schutte, K.

    2017-01-01

    High resolution imagery is of crucial importance for the performance on visual recognition tasks. Super-resolution (SR) reconstruction algorithms aim to enhance the image resolution beyond the capability of the image sensor being used. Traditional SR algorithms approach this inverse problem using

  9. A super-resolution approach for uncertainty estimation of PIV measurements

    NARCIS (Netherlands)

    Sciacchitano, A.; Wieneke, B.; Scarano, F.

    2012-01-01

    A super-resolution approach is proposed for the a posteriori uncertainty estimation of PIV measurements. The measured velocity field is employed to determine the displacement of individual particle images. A disparity set is built from the residual distance between paired particle images of

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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)

  16. Super-resolution biomolecular crystallography with low-resolution data.

    Science.gov (United States)

    Schröder, Gunnar F; Levitt, Michael; Brunger, Axel T

    2010-04-22

    -ray crystallography and cryo-electron microscopy: as optical imaging advances to subnanometre resolution, it can use similar tools.

  17. Advanced Microscopy of Microbial Cells

    DEFF Research Database (Denmark)

    Haagensen, Janus Anders Juul; Regenberg, Birgitte; Sternberg, Claus

    2011-01-01

    microscopy, super-resolution optical microscopy (STED, SIM, PALM) as well as atomic force microscopy and Raman spectroscopy. Using examples of bistability in microbial populations as well as biofilm development and differentiation in bacterial and yeast consortia, we demonstrate the importance of microscopy...

  18. Simulation of Far-Field Superresolution Fluorescence Imaging with Two-Color One-Photon Excitation of Reversible Photoactivatable Protein

    International Nuclear Information System (INIS)

    Wang Chen; Qiao Ling-Ling; Mao Zheng-Le

    2011-01-01

    We propose to achieve far-field super-resolution imaging by using offset two-color one-photon (2C1P) excitation of reversible photoactivatable fluorescence proteins. Due to the distinctive photoswitching performance of the proteins, such as dronpa, the fluorescence emission will only come from the overlapped region of activation beam and excitation beam. The analysis solution of rate equation shows that the resolution of offset 2C1P microscope is 'engineered' by laser power of excitation and activation beams and the power ratio between them. Superior lateral and transverse resolution is theoretically demonstrated compared with conventional fluorescence scanning microscopy. (fundamental areas of phenomenology(including applications))

  19. 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.

  20. Super-resolution using a light inception layer in convolutional neural network

    Science.gov (United States)

    Mou, Qinyang; Guo, Jun

    2018-04-01

    Recently, several models based on CNN architecture have achieved great result on Single Image Super-Resolution (SISR) problem. In this paper, we propose an image super-resolution method (SR) using a light inception layer in convolutional network (LICN). Due to the strong representation ability of our well-designed inception layer that can learn richer representation with less parameters, we can build our model with shallow architecture that can reduce the effect of vanishing gradients problem and save computational costs. Our model strike a balance between computational speed and the quality of the result. Compared with state-of-the-art result, we produce comparable or better results with faster computational speed.

  1. Optical super-resolution effect induced by nonlinear characteristics of graphene oxide films

    Science.gov (United States)

    Zhao, Yong-chuang; Nie, Zhong-quan; Zhai, Ai-ping; Tian, Yan-ting; Liu, Chao; Shi, Chang-kun; Jia, Bao-hua

    2018-01-01

    In this work, we focus on the optical super-resolution effect induced by strong nonlinear saturation absorption (NSA) of graphene oxide (GO) membranes. The third-order optical nonlinearities are characterized by the canonical Z-scan technique under femtosecond laser (wavelength: 800 nm, pulse width: 100 fs) excitation. Through controlling the applied femtosecond laser energy, NSA of the GO films can be tuned continuously. The GO film is placed at the focal plane as a unique amplitude filter to improve the resolution of the focused field. A multi-layer system model is proposed to present the generation of a deep sub-wavelength spot associated with the nonlinearity of GO films. Moreover, the parameter conditions to achieve the best resolution (˜λ/6) are determined entirely. The demonstrated results here are useful for high density optical recoding and storage, nanolithography, and super-resolution optical imaging.

  2. Multiple-image hiding using super resolution reconstruction in high-frequency domains

    Science.gov (United States)

    Li, Xiao-Wei; Zhao, Wu-Xiang; Wang, Jun; Wang, Qiong-Hua

    2017-12-01

    In this paper, a robust multiple-image hiding method using the computer-generated integral imaging and the modified super-resolution reconstruction algorithm is proposed. In our work, the host image is first transformed into frequency domains by cellular automata (CA), to assure the quality of the stego-image, the secret images are embedded into the CA high-frequency domains. The proposed method has the following advantages: (1) robustness to geometric attacks because of the memory-distributed property of elemental images, (2) increasing quality of the reconstructed secret images as the scheme utilizes the modified super-resolution reconstruction algorithm. The simulation results show that the proposed multiple-image hiding method outperforms other similar hiding methods and is robust to some geometric attacks, e.g., Gaussian noise and JPEG compression attacks.

  3. Single image super-resolution via regularized extreme learning regression for imagery from microgrid polarimeters

    Science.gov (United States)

    Sargent, Garrett C.; Ratliff, Bradley M.; Asari, Vijayan K.

    2017-08-01

    The advantage of division of focal plane imaging polarimeters is their ability to obtain temporally synchronized intensity measurements across a scene; however, they sacrifice spatial resolution in doing so due to their spatially modulated arrangement of the pixel-to-pixel polarizers and often result in aliased imagery. Here, we propose a super-resolution method based upon two previously trained extreme learning machines (ELM) that attempt to recover missing high frequency and low frequency content beyond the spatial resolution of the sensor. This method yields a computationally fast and simple way of recovering lost high and low frequency content from demosaicing raw microgrid polarimetric imagery. The proposed method outperforms other state-of-the-art single-image super-resolution algorithms in terms of structural similarity and peak signal-to-noise ratio.

  4. Application of regularization technique in image super-resolution algorithm via sparse representation

    Science.gov (United States)

    Huang, De-tian; Huang, Wei-qin; Huang, Hui; Zheng, Li-xin

    2017-11-01

    To make use of the prior knowledge of the image more effectively and restore more details of the edges and structures, a novel sparse coding objective function is proposed by applying the principle of the non-local similarity and manifold learning on the basis of super-resolution algorithm via sparse representation. Firstly, the non-local similarity regularization term is constructed by using the similar image patches to preserve the edge information. Then, the manifold learning regularization term is constructed by utilizing the locally linear embedding approach to enhance the structural information. The experimental results validate that the proposed algorithm has a significant improvement compared with several super-resolution algorithms in terms of the subjective visual effect and objective evaluation indices.

  5. 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.

  6. 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.

  7. 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.

  8. A simple model explaining super-resolution in absolute optical instruments

    Science.gov (United States)

    Leonhardt, Ulf; Sahebdivan, Sahar; Kogan, Alex; Tyc, Tomáš

    2015-05-01

    We develop a simple, one-dimensional model for super-resolution in absolute optical instruments that is able to describe the interplay between sources and detectors. Our model explains the subwavelength sensitivity of a point detector to a point source reported in previous computer simulations and experiments (Miñano 2011 New J. Phys.13 125009; Miñano 2014 New J. Phys.16 033015).

  9. Windowed time-reversal music technique for super-resolution ultrasound imaging

    Science.gov (United States)

    Huang, Lianjie; Labyed, Yassin

    2018-05-01

    Systems and methods for super-resolution ultrasound imaging using a windowed and generalized TR-MUSIC algorithm that divides the imaging region into overlapping sub-regions and applies the TR-MUSIC algorithm to the windowed backscattered ultrasound signals corresponding to each sub-region. The algorithm is also structured to account for the ultrasound attenuation in the medium and the finite-size effects of ultrasound transducer elements.

  10. 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

  11. Super-resolution for a point source better than λ/500 using positive refraction

    International Nuclear Information System (INIS)

    Miñano, Juan C; González, Juan C; Benítez, Pablo; Grabovickic, Dejan; Marqués, Ricardo; Delgado, Vicente; Freire, Manuel

    2011-01-01

    Leonhardt (2009 New J. Phys. 11 093040) demonstrated that the two-dimensional (2D) Maxwell fish eye (MFE) lens can focus perfectly 2D Helmholtz waves of arbitrary frequency; that is, it can transport perfectly an outward (monopole) 2D Helmholtz wave field, generated by a point source, towards a ‘perfect point drain’ located at the corresponding image point. Moreover, a prototype with λ/5 super-resolution property for one microwave frequency has been manufactured and tested (Ma et al 2010 arXiv:1007.2530v1; Ma et al 2010 New J. Phys. 13 033016). However, neither software simulations nor experimental measurements for a broad band of frequencies have yet been reported. Here, we present steady-state simulations with a non-perfect drain for a device equivalent to the MFE, called the spherical geodesic waveguide (SGW), which predicts up to λ/500 super-resolution close to discrete frequencies. Out of these frequencies, the SGW does not show super-resolution in the analysis carried out. (paper)

  12. A Novel Method to Implement the Matrix Pencil Super Resolution Algorithm for Indoor Positioning

    Directory of Open Access Journals (Sweden)

    Tariq Jamil Saifullah Khanzada

    2011-10-01

    Full Text Available This article highlights the estimation of the results for the algorithms implemented in order to estimate the delays and distances for the indoor positioning system. The data sets for the transmitted and received signals are captured at a typical outdoor and indoor area. The estimation super resolution algorithms are applied. Different state of art and super resolution techniques based algorithms are applied to avail the optimal estimates of the delays and distances between the transmitted and received signals and a novel method for matrix pencil algorithm is devised. The algorithms perform variably at different scenarios of transmitted and received positions. Two scenarios are experienced, for the single antenna scenario the super resolution techniques like ESPRIT (Estimation of Signal Parameters via Rotational Invariance Technique and theMatrix Pencil algorithms give optimal performance compared to the conventional techniques. In two antenna scenario RootMUSIC and Matrix Pencil algorithm performed better than other algorithms for the distance estimation, however, the accuracy of all the algorithms is worst than the single antenna scenario. In all cases our devised Matrix Pencil algorithm achieved the best estimation results.

  13. Super-resolution for a point source better than λ/500 using positive refraction

    Science.gov (United States)

    Miñano, Juan C.; Marqués, Ricardo; González, Juan C.; Benítez, Pablo; Delgado, Vicente; Grabovickic, Dejan; Freire, Manuel

    2011-12-01

    Leonhardt (2009 New J. Phys. 11 093040) demonstrated that the two-dimensional (2D) Maxwell fish eye (MFE) lens can focus perfectly 2D Helmholtz waves of arbitrary frequency; that is, it can transport perfectly an outward (monopole) 2D Helmholtz wave field, generated by a point source, towards a ‘perfect point drain’ located at the corresponding image point. Moreover, a prototype with λ/5 super-resolution property for one microwave frequency has been manufactured and tested (Ma et al 2010 arXiv:1007.2530v1; Ma et al 2010 New J. Phys. 13 033016). However, neither software simulations nor experimental measurements for a broad band of frequencies have yet been reported. Here, we present steady-state simulations with a non-perfect drain for a device equivalent to the MFE, called the spherical geodesic waveguide (SGW), which predicts up to λ/500 super-resolution close to discrete frequencies. Out of these frequencies, the SGW does not show super-resolution in the analysis carried out.

  14. Large-area super-resolution optical imaging by using core-shell microfibers

    Science.gov (United States)

    Liu, Cheng-Yang; Lo, Wei-Chieh

    2017-09-01

    We first numerically and experimentally report large-area super-resolution optical imaging achieved by using core-shell microfibers. The particular spatial electromagnetic waves for different core-shell microfibers are studied by using finite-difference time-domain and ray tracing calculations. The focusing properties of photonic nanojets are evaluated in terms of intensity profile and full width at half-maximum along propagation and transversal directions. In experiment, the general optical fiber is chemically etched down to 6 μm diameter and coated with different metallic thin films by using glancing angle deposition. The direct imaging of photonic nanojets for different core-shell microfibers is performed with a scanning optical microscope system. We show that the intensity distribution of a photonic nanojet is highly related to the metallic shell due to the surface plasmon polaritons. Furthermore, large-area super-resolution optical imaging is performed by using different core-shell microfibers placed over the nano-scale grating with 150 nm line width. The core-shell microfiber-assisted imaging is achieved with super-resolution and hundreds of times the field-of-view in contrast to microspheres. The possible applications of these core-shell optical microfibers include real-time large-area micro-fluidics and nano-structure inspections.

  15. Multi-frame super-resolution with quality self-assessment for retinal fundus videos.

    Science.gov (United States)

    Köhler, Thomas; Brost, Alexander; Mogalle, Katja; Zhang, Qianyi; Köhler, Christiane; Michelson, Georg; Hornegger, Joachim; Tornow, Ralf P

    2014-01-01

    This paper proposes a novel super-resolution framework to reconstruct high-resolution fundus images from multiple low-resolution video frames in retinal fundus imaging. Natural eye movements during an examination are used as a cue for super-resolution in a robust maximum a-posteriori scheme. In order to compensate heterogeneous illumination on the fundus, we integrate retrospective illumination correction for photometric registration to the underlying imaging model. Our method utilizes quality self-assessment to provide objective quality scores for reconstructed images as well as to select regularization parameters automatically. In our evaluation on real data acquired from six human subjects with a low-cost video camera, the proposed method achieved considerable enhancements of low-resolution frames and improved noise and sharpness characteristics by 74%. In terms of image analysis, we demonstrate the importance of our method for the improvement of automatic blood vessel segmentation as an example application, where the sensitivity was increased by 13% using super-resolution reconstruction.

  16. Super-resolution processing for pulsed neutron imaging system using a high-speed camera

    International Nuclear Information System (INIS)

    Ishizuka, Ken; Kai, Tetsuya; Shinohara, Takenao; Segawa, Mariko; Mochiki, Koichi

    2015-01-01

    Super-resolution and center-of-gravity processing improve the resolution of neutron-transmitted images. These processing methods calculate the center-of-gravity pixel or sub-pixel of the neutron point converted into light by a scintillator. The conventional neutron-transmitted image is acquired using a high-speed camera by integrating many frames when a transmitted image with one frame is not provided. It succeeds in acquiring the transmitted image and calculating a spectrum by integrating frames of the same energy. However, because a high frame rate is required for neutron resonance absorption imaging, the number of pixels of the transmitted image decreases, and the resolution decreases to the limit of the camera performance. Therefore, we attempt to improve the resolution by integrating the frames after applying super-resolution or center-of-gravity processing. The processed results indicate that center-of-gravity processing can be effective in pulsed-neutron imaging with a high-speed camera. In addition, the results show that super-resolution processing is effective indirectly. A project to develop a real-time image data processing system has begun, and this system will be used at J-PARC in JAEA. (author)

  17. 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...

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti

    CSIR Research Space (South Africa)

    Scholefield, Janine

    2016-09-01

    Full Text Available Boucharlat4, Emmanuel Laplantine5, Asma Smahi6, Alain Israe¨l5, Fabrice Agou4 & Musa M. Mhlanga1,2,7 The NF-kB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved...

  4. Restoration and Super-Resolution of Diffraction-Limited Imagery Data by Bayesian and Set-Theoretic Approaches

    National Research Council Canada - National Science Library

    Sundareshan, Malur

    2001-01-01

    This project was primarily aimed at the design of novel algorithms for the restoration and super-resolution processing of imagery data to improve the resolution in images acquired from practical sensing operations...

  5. 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.

  6. 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.

  7. 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.

  8. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

    Science.gov (United States)

    Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

    2014-05-01

    Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  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. Super-resolution by elliptical bubble formation with PtOx and AgInSbTe layers

    International Nuclear Information System (INIS)

    Kim, Jooho; Hwang, Inoh; Yoon, Duseop; Park, Insik; Shin, Dongho; Kikukawa, Takashi; Shima, Takayuki; Tominaga, Junji

    2003-01-01

    The recording and retrieval of signals below 100 nm mark length were attempted with elliptical bubble-type super-resolution technology with platinum oxide (PtO x ) and ductile AgInSbTe layers, using the same optical system as that of a digital versatile disk (a 635 nm wavelength red laser system). The carrier-to-noise ratio (CNR) of over 47 dB for 100 nm mark length signals (over 43 dB for 80 nm mark length signals) was obtained, which can be considered as a commercially acceptable level of CNR. The recording mechanism of the sample disk was shown through the transmission electron microscopy cross-section image observation to be by rigid elliptical bubble formation at the PtO x layer located between the AgInSbTe layers. The results of this report represent the potential for a much higher-density storage using the red laser system and a subterabyte optical storage using the blue laser system

  13. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-19

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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Ordinal Regression Based Subpixel Shift Estimation for Video Super-Resolution

    Directory of Open Access Journals (Sweden)

    Petrovic Nemanja

    2007-01-01

    Full Text Available We present a supervised learning-based approach for subpixel motion estimation which is then used to perform video super-resolution. The novelty of this work is the formulation of the problem of subpixel motion estimation in a ranking framework. The ranking formulation is a variant of classification and regression formulation, in which the ordering present in class labels namely, the shift between patches is explicitly taken into account. Finally, we demonstrate the applicability of our approach on superresolving synthetically generated images with global subpixel shifts and enhancing real video frames by accounting for both local integer and subpixel shifts.

  2. Accelerating cross-validation with total variation and its application to super-resolution imaging.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Obuchi

    Full Text Available We develop an approximation formula for the cross-validation error (CVE of a sparse linear regression penalized by ℓ1-norm and total variation terms, which is based on a perturbative expansion utilizing the largeness of both the data dimensionality and the model. The developed formula allows us to reduce the necessary computational cost of the CVE evaluation significantly. The practicality of the formula is tested through application to simulated black-hole image reconstruction on the event-horizon scale with super resolution. The results demonstrate that our approximation reproduces the CVE values obtained via literally conducted cross-validation with reasonably good precision.

  3. Video-to-Video Dynamic Super-Resolution for Grayscale and Color Sequences

    Directory of Open Access Journals (Sweden)

    Elad Michael

    2006-01-01

    Full Text Available We address the dynamic super-resolution (SR problem of reconstructing a high-quality set of monochromatic or color super-resolved images from low-quality monochromatic, color, or mosaiced frames. Our approach includes a joint method for simultaneous SR, deblurring, and demosaicing, this way taking into account practical color measurements encountered in video sequences. For the case of translational motion and common space-invariant blur, the proposed method is based on a very fast and memory efficient approximation of the Kalman filter (KF. Experimental results on both simulated and real data are supplied, demonstrating the presented algorithms, and their strength.

  4. Super-resolution with a positive epsilon multi-quantum-well super-lens

    International Nuclear Information System (INIS)

    Bak, A. O.; Giannini, V.; Maier, S. A.; Phillips, C. C.

    2013-01-01

    We design an anisotropic and dichroic quantum metamaterial that is able to achieve super-resolution without the need for a negative permittivity. When exploring the parameters of the structure, we take into account the limits of semiconductor fabrication technology based on quantum well stacks. By heavily doping the structure with free electrons, we infer an anisotropic effective medium with a prolate ellipsoid dispersion curve which allows for near-diffractionless propagation of light (similar to an epsilon-near-zero hyperbolic lens). This, coupled with low absorption, allows us to resolve images at the sub-wavelength scale at distances 6 times greater than equivalent natural materials

  5. Time reversal and phase coherent music techniques for super-resolution ultrasound imaging

    Science.gov (United States)

    Huang, Lianjie; Labyed, Yassin

    2018-05-01

    Systems and methods for super-resolution ultrasound imaging using a windowed and generalized TR-MUSIC algorithm that divides the imaging region into overlapping sub-regions and applies the TR-MUSIC algorithm to the windowed backscattered ultrasound signals corresponding to each sub-region. The algorithm is also structured to account for the ultrasound attenuation in the medium and the finite-size effects of ultrasound transducer elements. A modified TR-MUSIC imaging algorithm is used to account for ultrasound scattering from both density and compressibility contrasts. The phase response of ultrasound transducer elements is accounted for in a PC-MUSIC system.

  6. 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.

  7. 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.

  8. Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

    Science.gov (United States)

    Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

    2012-10-01

    The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

  9. 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.

  10. 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.

  11. 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...

  12. Stimulated emission depletion microscopy resolves individual nitrogen vacancy centers in diamond nanocrystals.

    Science.gov (United States)

    Arroyo-Camejo, Silvia; Adam, Marie-Pierre; Besbes, Mondher; Hugonin, Jean-Paul; Jacques, Vincent; Greffet, Jean-Jacques; Roch, Jean-François; Hell, Stefan W; Treussart, François

    2013-12-23

    Nitrogen-vacancy (NV) color centers in nanodiamonds are highly promising for bioimaging and sensing. However, resolving individual NV centers within nanodiamond particles and the controlled addressing and readout of their spin state has remained a major challenge. Spatially stochastic super-resolution techniques cannot provide this capability in principle, whereas coordinate-controlled super-resolution imaging methods, like stimulated emission depletion (STED) microscopy, have been predicted to fail in nanodiamonds. Here we show that, contrary to these predictions, STED can resolve single NV centers in 40-250 nm sized nanodiamonds with a resolution of ≈10 nm. Even multiple adjacent NVs located in single nanodiamonds can be imaged individually down to relative distances of ≈15 nm. Far-field optical super-resolution of NVs inside nanodiamonds is highly relevant for bioimaging applications of these fluorescent nanolabels. The targeted addressing and readout of individual NV(-) spins inside nanodiamonds by STED should also be of high significance for quantum sensing and information applications.

  13. 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.

  14. Resolution enhancement of tri-stereo remote sensing images by super resolution methods

    Science.gov (United States)

    Tuna, Caglayan; Akoguz, Alper; Unal, Gozde; Sertel, Elif

    2016-10-01

    Super resolution (SR) refers to generation of a High Resolution (HR) image from a decimated, blurred, low-resolution (LR) image set, which can be either a single frame or multi-frame that contains a collection of several images acquired from slightly different views of the same observation area. In this study, we propose a novel application of tri-stereo Remote Sensing (RS) satellite images to the super resolution problem. Since the tri-stereo RS images of the same observation area are acquired from three different viewing angles along the flight path of the satellite, these RS images are properly suited to a SR application. We first estimate registration between the chosen reference LR image and other LR images to calculate the sub pixel shifts among the LR images. Then, the warping, blurring and down sampling matrix operators are created as sparse matrices to avoid high memory and computational requirements, which would otherwise make the RS-SR solution impractical. Finally, the overall system matrix, which is constructed based on the obtained operator matrices is used to obtain the estimate HR image in one step in each iteration of the SR algorithm. Both the Laplacian and total variation regularizers are incorporated separately into our algorithm and the results are presented to demonstrate an improved quantitative performance against the standard interpolation method as well as improved qualitative results due expert evaluations.

  15. MAP-MRF-Based Super-Resolution Reconstruction Approach for Coded Aperture Compressive Temporal Imaging

    Directory of Open Access Journals (Sweden)

    Tinghua Zhang

    2018-02-01

    Full Text Available Coded Aperture Compressive Temporal Imaging (CACTI can afford low-cost temporal super-resolution (SR, but limits are imposed by noise and compression ratio on reconstruction quality. To utilize inter-frame redundant information from multiple observations and sparsity in multi-transform domains, a robust reconstruction approach based on maximum a posteriori probability and Markov random field (MAP-MRF model for CACTI is proposed. The proposed approach adopts a weighted 3D neighbor system (WNS and the coordinate descent method to perform joint estimation of model parameters, to achieve the robust super-resolution reconstruction. The proposed multi-reconstruction algorithm considers both total variation (TV and ℓ 2 , 1 norm in wavelet domain to address the minimization problem for compressive sensing, and solves it using an accelerated generalized alternating projection algorithm. The weighting coefficient for different regularizations and frames is resolved by the motion characteristics of pixels. The proposed approach can provide high visual quality in the foreground and background of a scene simultaneously and enhance the fidelity of the reconstruction results. Simulation results have verified the efficacy of our new optimization framework and the proposed reconstruction approach.

  16. Super-Resolution Reconstruction of Remote Sensing Images Using Multifractal Analysis

    Directory of Open Access Journals (Sweden)

    Mao-Gui Hu

    2009-10-01

    Full Text Available Satellite remote sensing (RS is an important contributor to Earth observation, providing various kinds of imagery every day, but low spatial resolution remains a critical bottleneck in a lot of applications, restricting higher spatial resolution analysis (e.g., intraurban. In this study, a multifractal-based super-resolution reconstruction method is proposed to alleviate this problem. The multifractal characteristic is common in Nature. The self-similarity or self-affinity presented in the image is useful to estimate details at larger and smaller scales than the original. We first look for the presence of multifractal characteristics in the images. Then we estimate parameters of the information transfer function and noise of the low resolution image. Finally, a noise-free, spatial resolutionenhanced image is generated by a fractal coding-based denoising and downscaling method. The empirical case shows that the reconstructed super-resolution image performs well indetail enhancement. This method is not only useful for remote sensing in investigating Earth, but also for other images with multifractal characteristics.

  17. Multi-example feature-constrained back-projection method for image super-resolution

    Institute of Scientific and Technical Information of China (English)

    Junlei Zhang; Dianguang Gai; Xin Zhang; Xuemei Li

    2017-01-01

    Example-based super-resolution algorithms,which predict unknown high-resolution image information using a relationship model learnt from known high- and low-resolution image pairs, have attracted considerable interest in the field of image processing. In this paper, we propose a multi-example feature-constrained back-projection method for image super-resolution. Firstly, we take advantage of a feature-constrained polynomial interpolation method to enlarge the low-resolution image. Next, we consider low-frequency images of different resolutions to provide an example pair. Then, we use adaptive k NN search to find similar patches in the low-resolution image for every image patch in the high-resolution low-frequency image, leading to a regression model between similar patches to be learnt. The learnt model is applied to the low-resolution high-frequency image to produce high-resolution high-frequency information. An iterative back-projection algorithm is used as the final step to determine the final high-resolution image.Experimental results demonstrate that our method improves the visual quality of the high-resolution image.

  18. Application of Super-Resolution Convolutional Neural Network for Enhancing Image Resolution in Chest CT.

    Science.gov (United States)

    Umehara, Kensuke; Ota, Junko; Ishida, Takayuki

    2017-10-18

    In this study, the super-resolution convolutional neural network (SRCNN) scheme, which is the emerging deep-learning-based super-resolution method for enhancing image resolution in chest CT images, was applied and evaluated using the post-processing approach. For evaluation, 89 chest CT cases were sampled from The Cancer Imaging Archive. The 89 CT cases were divided randomly into 45 training cases and 44 external test cases. The SRCNN was trained using the training dataset. With the trained SRCNN, a high-resolution image was reconstructed from a low-resolution image, which was down-sampled from an original test image. For quantitative evaluation, two image quality metrics were measured and compared to those of the conventional linear interpolation methods. The image restoration quality of the SRCNN scheme was significantly higher than that of the linear interpolation methods (p < 0.001 or p < 0.05). The high-resolution image reconstructed by the SRCNN scheme was highly restored and comparable to the original reference image, in particular, for a ×2 magnification. These results indicate that the SRCNN scheme significantly outperforms the linear interpolation methods for enhancing image resolution in chest CT images. The results also suggest that SRCNN may become a potential solution for generating high-resolution CT images from standard CT images.

  19. LFNet: A Novel Bidirectional Recurrent Convolutional Neural Network for Light-Field Image Super-Resolution.

    Science.gov (United States)

    Wang, Yunlong; Liu, Fei; Zhang, Kunbo; Hou, Guangqi; Sun, Zhenan; Tan, Tieniu

    2018-09-01

    The low spatial resolution of light-field image poses significant difficulties in exploiting its advantage. To mitigate the dependency of accurate depth or disparity information as priors for light-field image super-resolution, we propose an implicitly multi-scale fusion scheme to accumulate contextual information from multiple scales for super-resolution reconstruction. The implicitly multi-scale fusion scheme is then incorporated into bidirectional recurrent convolutional neural network, which aims to iteratively model spatial relations between horizontally or vertically adjacent sub-aperture images of light-field data. Within the network, the recurrent convolutions are modified to be more effective and flexible in modeling the spatial correlations between neighboring views. A horizontal sub-network and a vertical sub-network of the same network structure are ensembled for final outputs via stacked generalization. Experimental results on synthetic and real-world data sets demonstrate that the proposed method outperforms other state-of-the-art methods by a large margin in peak signal-to-noise ratio and gray-scale structural similarity indexes, which also achieves superior quality for human visual systems. Furthermore, the proposed method can enhance the performance of light field applications such as depth estimation.

  20. 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.

  1. 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.

  2. 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.

  3. 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....

  4. 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.

  5. 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.

  6. Stochastic Optical Reconstruction Microscopy (STORM).

    Science.gov (United States)

    Xu, Jianquan; Ma, Hongqiang; Liu, Yang

    2017-07-05

    Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  7. 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.

  8. 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

  9. 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

  10. 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...

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. A novel algorithm of super-resolution image reconstruction based on multi-class dictionaries for natural scene

    Science.gov (United States)

    Wu, Wei; Zhao, Dewei; Zhang, Huan

    2015-12-01

    Super-resolution image reconstruction is an effective method to improve the image quality. It has important research significance in the field of image processing. However, the choice of the dictionary directly affects the efficiency of image reconstruction. A sparse representation theory is introduced into the problem of the nearest neighbor selection. Based on the sparse representation of super-resolution image reconstruction method, a super-resolution image reconstruction algorithm based on multi-class dictionary is analyzed. This method avoids the redundancy problem of only training a hyper complete dictionary, and makes the sub-dictionary more representatives, and then replaces the traditional Euclidean distance computing method to improve the quality of the whole image reconstruction. In addition, the ill-posed problem is introduced into non-local self-similarity regularization. Experimental results show that the algorithm is much better results than state-of-the-art algorithm in terms of both PSNR and visual perception.

  16. 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.

  17. 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.

  18. 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).

  19. 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.

  20. 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.

  1. Convex relaxations of spectral sparsity for robust super-resolution and line spectrum estimation

    Science.gov (United States)

    Chi, Yuejie

    2017-08-01

    We consider recovering the amplitudes and locations of spikes in a point source signal from its low-pass spectrum that may suffer from missing data and arbitrary outliers. We first review and provide a unified view of several recently proposed convex relaxations that characterize and capitalize the spectral sparsity of the point source signal without discretization under the framework of atomic norms. Next we propose a new algorithm when the spikes are known a priori to be positive, motivated by applications such as neural spike sorting and fluorescence microscopy imaging. Numerical experiments are provided to demonstrate the effectiveness of the proposed approach.

  2. Three-dimensional live microscopy beyond the diffraction limit

    International Nuclear Information System (INIS)

    Fiolka, Reto

    2013-01-01

    In fluorescence microscopy it has become possible to fundamentally overcome the diffraction limited resolution in all three spatial dimensions. However, to have the most impact in biological sciences, new optical microscopy techniques need to be compatible with live cell imaging: image acquisition has to be fast enough to capture cellular dynamics at the new resolution limit while light exposure needs to be minimized to prevent photo-toxic effects. With increasing spatial resolution, these requirements become more difficult to meet, even more so when volumetric imaging is performed. In this review, techniques that have been successfully applied to three-dimensional, super-resolution live microscopy are presented and their relative strengths and weaknesses are discussed. (special issue article)

  3. 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.

  4. 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.

  5. 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.

  6. Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

    Energy Technology Data Exchange (ETDEWEB)

    Żurek-Biesiada, Dominika [Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków (Poland); Szczurek, Aleksander T. [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Prakash, Kirti [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg (Germany); Mohana, Giriram K. [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Lee, Hyun-Keun [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz (Germany); Roignant, Jean-Yves [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Birk, Udo J. [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz (Germany); Dobrucki, Jurek W., E-mail: jerzy.dobrucki@uj.edu.pl [Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków (Poland); Cremer, Christoph, E-mail: c.cremer@imb-mainz.de [Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz (Germany); Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg (Germany); Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz (Germany)

    2016-05-01

    Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.

  7. 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.

  8. 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

  9. 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.

  10. 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...

  11. 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.

  12. 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

  13. Super-resolution pupil filtering for visual performance enhancement using adaptive optics

    Science.gov (United States)

    Zhao, Lina; Dai, Yun; Zhao, Junlei; Zhou, Xiaojun

    2018-05-01

    Ocular aberration correction can significantly improve visual function of the human eye. However, even under ideal aberration correction conditions, pupil diffraction restricts the resolution of retinal images. Pupil filtering is a simple super-resolution (SR) method that can overcome this diffraction barrier. In this study, a 145-element piezoelectric deformable mirror was used as a pupil phase filter because of its programmability and high fitting accuracy. Continuous phase-only filters were designed based on Zernike polynomial series and fitted through closed-loop adaptive optics. SR results were validated using double-pass point spread function images. Contrast sensitivity was further assessed to verify the SR effect on visual function. An F-test was conducted for nested models to statistically compare different CSFs. These results indicated CSFs for the proposed SR filter were significantly higher than the diffraction correction (p vision optical correction of the human eye.

  14. Effects of whispering gallery mode in microsphere super-resolution imaging

    Science.gov (United States)

    Zhou, Song; Deng, Yongbo; Zhou, Wenchao; Yu, Muxin; Urbach, H. P.; Wu, Yihui

    2017-09-01

    Whispering Gallery modes have been presented in microscopic glass spheres or toruses with many applications. In this paper, the possible approaches to enhance the imaging resolution by Whispering Gallery modes are discussed, including evanescent waves coupling, transformed and illustration by Whispering Gallery modes. It shows that the high-order scattering modes play the dominant role in the reconstructed virtual image when the Whispering Gallery modes exist. Furthermore, we find that the high image resolution of electric dipoles can be achieved, when the out-of-phase components exist from the illustration of Whispering Gallery modes. Those results of our simulation could contribute to the knowledge of microsphere-assisted super-resolution imaging and its potential applications.

  15. Long-distance super-resolution imaging assisted by enhanced spatial Fourier transform.

    Science.gov (United States)

    Tang, Heng-He; Liu, Pu-Kun

    2015-09-07

    A new gradient-index (GRIN) lens that can realize enhanced spatial Fourier transform (FT) over optically long distances is demonstrated. By using an anisotropic GRIN metamaterial with hyperbolic dispersion, evanescent wave in free space can be transformed into propagating wave in the metamaterial and then focused outside due to negative-refraction. Both the results based on the ray tracing and the finite element simulation show that the spatial frequency bandwidth of the spatial FT can be extended to 2.7k(0) (k(0) is the wave vector in free space). Furthermore, assisted by the enhanced spatial FT, a new long-distance (in the optical far-field region) super-resolution imaging scheme is also proposed and the super resolved capability of λ/5 (λ is the wavelength in free space) is verified. The work may provide technical support for designing new-type high-speed microscopes with long working distances.

  16. A Total Variation Regularization Based Super-Resolution Reconstruction Algorithm for Digital Video

    Directory of Open Access Journals (Sweden)

    Zhang Liangpei

    2007-01-01

    Full Text Available Super-resolution (SR reconstruction technique is capable of producing a high-resolution image from a sequence of low-resolution images. In this paper, we study an efficient SR algorithm for digital video. To effectively deal with the intractable problems in SR video reconstruction, such as inevitable motion estimation errors, noise, blurring, missing regions, and compression artifacts, the total variation (TV regularization is employed in the reconstruction model. We use the fixed-point iteration method and preconditioning techniques to efficiently solve the associated nonlinear Euler-Lagrange equations of the corresponding variational problem in SR. The proposed algorithm has been tested in several cases of motion and degradation. It is also compared with the Laplacian regularization-based SR algorithm and other TV-based SR algorithms. Experimental results are presented to illustrate the effectiveness of the proposed algorithm.

  17. Remote classification from an airborne camera using image super-resolution.

    Science.gov (United States)

    Woods, Matthew; Katsaggelos, Aggelos

    2017-02-01

    The image processing technique known as super-resolution (SR), which attempts to increase the effective pixel sampling density of a digital imager, has gained rapid popularity over the last decade. The majority of literature focuses on its ability to provide results that are visually pleasing to a human observer. In this paper, we instead examine the ability of SR to improve the resolution-critical capability of an imaging system to perform a classification task from a remote location, specifically from an airborne camera. In order to focus the scope of the study, we address and quantify results for the narrow case of text classification. However, we expect the results generalize to a large set of related, remote classification tasks. We generate theoretical results through simulation, which are corroborated by experiments with a camera mounted on a DJI Phantom 3 quadcopter.

  18. Single image super-resolution based on approximated Heaviside functions and iterative refinement

    Science.gov (United States)

    Wang, Xin-Yu; Huang, Ting-Zhu; Deng, Liang-Jian

    2018-01-01

    One method of solving the single-image super-resolution problem is to use Heaviside functions. This has been done previously by making a binary classification of image components as “smooth” and “non-smooth”, describing these with approximated Heaviside functions (AHFs), and iteration including l1 regularization. We now introduce a new method in which the binary classification of image components is extended to different degrees of smoothness and non-smoothness, these components being represented by various classes of AHFs. Taking into account the sparsity of the non-smooth components, their coefficients are l1 regularized. In addition, to pick up more image details, the new method uses an iterative refinement for the residuals between the original low-resolution input and the downsampled resulting image. Experimental results showed that the new method is superior to the original AHF method and to four other published methods. PMID:29329298

  19. Deep Learning- and Transfer Learning-Based Super Resolution Reconstruction from Single Medical Image

    Directory of Open Access Journals (Sweden)

    YiNan Zhang

    2017-01-01

    Full Text Available Medical images play an important role in medical diagnosis and research. In this paper, a transfer learning- and deep learning-based super resolution reconstruction method is introduced. The proposed method contains one bicubic interpolation template layer and two convolutional layers. The bicubic interpolation template layer is prefixed by mathematics deduction, and two convolutional layers learn from training samples. For saving training medical images, a SIFT feature-based transfer learning method is proposed. Not only can medical images be used to train the proposed method, but also other types of images can be added into training dataset selectively. In empirical experiments, results of eight distinctive medical images show improvement of image quality and time reduction. Further, the proposed method also produces slightly sharper edges than other deep learning approaches in less time and it is projected that the hybrid architecture of prefixed template layer and unfixed hidden layers has potentials in other applications.

  20. Comparison between beamforming and super resolution imaging algorithms for non-destructive evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Chengguang [College of Mechatronic Engineering and Automation, National University of Defense Technology, Changsha 410073, PR China and Department of Mechanical Engineering, University of Bristol, Queen' s Building, University Walk, Bristol BS8 1TR (United Kingdom); Drinkwater, Bruce W. [Department of Mechanical Engineering, University of Bristol, Queen' s Building, University Walk, Bristol BS8 1TR (United Kingdom)

    2014-02-18

    In this paper the performance of total focusing method is compared with the widely used time-reversal MUSIC super resolution technique. The algorithms are tested with simulated and experimental ultrasonic array data, each containing different noise levels. The simulated time domain signals allow the effects of array geometry, frequency, scatterer location, scatterer size, scatterer separation and random noise to be carefully controlled. The performance of the imaging algorithms is evaluated in terms of resolution and sensitivity to random noise. It is shown that for the low noise situation, time-reversal MUSIC provides enhanced lateral resolution when compared to the total focusing method. However, for higher noise levels, the total focusing method shows robustness, whilst the performance of time-reversal MUSIC is significantly degraded.

  1. Development of a super-resolution optical microscope for directional dark matter search experiment

    International Nuclear Information System (INIS)

    Alexandrov, A.; Asada, T.; Consiglio, L.; D'Ambrosio, N.; De Lellis, G.; Di Crescenzo, A.; Di Marco, N.; Furuya, S.; Hakamata, K.; Ishikawa, M.; Katsuragawa, T.; Kuwabara, K.; Machii, S.; Naka, T.; Pupilli, F.; Sirignano, C.; Tawara, Y.; Tioukov, V.; Umemoto, A.; Yoshimoto, M.

    2016-01-01

    Nuclear emulsion is a perfect choice for a detector for directional DM search because of its high density and excellent position accuracy. The minimal detectable track length of a recoil nucleus in emulsion is required to be at least 100 nm, making the resolution of conventional optical microscopes insufficient to resolve them. Here we report about the R&D on a super-resolution optical microscope to be used in future directional DM search experiments with nuclear emulsion as a detector media. The microscope will be fully automatic, will use novel image acquisition and analysis techniques, will achieve the spatial resolution of the order of few tens of nm and will be capable of reconstructing recoil tracks with the length of at least 100 nm with high angular resolution.

  2. Comparison between beamforming and super resolution imaging algorithms for non-destructive evaluation

    International Nuclear Information System (INIS)

    Fan, Chengguang; Drinkwater, Bruce W.

    2014-01-01

    In this paper the performance of total focusing method is compared with the widely used time-reversal MUSIC super resolution technique. The algorithms are tested with simulated and experimental ultrasonic array data, each containing different noise levels. The simulated time domain signals allow the effects of array geometry, frequency, scatterer location, scatterer size, scatterer separation and random noise to be carefully controlled. The performance of the imaging algorithms is evaluated in terms of resolution and sensitivity to random noise. It is shown that for the low noise situation, time-reversal MUSIC provides enhanced lateral resolution when compared to the total focusing method. However, for higher noise levels, the total focusing method shows robustness, whilst the performance of time-reversal MUSIC is significantly degraded

  3. Robust isotropic super-resolution by maximizing a Laplace posterior for MRI volumes

    Science.gov (United States)

    Han, Xian-Hua; Iwamoto, Yutaro; Shiino, Akihiko; Chen, Yen-Wei

    2014-03-01

    Magnetic resonance imaging can only acquire volume data with finite resolution due to various factors. In particular, the resolution in one direction (such as the slice direction) is much lower than others (such as the in-plane direction), yielding un-realistic visualizations. This study explores to reconstruct MRI isotropic resolution volumes from three orthogonal scans. This proposed super- resolution reconstruction is formulated as a maximum a posterior (MAP) problem, which relies on the generation model of the acquired scans from the unknown high-resolution volumes. Generally, the deviation ensemble of the reconstructed high-resolution (HR) volume from the available LR ones in the MAP is represented as a Gaussian distribution, which usually results in some noise and artifacts in the reconstructed HR volume. Therefore, this paper investigates a robust super-resolution by formulating the deviation set as a Laplace distribution, which assumes sparsity in the deviation ensemble based on the possible insight of the appeared large values only around some unexpected regions. In addition, in order to achieve reliable HR MRI volume, we integrates the priors such as bilateral total variation (BTV) and non-local mean (NLM) into the proposed MAP framework for suppressing artifacts and enriching visual detail. We validate the proposed robust SR strategy using MRI mouse data with high-definition resolution in two direction and low-resolution in one direction, which are imaged in three orthogonal scans: axial, coronal and sagittal planes. Experiments verifies that the proposed strategy can achieve much better HR MRI volumes than the conventional MAP method even with very high-magnification factor: 10.

  4. New learning based super-resolution: use of DWT and IGMRF prior.

    Science.gov (United States)

    Gajjar, Prakash P; Joshi, Manjunath V

    2010-05-01

    In this paper, we propose a new learning-based approach for super-resolving an image captured at low spatial resolution. Given the low spatial resolution test image and a database consisting of low and high spatial resolution images, we obtain super-resolution for the test image. We first obtain an initial high-resolution (HR) estimate by learning the high-frequency details from the available database. A new discrete wavelet transform (DWT) based approach is proposed for learning that uses a set of low-resolution (LR) images and their corresponding HR versions. Since the super-resolution is an ill-posed problem, we obtain the final solution using a regularization framework. The LR image is modeled as the aliased and noisy version of the corresponding HR image, and the aliasing matrix entries are estimated using the test image and the initial HR estimate. The prior model for the super-resolved image is chosen as an Inhomogeneous Gaussian Markov random field (IGMRF) and the model parameters are estimated using the same initial HR estimate. A maximum a posteriori (MAP) estimation is used to arrive at the cost function which is minimized using a simple gradient descent approach. We demonstrate the effectiveness of the proposed approach by conducting the experiments on gray scale as well as on color images. The method is compared with the standard interpolation technique and also with existing learning-based approaches. The proposed approach can be used in applications such as wildlife sensor networks, remote surveillance where the memory, the transmission bandwidth, and the camera cost are the main constraints.

  5. Super Resolution and Interference Suppression Technique applied to SHARAD Radar Data

    Science.gov (United States)

    Raguso, M. C.; Mastrogiuseppe, M.; Seu, R.; Piazzo, L.

    2017-12-01

    We will present a super resolution and interference suppression technique applied to the data acquired by the SHAllow RADar (SHARAD) on board the NASA's 2005 Mars Reconnaissance Orbiter (MRO) mission, currently operating around Mars [1]. The algorithms allow to improve the range resolution roughly by a factor of 3 and the Signal to Noise Ratio (SNR) by a several decibels. Range compression algorithms usually adopt conventional Fourier transform techniques, which are limited in the resolution by the transmitted signal bandwidth, analogous to the Rayleigh's criterion in optics. In this work, we investigate a super resolution method based on autoregressive models and linear prediction techniques [2]. Starting from the estimation of the linear prediction coefficients from the spectral data, the algorithm performs the radar bandwidth extrapolation (BWE), thereby improving the range resolution of the pulse-compressed coherent radar data. Moreover, the EMIs (ElectroMagnetic Interferences) are detected and the spectra is interpolated in order to reconstruct an interference free spectrum, thereby improving the SNR. The algorithm can be applied to the single complex look image after synthetic aperture processing (SAR). We apply the proposed algorithm to simulated as well as to real radar data. We will demonstrate the effective enhancement on vertical resolution with respect to the classical spectral estimator. We will show that the imaging of the subsurface layered structures observed in radargrams is improved, allowing additional insights for the scientific community in the interpretation of the SHARAD radar data, which will help to further our understanding of the formation and evolution of known geological features on Mars. References: [1] Seu et al. 2007, Science, 2007, 317, 1715-1718 [2] K.M. Cuomo, "A Bandwidth Extrapolation Technique for Improved Range Resolution of Coherent Radar Data", Project Report CJP-60, Revision 1, MIT Lincoln Laboratory (4 Dec. 1992).

  6. Hyperspectral Super-Resolution of Locally Low Rank Images From Complementary Multisource Data.

    Science.gov (United States)

    Veganzones, Miguel A; Simoes, Miguel; Licciardi, Giorgio; Yokoya, Naoto; Bioucas-Dias, Jose M; Chanussot, Jocelyn

    2016-01-01

    Remote sensing hyperspectral images (HSIs) are quite often low rank, in the sense that the data belong to a low dimensional subspace/manifold. This has been recently exploited for the fusion of low spatial resolution HSI with high spatial resolution multispectral images in order to obtain super-resolution HSI. Most approaches adopt an unmixing or a matrix factorization perspective. The derived methods have led to state-of-the-art results when the spectral information lies in a low-dimensional subspace/manifold. However, if the subspace/manifold dimensionality spanned by the complete data set is large, i.e., larger than the number of multispectral bands, the performance of these methods mainly decreases because the underlying sparse regression problem is severely ill-posed. In this paper, we propose a local approach to cope with this difficulty. Fundamentally, we exploit the fact that real world HSIs are locally low rank, that is, pixels acquired from a given spatial neighborhood span a very low-dimensional subspace/manifold, i.e., lower or equal than the number of multispectral bands. Thus, we propose to partition the image into patches and solve the data fusion problem independently for each patch. This way, in each patch the subspace/manifold dimensionality is low enough, such that the problem is not ill-posed anymore. We propose two alternative approaches to define the hyperspectral super-resolution through local dictionary learning using endmember induction algorithms. We also explore two alternatives to define the local regions, using sliding windows and binary partition trees. The effectiveness of the proposed approaches is illustrated with synthetic and semi real data.

  7. Droplet Image Super Resolution Based on Sparse Representation and Kernel Regression

    Science.gov (United States)

    Zou, Zhenzhen; Luo, Xinghong; Yu, Qiang

    2018-05-01

    Microgravity and containerless conditions, which are produced via electrostatic levitation combined with a drop tube, are important when studying the intrinsic properties of new metastable materials. Generally, temperature and image sensors can be used to measure the changes of sample temperature, morphology and volume. Then, the specific heat, surface tension, viscosity changes and sample density can be obtained. Considering that the falling speed of the material sample droplet is approximately 31.3 m/s when it reaches the bottom of a 50-meter-high drop tube, a high-speed camera with a collection rate of up to 106 frames/s is required to image the falling droplet. However, at the high-speed mode, very few pixels, approximately 48-120, will be obtained in each exposure time, which results in low image quality. Super-resolution image reconstruction is an algorithm that provides finer details than the sampling grid of a given imaging device by increasing the number of pixels per unit area in the image. In this work, we demonstrate the application of single image-resolution reconstruction in the microgravity and electrostatic levitation for the first time. Here, using the image super-resolution method based on sparse representation, a low-resolution droplet image can be reconstructed. Employed Yang's related dictionary model, high- and low-resolution image patches were combined with dictionary training, and high- and low-resolution-related dictionaries were obtained. The online double-sparse dictionary training algorithm was used in the study of related dictionaries and overcome the shortcomings of the traditional training algorithm with small image patch. During the stage of image reconstruction, the algorithm of kernel regression is added, which effectively overcomes the shortcomings of the Yang image's edge blurs.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. A Brief Introduction to Single-Molecule Fluorescence Methods.

    Science.gov (United States)

    van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

    2018-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

  17. 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.

  18. 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.

  19. 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.

  20. Tryptophan and ATTO 590: mutual fluorescence quenching and exciplex formation.

    Science.gov (United States)

    Bhattacharjee, Ujjal; Beck, Christie; Winter, Arthur; Wells, Carson; Petrich, Jacob W

    2014-07-24

    Investigation of fluorescence quenching of probes, such as ATTO dyes, is becoming an increasingly important topic owing to the use of these dyes in super-resolution microscopies and in single-molecule studies. Photoinduced electron transfer is their most important nonradiative pathway. Because of the increasing frequency of the use of ATTO and related dyes to investigate biological systems, studies are presented for inter- and intramolecular quenching of ATTO 590 with tryptophan. In order to examine intramolecular quenching, an ATTO 590-tryptophan conjugate was synthesized. It was determined that tryptophan is efficiently quenching ATTO 590 fluorescence by excited-state charge transfer and two charge transfer complexes are forming. In addition, it was discovered that an exciplex (whose lifetime is 5.6 ns) can be formed between tryptophan and ATTO 590, and it is suggested that the possibility of such exciplex formation should be taken into account when protein fluorescence is monitored in a system tagged with ATTO dyes.

  1. Synthetic biology's tall order: Reconstruction of 3D, super resolution images of single molecules in real-time

    CSIR Research Space (South Africa)

    Henriques, R

    2010-08-31

    Full Text Available -to-use reconstruction software coupled with image acquisition. Here, we present QuickPALM, an Image plugin, enabling real-time reconstruction of 3D super-resolution images during acquisition and drift correction. We illustrate its application by reconstructing Cy5...

  2. Detecting breast microcalcifications using super-resolution and wave-equation ultrasound imaging: a numerical phantom study

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Lianjie [Los Alamos National Laboratory; Simonetti, Francesco [IMPERIAL COLLEGE LONDON; Huthwaite, Peter [IMPERIAL COLLEGE LONDON; Rosenberg, Robert [UNM; Williamson, Michael [UNM

    2010-01-01

    Ultrasound image resolution and quality need to be significantly improved for breast microcalcification detection. Super-resolution imaging with the factorization method has recently been developed as a promising tool to break through the resolution limit of conventional imaging. In addition, wave-equation reflection imaging has become an effective method to reduce image speckles by properly handling ultrasound scattering/diffraction from breast heterogeneities during image reconstruction. We explore the capabilities of a novel super-resolution ultrasound imaging method and a wave-equation reflection imaging scheme for detecting breast microcalcifications. Super-resolution imaging uses the singular value decomposition and a factorization scheme to achieve an image resolution that is not possible for conventional ultrasound imaging. Wave-equation reflection imaging employs a solution to the acoustic-wave equation in heterogeneous media to backpropagate ultrasound scattering/diffraction waves to scatters and form images of heterogeneities. We construct numerical breast phantoms using in vivo breast images, and use a finite-difference wave-equation scheme to generate ultrasound data scattered from inclusions that mimic microcalcifications. We demonstrate that microcalcifications can be detected at full spatial resolution using the super-resolution ultrasound imaging and wave-equation reflection imaging methods.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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...

  10. 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 ...

  11. 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....

  12. Super-Resolution of Plant Disease Images for the Acceleration of Image-based Phenotyping and Vigor Diagnosis in Agriculture.

    Science.gov (United States)

    Yamamoto, Kyosuke; Togami, Takashi; Yamaguchi, Norio

    2017-11-06

    Unmanned aerial vehicles (UAVs or drones) are a very promising branch of technology, and they have been utilized in agriculture-in cooperation with image processing technologies-for phenotyping and vigor diagnosis. One of the problems in the utilization of UAVs for agricultural purposes is the limitation in flight time. It is necessary to fly at a high altitude to capture the maximum number of plants in the limited time available, but this reduces the spatial resolution of the captured images. In this study, we applied a super-resolution method to the low-resolution images of tomato diseases to recover detailed appearances, such as lesions on plant organs. We also conducted disease classification using high-resolution, low-resolution, and super-resolution images to evaluate the effectiveness of super-resolution methods in disease classification. Our results indicated that the super-resolution method outperformed conventional image scaling methods in spatial resolution enhancement of tomato disease images. The results of disease classification showed that the accuracy attained was also better by a large margin with super-resolution images than with low-resolution images. These results indicated that our approach not only recovered the information lost in low-resolution images, but also exerted a beneficial influence on further image analysis. The proposed approach will accelerate image-based phenotyping and vigor diagnosis in the field, because it not only saves time to capture images of a crop in a cultivation field but also secures the accuracy of these images for further analysis.

  13. Super-Resolution of Plant Disease Images for the Acceleration of Image-based Phenotyping and Vigor Diagnosis in Agriculture

    Directory of Open Access Journals (Sweden)

    Kyosuke Yamamoto

    2017-11-01

    Full Text Available Unmanned aerial vehicles (UAVs or drones are a very promising branch of technology, and they have been utilized in agriculture—in cooperation with image processing technologies—for phenotyping and vigor diagnosis. One of the problems in the utilization of UAVs for agricultural purposes is the limitation in flight time. It is necessary to fly at a high altitude to capture the maximum number of plants in the limited time available, but this reduces the spatial resolution of the captured images. In this study, we applied a super-resolution method to the low-resolution images of tomato diseases to recover detailed appearances, such as lesions on plant organs. We also conducted disease classification using high-resolution, low-resolution, and super-resolution images to evaluate the effectiveness of super-resolution methods in disease classification. Our results indicated that the super-resolution method outperformed conventional image scaling methods in spatial resolution enhancement of tomato disease images. The results of disease classification showed that the accuracy attained was also better by a large margin with super-resolution images than with low-resolution images. These results indicated that our approach not only recovered the information lost in low-resolution images, but also exerted a beneficial influence on further image analysis. The proposed approach will accelerate image-based phenotyping and vigor diagnosis in the field, because it not only saves time to capture images of a crop in a cultivation field but also secures the accuracy of these images for further analysis.

  14. 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.

  15. 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.

  16. 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.)

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Single Image Super-Resolution Using Global Regression Based on Multiple Local Linear Mappings.

    Science.gov (United States)

    Choi, Jae-Seok; Kim, Munchurl

    2017-03-01

    Super-resolution (SR) has become more vital, because of its capability to generate high-quality ultra-high definition (UHD) high-resolution (HR) images from low-resolution (LR) input images. Conventional SR methods entail high computational complexity, which makes them difficult to be implemented for up-scaling of full-high-definition input images into UHD-resolution images. Nevertheless, our previous super-interpolation (SI) method showed a good compromise between Peak-Signal-to-Noise Ratio (PSNR) performances and computational complexity. However, since SI only utilizes simple linear mappings, it may fail to precisely reconstruct HR patches with complex texture. In this paper, we present a novel SR method, which inherits the large-to-small patch conversion scheme from SI but uses global regression based on local linear mappings (GLM). Thus, our new SR method is called GLM-SI. In GLM-SI, each LR input patch is divided into 25 overlapped subpatches. Next, based on the local properties of these subpatches, 25 different local linear mappings are applied to the current LR input patch to generate 25 HR patch candidates, which are then regressed into one final HR patch using a global regressor. The local linear mappings are learned cluster-wise in our off-line training phase. The main contribution of this paper is as follows: Previously, linear-mapping-based conventional SR methods, including SI only used one simple yet coarse linear mapping to each patch to reconstruct its HR version. On the contrary, for each LR input patch, our GLM-SI is the first to apply a combination of multiple local linear mappings, where each local linear mapping is found according to local properties of the current LR patch. Therefore, it can better approximate nonlinear LR-to-HR mappings for HR patches with complex texture. Experiment results show that the proposed GLM-SI method outperforms most of the state-of-the-art methods, and shows comparable PSNR performance with much lower

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. From local pixel structure to global image super-resolution: a new face hallucination framework.

    Science.gov (United States)

    Hu, Yu; Lam, Kin-Man; Qiu, Guoping; Shen, Tingzhi

    2011-02-01

    We have developed a new face hallucination framework termed from local pixel structure to global image super-resolution (LPS-GIS). Based on the assumption that two similar face images should have similar local pixel structures, the new framework first uses the input low-resolution (LR) face image to search a face database for similar example high-resolution (HR) faces in order to learn the local pixel structures for the target HR face. It then uses the input LR face and the learned pixel structures as priors to estimate the target HR face. We present a three-step implementation procedure for the framework. Step 1 searches the database for K example faces that are the most similar to the input, and then warps the K example images to the input using optical flow. Step 2 uses the warped HR version of the K example faces to learn the local pixel structures for the target HR face. An effective method for learning local pixel structures from an individual face, and an adaptive procedure for fusing the local pixel structures of different example faces to reduce the influence of warping errors, have been developed. Step 3 estimates the target HR face by solving a constrained optimization problem by means of an iterative procedure. Experimental results show that our new method can provide good performances for face hallucination, both in terms of reconstruction error and visual quality; and that it is competitive with existing state-of-the-art methods.

  9. An Example-Based Super-Resolution Algorithm for Selfie Images

    Directory of Open Access Journals (Sweden)

    Jino Hans William

    2016-01-01

    Full Text Available A selfie is typically a self-portrait captured using the front camera of a smartphone. Most state-of-the-art smartphones are equipped with a high-resolution (HR rear camera and a low-resolution (LR front camera. As selfies are captured by front camera with limited pixel resolution, the fine details in it are explicitly missed. This paper aims to improve the resolution of selfies by exploiting the fine details in HR images captured by rear camera using an example-based super-resolution (SR algorithm. HR images captured by rear camera carry significant fine details and are used as an exemplar to train an optimal matrix-value regression (MVR operator. The MVR operator serves as an image-pair priori which learns the correspondence between the LR-HR patch-pairs and is effectively used to super-resolve LR selfie images. The proposed MVR algorithm avoids vectorization of image patch-pairs and preserves image-level information during both learning and recovering process. The proposed algorithm is evaluated for its efficiency and effectiveness both qualitatively and quantitatively with other state-of-the-art SR algorithms. The results validate that the proposed algorithm is efficient as it requires less than 3 seconds to super-resolve LR selfie and is effective as it preserves sharp details without introducing any counterfeit fine details.

  10. Impact of detector-element active-area shape and fill factor on super-resolution

    Directory of Open Access Journals (Sweden)

    Russell Craig Hardie

    2015-05-01

    Full Text Available In many undersampled imaging systems, spatial integration from the individual detector elements is the dominant component of the system point spread function (PSF. Conventional focal plane arrays (FPAs utilize square detector elements with a nearly 100% fill factor, where fill factor is defined as the fraction of the detector element area that is active in light detection. A large fill factor is generally considered to be desirable because more photons are collected for a given pitch, and this leads to a higher signal-to-noise-ratio (SNR. However, the large active area works against super-resolution (SR image restoration by acting as an additional low pass filter in the overall PSF when modeled on the SR sampling grid. A high fill factor also tends to increase blurring from pixel cross-talk. In this paper, we study the impact of FPA detector-element shape and fill factor on SR. A detailed modulation transfer function analysis is provided along with a number of experimental results with both simulated data and real data acquired with a midwave infrared (MWIR imaging system. We demonstrate the potential advantage of low fill factor detector elements when combined with SR image restoration. Our results suggest that low fill factor circular detector elements may be the best choice. New video results are presented using robust adaptive Wiener filter SR processing applied to data from a commercial MWIR imaging system with both high and low detector element fill factors.

  11. Image Super-Resolution Algorithm Based on an Improved Sparse Autoencoder

    Directory of Open Access Journals (Sweden)

    Detian Huang

    2018-01-01

    Full Text Available Due to the limitations of the resolution of the imaging system and the influence of scene changes and other factors, sometimes only low-resolution images can be acquired, which cannot satisfy the practical application’s requirements. To improve the quality of low-resolution images, a novel super-resolution algorithm based on an improved sparse autoencoder is proposed. Firstly, in the training set preprocessing stage, the high- and low-resolution image training sets are constructed, respectively, by using high-frequency information of the training samples as the characterization, and then the zero-phase component analysis whitening technique is utilized to decorrelate the formed joint training set to reduce its redundancy. Secondly, a constructed sparse regularization term is added to the cost function of the traditional sparse autoencoder to further strengthen the sparseness constraint on the hidden layer. Finally, in the dictionary learning stage, the improved sparse autoencoder is adopted to achieve unsupervised dictionary learning to improve the accuracy and stability of the dictionary. Experimental results validate that the proposed algorithm outperforms the existing algorithms both in terms of the subjective visual perception and the objective evaluation indices, including the peak signal-to-noise ratio and the structural similarity measure.

  12. Robust Single Image Super-Resolution via Deep Networks With Sparse Prior.

    Science.gov (United States)

    Liu, Ding; Wang, Zhaowen; Wen, Bihan; Yang, Jianchao; Han, Wei; Huang, Thomas S

    2016-07-01

    Single image super-resolution (SR) is an ill-posed problem, which tries to recover a high-resolution image from its low-resolution observation. To regularize the solution of the problem, previous methods have focused on designing good priors for natural images, such as sparse representation, or directly learning the priors from a large data set with models, such as deep neural networks. In this paper, we argue that domain expertise from the conventional sparse coding model can be combined with the key ingredients of deep learning to achieve further improved results. We demonstrate that a sparse coding model particularly designed for SR can be incarnated as a neural network with the merit of end-to-end optimization over training data. The network has a cascaded structure, which boosts the SR performance for both fixed and incremental scaling factors. The proposed training and testing schemes can be extended for robust handling of images with additional degradation, such as noise and blurring. A subjective assessment is conducted and analyzed in order to thoroughly evaluate various SR techniques. Our proposed model is tested on a wide range of images, and it significantly outperforms the existing state-of-the-art methods for various scaling factors both quantitatively and perceptually.

  13. Image super-resolution reconstruction based on regularization technique and guided filter

    Science.gov (United States)

    Huang, De-tian; Huang, Wei-qin; Gu, Pei-ting; Liu, Pei-zhong; Luo, Yan-min

    2017-06-01

    In order to improve the accuracy of sparse representation coefficients and the quality of reconstructed images, an improved image super-resolution algorithm based on sparse representation is presented. In the sparse coding stage, the autoregressive (AR) regularization and the non-local (NL) similarity regularization are introduced to improve the sparse coding objective function. A group of AR models which describe the image local structures are pre-learned from the training samples, and one or several suitable AR models can be adaptively selected for each image patch to regularize the solution space. Then, the image non-local redundancy is obtained by the NL similarity regularization to preserve edges. In the process of computing the sparse representation coefficients, the feature-sign search algorithm is utilized instead of the conventional orthogonal matching pursuit algorithm to improve the accuracy of the sparse coefficients. To restore image details further, a global error compensation model based on weighted guided filter is proposed to realize error compensation for the reconstructed images. Experimental results demonstrate that compared with Bicubic, L1SR, SISR, GR, ANR, NE + LS, NE + NNLS, NE + LLE and A + (16 atoms) methods, the proposed approach has remarkable improvement in peak signal-to-noise ratio, structural similarity and subjective visual perception.

  14. Single image super-resolution based on compressive sensing and improved TV minimization sparse recovery

    Science.gov (United States)

    Vishnukumar, S.; Wilscy, M.

    2017-12-01

    In this paper, we propose a single image Super-Resolution (SR) method based on Compressive Sensing (CS) and Improved Total Variation (TV) Minimization Sparse Recovery. In the CS framework, low-resolution (LR) image is treated as the compressed version of high-resolution (HR) image. Dictionary Training and Sparse Recovery are the two phases of the method. K-Singular Value Decomposition (K-SVD) method is used for dictionary training and the dictionary represents HR image patches in a sparse manner. Here, only the interpolated version of the LR image is used for training purpose and thereby the structural self similarity inherent in the LR image is exploited. In the sparse recovery phase the sparse representation coefficients with respect to the trained dictionary for LR image patches are derived using Improved TV Minimization method. HR image can be reconstructed by the linear combination of the dictionary and the sparse coefficients. The experimental results show that the proposed method gives better results quantitatively as well as qualitatively on both natural and remote sensing images. The reconstructed images have better visual quality since edges and other sharp details are preserved.

  15. Machine Learning Based Single-Frame Super-Resolution Processing for Lensless Blood Cell Counting

    Directory of Open Access Journals (Sweden)

    Xiwei Huang

    2016-11-01

    Full Text Available A lensless blood cell counting system integrating microfluidic channel and a complementary metal oxide semiconductor (CMOS image sensor is a promising technique to miniaturize the conventional optical lens based imaging system for point-of-care testing (POCT. However, such a system has limited resolution, making it imperative to improve resolution from the system-level using super-resolution (SR processing. Yet, how to improve resolution towards better cell detection and recognition with low cost of processing resources and without degrading system throughput is still a challenge. In this article, two machine learning based single-frame SR processing types are proposed and compared for lensless blood cell counting, namely the Extreme Learning Machine based SR (ELMSR and Convolutional Neural Network based SR (CNNSR. Moreover, lensless blood cell counting prototypes using commercial CMOS image sensors and custom designed backside-illuminated CMOS image sensors are demonstrated with ELMSR and CNNSR. When one captured low-resolution lensless cell image is input, an improved high-resolution cell image will be output. The experimental results show that the cell resolution is improved by 4×, and CNNSR has 9.5% improvement over the ELMSR on resolution enhancing performance. The cell counting results also match well with a commercial flow cytometer. Such ELMSR and CNNSR therefore have the potential for efficient resolution improvement in lensless blood cell counting systems towards POCT applications.

  16. Cell-specific STORM super-resolution imaging reveals nanoscale organization of cannabinoid signaling.

    Science.gov (United States)

    Dudok, Barna; Barna, László; Ledri, Marco; Szabó, Szilárd I; Szabadits, Eszter; Pintér, Balázs; Woodhams, Stephen G; Henstridge, Christopher M; Balla, Gyula Y; Nyilas, Rita; Varga, Csaba; Lee, Sang-Hun; Matolcsi, Máté; Cervenak, Judit; Kacskovics, Imre; Watanabe, Masahiko; Sagheddu, Claudia; Melis, Miriam; Pistis, Marco; Soltesz, Ivan; Katona, István

    2015-01-01

    A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type- and subcellular compartment-specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ(9)-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ(9)-tetrahydrocannabinol treatment. These findings indicate that cell type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.

  17. 3D Super-Resolution Motion-Corrected MRI: Validation of Fetal Posterior Fossa Measurements.

    Science.gov (United States)

    Pier, Danielle B; Gholipour, Ali; Afacan, Onur; Velasco-Annis, Clemente; Clancy, Sean; Kapur, Kush; Estroff, Judy A; Warfield, Simon K

    2016-09-01

    Current diagnosis of fetal posterior fossa anomalies by sonography and conventional MRI is limited by fetal position, motion, and by two-dimensional (2D), rather than three-dimensional (3D), representation. In this study, we aimed to validate the use of a novel magnetic resonance imaging (MRI) technique, 3D super-resolution motion-corrected MRI, to image the fetal posterior fossa. From a database of pregnant women who received fetal MRIs at our institution, images of 49 normal fetal brains were reconstructed. Six measurements of the cerebellum, vermis, and pons were obtained for all cases on 2D conventional and 3D reconstructed MRI, and the agreement between the two methods was determined using concordance correlation coefficients. Concordance of axial and coronal measurements of the transcerebellar diameter was also assessed within each method. Between the two methods, the concordance of measurements was high for all six structures (P fetal motion and orthogonal slice acquisition. This technique will facilitate further study of fetal abnormalities of the posterior fossa. Copyright © 2016 by the American Society of Neuroimaging.

  18. All-passive pixel super-resolution of time-stretch imaging

    Science.gov (United States)

    Chan, Antony C. S.; Ng, Ho-Cheung; Bogaraju, Sharat C. V.; So, Hayden K. H.; Lam, Edmund Y.; Tsia, Kevin K.

    2017-03-01

    Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the-art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate — hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image frames inherently introduced by asynchronous digital sampling of the continuous time-stretch imaging process. Precise pixel registration is thus accomplished without any active opto-mechanical subpixel-shift control or other additional hardware. Here, we present the experimental pixel-SR image reconstruction pipeline that restores high-resolution time-stretch images of microparticles and biological cells (phytoplankton) at a relaxed sampling rate (≈2-5 GSa/s)—more than four times lower than the originally required readout rate (20 GSa/s) — is thus effective for high-throughput label-free, morphology-based cellular classification down to single-cell precision. Upon integration with the high-throughput image processing technology, this pixel-SR time-stretch imaging technique represents a cost-effective and practical solution for large scale cell-based phenotypic screening in biomedical diagnosis and machine vision for quality control in manufacturing.

  19. Multisensor Super Resolution Using Directionally-Adaptive Regularization for UAV Images.

    Science.gov (United States)

    Kang, Wonseok; Yu, Soohwan; Ko, Seungyong; Paik, Joonki

    2015-05-22

    In various unmanned aerial vehicle (UAV) imaging applications, the multisensor super-resolution (SR) technique has become a chronic problem and attracted increasing attention. Multisensor SR algorithms utilize multispectral low-resolution (LR) images to make a higher resolution (HR) image to improve the performance of the UAV imaging system. The primary objective of the paper is to develop a multisensor SR method based on the existing multispectral imaging framework instead of using additional sensors. In order to restore image details without noise amplification or unnatural post-processing artifacts, this paper presents an improved regularized SR algorithm by combining the directionally-adaptive constraints and multiscale non-local means (NLM) filter. As a result, the proposed method can overcome the physical limitation of multispectral sensors by estimating the color HR image from a set of multispectral LR images using intensity-hue-saturation (IHS) image fusion. Experimental results show that the proposed method provides better SR results than existing state-of-the-art SR methods in the sense of objective measures.

  20. Spatiotemporal Super-Resolution Reconstruction Based on Robust Optical Flow and Zernike Moment for Video Sequences

    Directory of Open Access Journals (Sweden)

    Meiyu Liang

    2013-01-01

    Full Text Available In order to improve the spatiotemporal resolution of the video sequences, a novel spatiotemporal super-resolution reconstruction model (STSR based on robust optical flow and Zernike moment is proposed in this paper, which integrates the spatial resolution reconstruction and temporal resolution reconstruction into a unified framework. The model does not rely on accurate estimation of subpixel motion and is robust to noise and rotation. Moreover, it can effectively overcome the problems of hole and block artifacts. First we propose an efficient robust optical flow motion estimation model based on motion details preserving, then we introduce the biweighted fusion strategy to implement the spatiotemporal motion compensation. Next, combining the self-adaptive region correlation judgment strategy, we construct a fast fuzzy registration scheme based on Zernike moment for better STSR with higher efficiency, and then the final video sequences with high spatiotemporal resolution can be obtained by fusion of the complementary and redundant information with nonlocal self-similarity between the adjacent video frames. Experimental results demonstrate that the proposed method outperforms the existing methods in terms of both subjective visual and objective quantitative evaluations.

  1. All-optical control and super-resolution imaging of quantum emitters in layered materials.

    Science.gov (United States)

    Kianinia, Mehran; Bradac, Carlo; Sontheimer, Bernd; Wang, Fan; Tran, Toan Trong; Nguyen, Minh; Kim, Sejeong; Xu, Zai-Quan; Jin, Dayong; Schell, Andreas W; Lobo, Charlene J; Aharonovich, Igor; Toth, Milos

    2018-02-28

    Layered van der Waals materials are emerging as compelling two-dimensional platforms for nanophotonics, polaritonics, valleytronics and spintronics, and have the potential to transform applications in sensing, imaging and quantum information processing. Among these, hexagonal boron nitride (hBN) is known to host ultra-bright, room-temperature quantum emitters, whose nature is yet to be fully understood. Here we present a set of measurements that give unique insight into the photophysical properties and level structure of hBN quantum emitters. Specifically, we report the existence of a class of hBN quantum emitters with a fast-decaying intermediate and a long-lived metastable state accessible from the first excited electronic state. Furthermore, by means of a two-laser repumping scheme, we show an enhanced photoluminescence and emission intensity, which can be utilized to realize a new modality of far-field super-resolution imaging. Our findings expand current understanding of quantum emitters in hBN and show new potential ways of harnessing their nonlinear optical properties in sub-diffraction nanoscopy.

  2. Improved Wallis Dodging Algorithm for Large-Scale Super-Resolution Reconstruction Remote Sensing Images

    Directory of Open Access Journals (Sweden)

    Chong Fan

    2017-03-01

    Full Text Available A sub-block algorithm is usually applied in the super-resolution (SR reconstruction of images because of limitations in computer memory. However, the sub-block SR images can hardly achieve a seamless image mosaicking because of the uneven distribution of brightness and contrast among these sub-blocks. An effectively improved weighted Wallis dodging algorithm is proposed, aiming at the characteristic that SR reconstructed images are gray images with the same size and overlapping region. This algorithm can achieve consistency of image brightness and contrast. Meanwhile, a weighted adjustment sequence is presented to avoid the spatial propagation and accumulation of errors and the loss of image information caused by excessive computation. A seam line elimination method can share the partial dislocation in the seam line to the entire overlapping region with a smooth transition effect. Subsequently, the improved method is employed to remove the uneven illumination for 900 SR reconstructed images of ZY-3. Then, the overlapping image mosaic method is adopted to accomplish a seamless image mosaic based on the optimal seam line.

  3. Newmark-Beta-FDTD method for super-resolution analysis of time reversal waves

    Science.gov (United States)

    Shi, Sheng-Bing; Shao, Wei; Ma, Jing; Jin, Congjun; Wang, Xiao-Hua

    2017-09-01

    In this work, a new unconditionally stable finite-difference time-domain (FDTD) method with the split-field perfectly matched layer (PML) is proposed for the analysis of time reversal (TR) waves. The proposed method is very suitable for multiscale problems involving microstructures. The spatial and temporal derivatives in this method are discretized by the central difference technique and Newmark-Beta algorithm, respectively, and the derivation results in the calculation of a banded-sparse matrix equation. Since the coefficient matrix keeps unchanged during the whole simulation process, the lower-upper (LU) decomposition of the matrix needs to be performed only once at the beginning of the calculation. Moreover, the reverse Cuthill-Mckee (RCM) technique, an effective preprocessing technique in bandwidth compression of sparse matrices, is used to improve computational efficiency. The super-resolution focusing of TR wave propagation in two- and three-dimensional spaces is included to validate the accuracy and efficiency of the proposed method.

  4. Super-resolution reconstruction of MR image with a novel residual learning network algorithm

    Science.gov (United States)

    Shi, Jun; Liu, Qingping; Wang, Chaofeng; Zhang, Qi; Ying, Shihui; Xu, Haoyu

    2018-04-01

    Spatial resolution is one of the key parameters of magnetic resonance imaging (MRI). The image super-resolution (SR) technique offers an alternative approach to improve the spatial resolution of MRI due to its simplicity. Convolutional neural networks (CNN)-based SR algorithms have achieved state-of-the-art performance, in which the global residual learning (GRL) strategy is now commonly used due to its effectiveness for learning image details for SR. However, the partial loss of image details usually happens in a very deep network due to the degradation problem. In this work, we propose a novel residual learning-based SR algorithm for MRI, which combines both multi-scale GRL and shallow network block-based local residual learning (LRL). The proposed LRL module works effectively in capturing high-frequency details by learning local residuals. One simulated MRI dataset and two real MRI datasets have been used to evaluate our algorithm. The experimental results show that the proposed SR algorithm achieves superior performance to all of the other compared CNN-based SR algorithms in this work.

  5. Ensemble variational Bayes tensor factorization for super resolution of CFRP debond detection

    Science.gov (United States)

    Lu, Peng; Gao, Bin; Feng, Qizhi; Yang, Yang; Woo, W. L.; Tian, Gui Yun

    2017-09-01

    The carbon fiber reinforced polymer (CFRP) is widely used in aircraft and wind turbine blades. The common type of CFRP defect is debond. Optical pulse thermographic nondestructive evaluation (OPTNDE) and relevant thermal feature extraction algorithms are generally used to detect the debond. However, the resolution of detection performance remain as challenges. In this paper, the ensemble variational Bayes tensor factorization has been proposed to conduct super resolution of the debond detection. The algorithm is based on the framework of variational Bayes tensor factorization and it constructs spatial-transient multi-layer mining structure which can significantly enhance the contrast ratio between the defective regions and sound regions. In order to quantitatively evaluate the results, the event based F-score is computed. The different information regions of the extracted thermal patterns are considered as different events and the purpose is to objectively evaluate the detectability for different algorithms. Experimental tests and comparative studies have been conducted to prove the efficacy of the proposed method.

  6. Single-Image Super-Resolution Based on Rational Fractal Interpolation.

    Science.gov (United States)

    Zhang, Yunfeng; Fan, Qinglan; Bao, Fangxun; Liu, Yifang; Zhang, Caiming

    2018-08-01

    This paper presents a novel single-image super-resolution (SR) procedure, which upscales a given low-resolution (LR) input image to a high-resolution image while preserving the textural and structural information. First, we construct a new type of bivariate rational fractal interpolation model and investigate its analytical properties. This model has different forms of expression with various values of the scaling factors and shape parameters; thus, it can be employed to better describe image features than current interpolation schemes. Furthermore, this model combines the advantages of rational interpolation and fractal interpolation, and its effectiveness is validated through theoretical analysis. Second, we develop a single-image SR algorithm based on the proposed model. The LR input image is divided into texture and non-texture regions, and then, the image is interpolated according to the characteristics of the local structure. Specifically, in the texture region, the scaling factor calculation is the critical step. We present a method to accurately calculate scaling factors based on local fractal analysis. Extensive experiments and comparisons with the other state-of-the-art methods show that our algorithm achieves competitive performance, with finer details and sharper edges.

  7. Extending the Capture Volume of an Iris Recognition System Using Wavefront Coding and Super-Resolution.

    Science.gov (United States)

    Hsieh, Sheng-Hsun; Li, Yung-Hui; Tien, Chung-Hao; Chang, Chin-Chen

    2016-12-01

    Iris recognition has gained increasing popularity over the last few decades; however, the stand-off distance in a conventional iris recognition system is too short, which limits its application. In this paper, we propose a novel hardware-software hybrid method to increase the stand-off distance in an iris recognition system. When designing the system hardware, we use an optimized wavefront coding technique to extend the depth of field. To compensate for the blurring of the image caused by wavefront coding, on the software side, the proposed system uses a local patch-based super-resolution method to restore the blurred image to its clear version. The collaborative effect of the new hardware design and software post-processing showed great potential in our experiment. The experimental results showed that such improvement cannot be achieved by using a hardware-or software-only design. The proposed system can increase the capture volume of a conventional iris recognition system by three times and maintain the system's high recognition rate.

  8. Super-Resolution for “Jilin-1” Satellite Video Imagery via a Convolutional Network

    Directory of Open Access Journals (Sweden)

    Aoran Xiao

    2018-04-01

    Full Text Available Super-resolution for satellite video attaches much significance to earth observation accuracy, and the special imaging and transmission conditions on the video satellite pose great challenges to this task. The existing deep convolutional neural-network-based methods require pre-processing or post-processing to be adapted to a high-resolution size or pixel format, leading to reduced performance and extra complexity. To this end, this paper proposes a five-layer end-to-end network structure without any pre-processing and post-processing, but imposes a reshape or deconvolution layer at the end of the network to retain the distribution of ground objects within the image. Meanwhile, we formulate a joint loss function by combining the output and high-dimensional features of a non-linear mapping network to precisely learn the desirable mapping relationship between low-resolution images and their high-resolution counterparts. Also, we use satellite video data itself as a training set, which favors consistency between training and testing images and promotes the method’s practicality. Experimental results on “Jilin-1” satellite video imagery show that this method demonstrates a superior performance in terms of both visual effects and measure metrics over competing methods.

  9. Super-Resolution for "Jilin-1" Satellite Video Imagery via a Convolutional Network.

    Science.gov (United States)

    Xiao, Aoran; Wang, Zhongyuan; Wang, Lei; Ren, Yexian

    2018-04-13

    Super-resolution for satellite video attaches much significance to earth observation accuracy, and the special imaging and transmission conditions on the video satellite pose great challenges to this task. The existing deep convolutional neural-network-based methods require pre-processing or post-processing to be adapted to a high-resolution size or pixel format, leading to reduced performance and extra complexity. To this end, this paper proposes a five-layer end-to-end network structure without any pre-processing and post-processing, but imposes a reshape or deconvolution layer at the end of the network to retain the distribution of ground objects within the image. Meanwhile, we formulate a joint loss function by combining the output and high-dimensional features of a non-linear mapping network to precisely learn the desirable mapping relationship between low-resolution images and their high-resolution counterparts. Also, we use satellite video data itself as a training set, which favors consistency between training and testing images and promotes the method's practicality. Experimental results on "Jilin-1" satellite video imagery show that this method demonstrates a superior performance in terms of both visual effects and measure metrics over competing methods.

  10. 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

  11. 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.

  12. 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.

  13. Live-cell and super-resolution imaging reveal that the distribution of wall-associated protein A is correlated with the cell chain integrity of Streptococcus mutans.

    Science.gov (United States)

    Li, Y; Liu, Z; Zhang, Y; Su, Q P; Xue, B; Shao, S; Zhu, Y; Xu, X; Wei, S; Sun, Y

    2015-10-01

    Streptococcus mutans is a primary pathogen responsible for dental caries. It has an outstanding ability to form biofilm, which is vital for virulence. Previous studies have shown that knockout of Wall-associated protein A (WapA) affects cell chain and biofilm formation of S. mutans. As a surface protein, the distribution of WapA remains unknown, but it is important to understand the mechanism underlying the function of WapA. This study applied the fluorescence protein mCherry as a reporter gene to characterize the dynamic distribution of WapA in S. mutans via time-lapse and super-resolution fluorescence imaging. The results revealed interesting subcellular distribution patterns of WapA in single, dividing and long chains of S. mutans cells. It appears at the middle of the cell and moves to the poles as the cell grows and divides. In a cell chain, after each round of cell division, such dynamic relocation results in WapA distribution at the previous cell division sites, resulting in a pattern where WapA is located at the boundary of two adjacent cell pairs. This WapA distribution pattern corresponds to the breaking segmentation of wapA deletion cell chains. The dynamic relocation of WapA through the cell cycle increases our understanding of the mechanism of WapA in maintaining cell chain integrity and biofilm formation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. 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

  15. 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.

  16. 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)

  17. 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...

  18. 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...

  19. 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

  20. 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

  1. 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

  2. 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...

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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...

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Brain Atlas Fusion from High-Thickness Diagnostic Magnetic Resonance Images by Learning-Based Super-Resolution.

    Science.gov (United States)

    Zhang, Jinpeng; Zhang, Lichi; Xiang, Lei; Shao, Yeqin; Wu, Guorong; Zhou, Xiaodong; Shen, Dinggang; Wang, Qian

    2017-03-01

    It is fundamentally important to fuse the brain atlas from magnetic resonance (MR) images for many imaging-based studies. Most existing works focus on fusing the atlases from high-quality MR images. However, for low-quality diagnostic images (i.e., with high inter-slice thickness), the problem of atlas fusion has not been addressed yet. In this paper, we intend to fuse the brain atlas from the high-thickness diagnostic MR images that are prevalent for clinical routines. The main idea of our works is to extend the conventional groupwise registration by incorporating a novel super-resolution strategy. The contribution of the proposed super-resolution framework is two-fold. First, each high-thickness subject image is reconstructed to be isotropic by the patch-based sparsity learning. Then, the reconstructed isotropic image is enhanced for better quality through the random-forest-based regression model. In this way, the images obtained by the super-resolution strategy can be fused together by applying the groupwise registration method to construct the required atlas. Our experiments have shown that the proposed framework can effectively solve the problem of atlas fusion from the low-quality brain MR images.

  16. Image inpainting and super-resolution using non-local recursive deep convolutional network with skip connections

    Science.gov (United States)

    Liu, Miaofeng

    2017-07-01

    In recent years, deep convolutional neural networks come into use in image inpainting and super-resolution in many fields. Distinct to most of the former methods requiring to know beforehand the local information for corrupted pixels, we propose a 20-depth fully convolutional network to learn an end-to-end mapping a dataset of damaged/ground truth subimage pairs realizing non-local blind inpainting and super-resolution. As there often exist image with huge corruptions or inpainting on a low-resolution image that the existing approaches unable to perform well, we also share parameters in local area of layers to achieve spatial recursion and enlarge the receptive field. To avoid the difficulty of training this deep neural network, skip-connections between symmetric convolutional layers are designed. Experimental results shows that the proposed method outperforms state-of-the-art methods for diverse corrupting and low-resolution conditions, it works excellently when realizing super-resolution and image inpainting simultaneously

  17. 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...

  18. 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...

  19. 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 ...

  20. Characterization and improvement of highly inclined optical sheet microscopy

    Science.gov (United States)

    Vignolini, T.; Curcio, V.; Gardini, L.; Capitanio, M.; Pavone, F. S.

    2018-02-01

    Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume1 . HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging2-4. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.