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Sample records for super-resolution fluorescence microscopy

  1. The Principles of Super-Resolution Fluorescence Microscopy (Review)

    OpenAIRE

    N.V. Klementieva; E.V. Zagaynova; К.А. Lukyanov; A.S. Mishin

    2016-01-01

    Diffraction limit of optical microscopy impedes imaging of biological objects much smaller than the wavelength of light. Conventional fluorescence microscopy does not enable to study fine structure and processes in a living cell at the macromolecular level. Super-resolution fluorescence microscopy techniques that overcome the diffraction barrier have opened up new opportunities for biological and biomedical research. These methods combine the resolution power comparable to electron microscopy...

  2. Spatial covariance reconstructive (SCORE) super-resolution fluorescence microscopy.

    Science.gov (United States)

    Deng, Yi; Sun, Mingzhai; Lin, Pei-Hui; Ma, Jianjie; Shaevitz, Joshua W

    2014-01-01

    Super-resolution fluorescence microscopy has become a powerful tool to resolve structural information that is not accessible to traditional diffraction-limited imaging techniques such as confocal microscopy. Stochastic optical reconstruction microscopy (STORM) and photoactivation localization microscopy (PALM) are promising super-resolution techniques due to their relative ease of implementation and instrumentation on standard microscopes. However, the application of STORM is critically limited by its long sampling time. Several recent works have been focused on improving the STORM imaging speed by making use of the information from emitters with overlapping point spread functions (PSF). In this work, we present a fast and efficient algorithm that takes into account the blinking statistics of independent fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging. Our method is insensitive to background and can be applied to different types of fluorescence sources, including but not limited to the organic dyes and quantum dots that we demonstrate in this work.

  3. Spatial covariance reconstructive (SCORE super-resolution fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Yi Deng

    Full Text Available Super-resolution fluorescence microscopy has become a powerful tool to resolve structural information that is not accessible to traditional diffraction-limited imaging techniques such as confocal microscopy. Stochastic optical reconstruction microscopy (STORM and photoactivation localization microscopy (PALM are promising super-resolution techniques due to their relative ease of implementation and instrumentation on standard microscopes. However, the application of STORM is critically limited by its long sampling time. Several recent works have been focused on improving the STORM imaging speed by making use of the information from emitters with overlapping point spread functions (PSF. In this work, we present a fast and efficient algorithm that takes into account the blinking statistics of independent fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging. Our method is insensitive to background and can be applied to different types of fluorescence sources, including but not limited to the organic dyes and quantum dots that we demonstrate in this work.

  4. Multiple signal classification algorithm for super-resolution fluorescence microscopy

    Science.gov (United States)

    Agarwal, Krishna; Macháň, Radek

    2016-12-01

    Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Here we propose a statistical super-resolution technique of wide-field fluorescence microscopy we call the multiple signal classification algorithm which has several advantages. It provides resolution down to at least 50 nm, requires fewer frames and lower excitation power and works even at high fluorophore concentrations. Further, it works with any fluorophore that exhibits blinking on the timescale of the recording. The multiple signal classification algorithm shows comparable or better performance in comparison with single-molecule localization techniques and four contemporary statistical super-resolution methods for experiments of in vitro actin filaments and other independently acquired experimental data sets. We also demonstrate super-resolution at timescales of 245 ms (using 49 frames acquired at 200 frames per second) in samples of live-cell microtubules and live-cell actin filaments imaged without imaging buffers.

  5. Quantitative super-resolution microscopy

    NARCIS (Netherlands)

    Harkes, Rolf

    2016-01-01

    Super-Resolution Microscopy is an optical fluorescence technique. In this thesis we focus on single molecule super-resolution, where the position of single molecules is determined. Typically these molecules can be localized with a 10 to 30nm precision. This technique is applied in four different s

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-07

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

  7. Multiple Signal Classification Algorithm (MUSICAL) for super-resolution fluorescence microscopy

    CERN Document Server

    Agarwal, Krishna

    2016-01-01

    Super-resolution microscopy is providing unprecedented insights into biology by resolving details much below the diffraction limit. State-of-the-art Single Molecule Localization Microscopy (SMLM) techniques for super-resolution are restricted by long acquisition and computational times, or the need of special fluorophores or chemical environments. Here, we propose a novel statistical super-resolution technique of wide-field fluorescence microscopy called MUltiple SIgnal Classification ALgorithm (MUSICAL) which has several advantages over SMLM techniques. MUSICAL provides resolution down to at least 50 nm, has low requirements on number of frames and excitation power and works even at high fluorophore concentrations. Further, it works with any fluorophore that exhibits blinking on the time scale of the recording. We compare imaging results of MUSICAL with SMLM and four contemporary statistical super-resolution methods for experiments of in-vitro actin filaments and datasets provided by independent research gro...

  8. Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

    Science.gov (United States)

    Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

    2015-03-01

    Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

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

    Directory of Open Access Journals (Sweden)

    Shangting You

    2015-08-01

    Full Text Available 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.

  10. Interrogating Surface Functional Group Heterogeneity of Activated Thermoplastics Using Super-Resolution Fluorescence Microscopy.

    Science.gov (United States)

    ONeil, Colleen E; Jackson, Joshua M; Shim, Sang-Hee; Soper, Steven A

    2016-04-01

    We present a novel approach for characterizing surfaces utilizing super-resolution fluorescence microscopy with subdiffraction limit spatial resolution. Thermoplastic surfaces were activated by UV/O3 or O2 plasma treatment under various conditions to generate pendant surface-confined carboxylic acids (-COOH). These surface functional groups were then labeled with a photoswitchable dye and interrogated using single-molecule, localization-based, super-resolution fluorescence microscopy to elucidate the surface heterogeneity of these functional groups across the activated surface. Data indicated nonuniform distributions of these functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of -COOH groups secured from super-resolution imaging were used to simulate nonuniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process.

  11. Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

    Science.gov (United States)

    Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

    2013-01-01

    Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry.

  12. Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing.

    Science.gov (United States)

    Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G; Menon, Rajesh; Gerton, Jordan M; Jorgensen, Erik M; Zalevsky, Zeev

    2013-12-16

    Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

  13. Single-molecule analysis of fluorescent carbon dots towards localization-based super-resolution microscopy

    Science.gov (United States)

    Verma, Navneet C.; Khan, Syamantak; Nandi, Chayan K.

    2016-12-01

    The advancement of high-resolution bioimaging has always been dependent on the discovery of bright and easily available fluorescent probes. Fluorescent carbon nanodots, an interesting class of relatively new nanomaterials, have emerged as a versatile alternative due to their superior optical properties, non-toxicity, cell penetrability and easy routes to synthesis. Although a plethora of reports is available on bioimaging using carbon dots, single-molecule-based super-resolution imaging is rare in the literature. In this study, we have systematically characterized the single-molecule fluorescence of three carbon dots and compared them with a standard fluorescent probe. Each of these carbon dots showed a long-lived dark state in the presence of an electron acceptor. The electron transfer mechanism was investigated in single-molecule as well as in ensemble experiments. The average on-off rate between the fluorescent bright and dark states, which is one of the important parameters for single-molecule localization-based super-resolution microscopy, was measured by changing the laser power. We report that the photon budget and on-off rate of these carbon dots were good enough to achieve single-molecule localization with a precision of ~35 nm.

  14. Multimodal combinational holographic and fluorescence fluctuation microscopy to obtain spatial super-resolution

    Science.gov (United States)

    Dudenkova, V. V.; Zakharov, Yu N.

    2016-08-01

    Ways of combination of holographic and super-resolution fluorescent techniques in the same optical scheme are described. The key parameters influencing achievement of maximum possible resolution are considered. The possibility to choose different fluorescence technic for different types of fluorophores without any optical scheme changes is presented. As a result in case of visualization of the samples, which transparent in optical band, three-dimensional super resolution is received that significantly expands possibilities of the noninvasive analysis of biological samples.

  15. Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

    Science.gov (United States)

    Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus

    2014-10-15

    Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

  16. Mirror-enhanced super-resolution microscopy

    OpenAIRE

    2016-01-01

    Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell a...

  17. Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns.

    Science.gov (United States)

    Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus

    2015-08-25

    We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

  18. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.

    Science.gov (United States)

    Liss, Viktoria; Barlag, Britta; Nietschke, Monika; Hensel, Michael

    2015-12-08

    Research in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches.

  19. 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.

  20. Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

    Science.gov (United States)

    Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

    2017-02-01

    Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

  1. The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging.

    Science.gov (United States)

    Backlund, Mikael P; Lew, Matthew D; Backer, Adam S; Sahl, Steffen J; Moerner, W E

    2014-03-17

    Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

  2. 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.

  3. Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization?

    Science.gov (United States)

    MacDonald, Laura; Baldini, Giulia; Storrie, Brian

    2015-01-01

    Conventional microscopy techniques, namely, the confocal microscope or deconvolution processes, are resolution limited to approximately 200-250 nm by the diffraction properties of light as developed by Ernst Abbe in 1873. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20-50 nm in diameter. In the mid-2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular co-localization questions. This "breaking" of the diffraction barrier, affording resolution beyond 200 nm, is termed super-resolution microscopy. More recent approaches include single-molecule localization (such as photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM)) and point spread function engineering (such as stimulated emission depletion (STED) microscopy). In this review, we explain basic principles behind currently commercialized super-resolution setups and address advantages and considerations in applying these techniques to protein co-localization in biological systems.

  4. The 2015 super-resolution microscopy roadmap

    Science.gov (United States)

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

    2015-11-01

    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

  5. 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.

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

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

    2015-12-01

    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.

  7. Aptamer Stainings for Super-resolution Microscopy.

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    de Castro, Maria Angela Gomes; Rammner, Burkhard; Opazo, Felipe

    2016-01-01

    Fluorescence microscopy is an invaluable tool to visualize molecules in their biological context with ease and flexibility. However, studies using conventional light microscopy have been limited to the resolution that light diffraction allows (i.e., ~200 nm). This limitation has been recently circumvented by several types of advanced fluorescence microscopy techniques, which have achieved resolutions of up to ~10 nm. The resulting enhanced imaging precision has helped to find important cellular details that were not visible using diffraction-limited instruments. However, it has also revealed that conventional stainings using large affinity tags, such as antibodies, are not accurate enough for these imaging techniques. Since aptamers are substantially smaller than antibodies, they could provide a real advantage in super-resolution imaging. Here we compare the live staining of transferrin receptors (TfnR) obtained with different fluorescently labeled affinity probes: aptamers, specific monoclonal antibodies, or the natural receptor ligand transferrin. We observed negligible differences between these staining strategies when imaging is performed with conventional light microscopy (i.e., laser scanning confocal microscopy). However, a clear superiority of the aptamer tag over antibodies became apparent in super-resolved images obtained with stimulated emission depletion (STED) microscopy.

  8. Where Do We Stand with Super-Resolution Optical Microscopy?

    Science.gov (United States)

    Nienhaus, Karin; Nienhaus, G Ulrich

    2016-01-29

    Super-resolution fluorescence microscopy has become an invaluable, powerful approach to study biomolecular dynamics and interactions via selective labeling and observation of specific molecules in living cells, tissues and even entire organisms. In this perspective, we present a brief overview of the main techniques and their application to cellular biophysics. We place special emphasis on super-resolution imaging via single-molecule localization microscopy and stimulated emission depletion/reversible saturable optical fluorescence transitions microscopy, and we also briefly address fluorescence fluctuation approaches, notably raster image correlation spectroscopy, as tools to record fast diffusion and transport.

  9. Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams

    Science.gov (United States)

    Rong, Zihao; Kuang, Cuifang; Fang, Yue; Zhao, Guangyuan; Xu, Yingke; Liu, Xu

    2015-11-01

    We propose a novel fluorescence emission difference microscopy (FED) system based on focusing cylindrical vector beams. In conventional FED, a Gaussian beam and a 0-2π vortex phase plate are used to generate solid and hollow spots. We focus radially polarized and azimuthally polarized cylindrical vector beams to obtain an expanded solid spot and a shrunken hollow spot, taking advantage of the optical properties of cylindrical vector beams to improve the conventional FED performance. Our novel method enhances FED performance because the hollow spot size determines the FED resolution and an expanded solid spot effectively reduces negative side-lobe emergence during image processing. We demonstrate improved performance theoretically and experimentally using an in-house built FED. Our FED achieved resolution of less than λ/4 in test images of 100 nm nanoparticles, better than the confocal image resolution by a factor of approximately 1/3. We also discuss detailed simulation analyses and FED imaging of biological cells.

  10. 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.

  11. Motion Analysis of Live Objects by Super-Resolution Fluorescence Microscopy

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    Chunyan Yao

    2012-01-01

    Full Text Available Motion analysis plays an important role in studing activities or behaviors of live objects in medicine, biotechnology, chemistry, physics, spectroscopy, nanotechnology, enzymology, and biological engineering. This paper briefly reviews the developments in this area mostly in the recent three years, especially for cellular analysis in fluorescence microscopy. The topic has received much attention with the increasing demands in biomedical applications. The tasks of motion analysis include detection and tracking of objects, as well as analysis of motion behavior, living activity, events, motion statistics, and so forth. In the last decades, hundreds of papers have been published in this research topic. They cover a wide area, such as investigation of cell, cancer, virus, sperm, microbe, karyogram, and so forth. These contributions are summarized in this review. Developed methods and practical examples are also introduced. The review is useful to people in the related field for easy referral of the state of the art.

  12. Clustered localization of STAT3 during the cell cycle detected by super-resolution fluorescence microscopy

    Science.gov (United States)

    Gao, Jing; Chen, Junling; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tong, Ti; Wang, Hongda

    2017-06-01

    Signal transducer and activator of transcription 3 (STAT3) plays a key role in various cellular processes such as cell proliferation, differentiation, apoptosis and immune responses. In particular, STAT3 has emerged as a potential molecular target for cancer therapy. The functional role and standard activation mechanism of STAT3 have been well studied, however, the spatial distribution of STAT3 during the cell cycle is poorly known. Therefore, it is indispensable to study STAT3 spatial arrangement and nuclear-cytoplasimic localization at the different phase of cell cycle in cancer cells. By direct stochastic optical reconstruction microscopy imaging, we find that STAT3 forms various number and size of clusters at the different cell-cycle stage, which could not be clearly observed by conventional fluorescent microscopy. STAT3 clusters get more and larger gradually from G1 to G2 phase, during which time transcription and other related activities goes on consistently. The results suggest that there is an intimate relationship between the clustered characteristic of STAT3 and the cell-cycle behavior. Meanwhile, clustering would facilitate STAT3 rapid response to activating signals due to short distances between molecules. Our data might open a new door to develop an antitumor drug for inhibiting STAT3 signaling pathway by destroying its clusters.

  13. Super-resolution Localization and Defocused Fluorescence Microscopy on Resonantly Coupled Single-Molecule, Single-Nanorod Hybrids.

    Science.gov (United States)

    Su, Liang; Yuan, Haifeng; Lu, Gang; Rocha, Susana; Orrit, Michel; Hofkens, Johan; Uji-i, Hiroshi

    2016-02-23

    Optical antennas made of metallic nanostructures dramatically enhance single-molecule fluorescence to boost the detection sensitivity. Moreover, emission properties detected at the optical far field are dictated by the antenna. Here we study the emission from molecule-antenna hybrids by means of super-resolution localization and defocused imaging. Whereas gold nanorods make single-crystal violet molecules in the tip's vicinity visible in fluorescence, super-resolution localization on the enhanced molecular fluorescence reveals geometrical centers of the nanorod antenna instead. Furthermore, emission angular distributions of dyes linked to the nanorod surface resemble that of nanorods in defocused imaging. The experimental observations are consistent with numerical calculations using the finite-difference time-domain method.

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  15. 4-Trifluoromethyl-substituted coumarins with large Stokes shifts: synthesis, bioconjugates, and their use in super-resolution fluorescence microscopy.

    Science.gov (United States)

    Schill, Heiko; Nizamov, Shamil; Bottanelli, Francesca; Bierwagen, Jakob; Belov, Vladimir N; Hell, Stefan W

    2013-12-02

    Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

  16. Super-resolution optical microscopy: multiple choices.

    Science.gov (United States)

    Huang, Bo

    2010-02-01

    The recent invention of super-resolution optical microscopy enables the visualization of fine features in biological samples with unprecedented clarity. It creates numerous opportunities in biology because vast amount of previously obscured subcellular processes now can be directly observed. Rapid development in this field in the past two years offers many imaging modalities that address different needs but they also complicates the choice of the 'perfect' method for answering a specific question. Here I will briefly describe the principles of super-resolution optical microscopy techniques and then focus on comparing their characteristics in various aspects of practical applications.

  17. Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

    Science.gov (United States)

    Butkevich, Alexey N; Mitronova, Gyuzel Yu; Sidenstein, Sven C; Klocke, Jessica L; Kamin, Dirk; Meineke, Dirk N H; D'Este, Elisa; Kraemer, Philip-Tobias; Danzl, Johann G; Belov, Vladimir N; Hell, Stefan W

    2016-03-01

    A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.

  18. Photostable and photoswitching fluorescent dyes for super-resolution imaging.

    Science.gov (United States)

    Minoshima, Masafumi; Kikuchi, Kazuya

    2017-01-12

    Super-resolution fluorescence microscopy is a recently developed imaging tool for biological researches. Several methods have been developed for detection of fluorescence signals from molecules in a subdiffraction-limited area, breaking the diffraction limit of the conventional optical microscopies and allowing visualization of detailed macromolecular structures in cells. As objectives are exposed to intense laser in the optical systems, fluorophores for super-resolution microscopy must be tolerated even under severe light irradiation conditions. The fluorophores must also be photoactivatable and photoswitchable for single-molecule localization-based super-resolution microscopy, because the number of active fluorophores must be controlled by light irradiation. This has led to growing interest in these properties in the development of fluorophores. In this mini-review, we focus on the development of photostable and photoswitching fluorescent dyes for super-resolution microscopy. We introduce recent efforts, including improvement of fluorophore photostability and control of photoswitching behaviors of fluorophores based on photochemical and photophysical processes. Understanding and manipulation of chemical reactions in excited fluorophores can develop highly photostable and efficiently photoswitchable fluorophores that are suitable for super-resolution imaging applications.

  19. Optical super-resolution microscopy in neurobiology.

    Science.gov (United States)

    Sigrist, Stephan J; Sabatini, Bernardo L

    2012-02-01

    Understanding the highly plastic nature of neurons requires the dynamic visualization of their molecular and cellular organization in a native context. However, due to the limited resolution of standard light microscopy, many of the structural specializations of neurons cannot be resolved. A recent revolution in light microscopy has given rise to several super-resolution light microscopy methods yielding 2-10-fold higher resolution than conventional microscopy. We here describe the principles behind these techniques as well as their application to the analysis of the molecular architecture of the synapse. Furthermore, we discuss the potential for continued development of super-resolution microscopy as necessary for live imaging of neuronal structure and function in the brain.

  20. Make them Blink : Probes for Super-Resolution Microscopy

    NARCIS (Netherlands)

    Vogelsang, Jan; Steinhauer, Christian; Forthmann, Carsten; Stein, Ingo H.; Person-Skegro, Britta; Cordes, Thorben; Tinnefeld, Philip

    2010-01-01

    In recent years, a number of approaches have emerged that enable far-field fluorescence imaging beyond the diffraction limit of light, namely super-resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to s

  1. Super-resolution microscopy: a comparative treatment.

    Science.gov (United States)

    Kasuboski, James M; Sigal, Yury J; Joens, Matthew S; Lillemeier, Bjorn F; Fitzpatrick, James A J

    2012-10-01

    One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.

  2. Aberrations and adaptive optics in super-resolution microscopy.

    Science.gov (United States)

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

    2015-08-01

    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.

  3. Super-resolution spectroscopic microscopy via photon localization

    Science.gov (United States)

    Dong, Biqin; Almassalha, Luay; Urban, Ben E.; Nguyen, The-Quyen; Khuon, Satya; Chew, Teng-Leong; Backman, Vadim; Sun, Cheng; Zhang, Hao F.

    2016-07-01

    Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm--a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

  4. Correlative atomic force microscopy and localization-based super-resolution microscopy: revealing labelling and image reconstruction artefacts.

    Science.gov (United States)

    Monserrate, Aitor; Casado, Santiago; Flors, Cristina

    2014-03-17

    Hybrid microscopy: A correlative microscopy tool that combines in situ super-resolution fluorescence microscopy based on single-molecule localization and atomic force microscopy is presented. Direct comparison with high- resolution topography allows the authors to improve fluorescence labeling and image analysis in super-resolution imaging.

  5. 3D super-resolution imaging by localization microscopy.

    Science.gov (United States)

    Magenau, Astrid; Gaus, Katharina

    2015-01-01

    Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.

  6. Super-resolution microscopy of the synaptic active zone

    Directory of Open Access Journals (Sweden)

    Nadine eEhmann

    2015-01-01

    Full Text Available Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ a variety of specialised proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission.Calcium (Ca2+ channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modelling approaches has provided predictions of channel properties, numbers and even positions on the nanometre scale. However, elucidating the nanoscopic organisation of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how super-resolution microscopy can be used to obtain information on the organisation of AZ proteins.

  7. Correcting chromatic offset in multicolor super-resolution localization microscopy.

    Science.gov (United States)

    Erdelyi, Miklos; Rees, Eric; Metcalf, Daniel; Schierle, Gabriele S Kaminski; Dudas, Laszlo; Sinko, Jozsef; Knight, Alex E; Kaminski, Clemens F

    2013-05-06

    Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

  8. 3D super-resolution microscopy of bacterial division machinery

    Science.gov (United States)

    Vedyaykin, A. D.; Sabantsev, A. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.

    2016-08-01

    Super-resolution microscopy is a promising tool for the field of microbiology, as bacteria sizes are comparable to the resolution limit of light microscopy. Bacterial division machinery and FtsZ protein in particular attract much attention of scientists who use different super-resolution microscopy techniques, but most of the available data on FtsZ structures was obtained using two-dimensional (2D) super-resolution microscopy. Using 3D single-molecule localization microscopy (SMLM, namely dSTORM) to visualize FtsZ, we demonstrate that this approach allows more accurate interpretation of super-resolution images and provides new opportunities for the study of complex structures like bacterial divisome.

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

    Directory of Open Access Journals (Sweden)

    Dominika Żurek-Biesiada

    2016-06-01

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

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

    Science.gov (United States)

    Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Best, Gerrit; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Dobrucki, Jurek W; Cremer, Christoph; Birk, Udo

    2016-06-01

    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.

  11. Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation.

    Science.gov (United States)

    Lukeš, Tomáš; Křížek, Pavel; Švindrych, Zdeněk; Benda, Jakub; Ovesný, Martin; Fliegel, Karel; Klíma, Miloš; Hagen, Guy M

    2014-12-01

    We introduce and demonstrate a new high performance image reconstruction method for super-resolution structured illumination microscopy based on maximum a posteriori probability estimation (MAP-SIM). Imaging performance is demonstrated on a variety of fluorescent samples of different thickness, labeling density and noise levels. The method provides good suppression of out of focus light, improves spatial resolution, and allows reconstruction of both 2D and 3D images of cells even in the case of weak signals. The method can be used to process both optical sectioning and super-resolution structured illumination microscopy data to create high quality super-resolution images.

  12. Super-resolution microscopy of the synaptic active zone.

    Science.gov (United States)

    Ehmann, Nadine; Sauer, Markus; Kittel, Robert J

    2015-01-01

    Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.

  13. Super-resolution microscopy of single atoms in optical lattices

    Science.gov (United States)

    Alberti, Andrea; Robens, Carsten; Alt, Wolfgang; Brakhane, Stefan; Karski, Michał; Reimann, René; Widera, Artur; Meschede, Dieter

    2016-05-01

    We report on image processing techniques and experimental procedures to determine the lattice-site positions of single atoms in an optical lattice with high reliability, even for limited acquisition time or optical resolution. Determining the positions of atoms beyond the diffraction limit relies on parametric deconvolution in close analogy to methods employed in super-resolution microscopy. We develop a deconvolution method that makes effective use of the prior knowledge of the optical transfer function, noise properties, and discreteness of the optical lattice. We show that accurate knowledge of the image formation process enables a dramatic improvement on the localization reliability. This allows us to demonstrate super-resolution of the atoms’ position in closely packed ensembles where the separation between particles cannot be directly optically resolved. Furthermore, we demonstrate experimental methods to precisely reconstruct the point spread function with sub-pixel resolution from fluorescence images of single atoms, and we give a mathematical foundation thereof. We also discuss discretized image sampling in pixel detectors and provide a quantitative model of noise sources in electron multiplying CCD cameras. The techniques developed here are not only beneficial to neutral atom experiments, but could also be employed to improve the localization precision of trapped ions for ultra precise force sensing.

  14. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    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.

  15. Super-resolution microscopy of single atoms in optical lattices

    CERN Document Server

    Alberti, Andrea; Alt, Wolfgang; Brakhane, Stefan; Karski, Michał; Reimann, René; Widera, Artur; Meschede, Dieter

    2015-01-01

    We report on image processing techniques and experimental procedures to determine the lattice-site positions of single atoms in an optical lattice with high reliability, even for limited acquisition time or optical resolution. Determining the positions of atoms beyond the diffraction limit relies on parametric deconvolution in close analogy to methods employed in super-resolution microscopy. We develop a deconvolution method that makes effective use of the prior knowledge of the optical transfer function, noise properties, and discreteness of the optical lattice. We show that accurate knowledge of the image formation process enables a dramatic improvement on the localization reliability. This is especially relevant for closely packed ensembles of atoms where the separation between particles cannot be directly optically resolved. Furthermore, we demonstrate experimental methods to precisely reconstruct the point spread function with sub-pixel resolution from fluorescence images of single atoms, and we give a m...

  16. Super-resolution optical microscopy of lipid plasma membrane dynamics.

    Science.gov (United States)

    Eggeling, Christian

    2015-01-01

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory.

  17. 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.

  18. 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.

  19. 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.

  20. Super-resolution fluorescence imaging of chromosomal DNA.

    Science.gov (United States)

    Zessin, Patrick J M; Finan, Kieran; Heilemann, Mike

    2012-02-01

    Super-resolution microscopy is a powerful tool for understanding cellular function. However one of the most important biomolecules - DNA - remains somewhat inaccessible because it cannot be effectively and appropriately labeled. Here, we demonstrate that robust and detailed super-resolution images of DNA can be produced by combining 5-ethynyl-2'-deoxyuridine (EdU) labeling using the 'click chemistry' approach and direct stochastic optical reconstruction microscopy (dSTORM). This method can resolve fine chromatin structure, and - when used in conjunction with pulse labeling - can reveal the paths taken by individual fibers through the nucleus. This technique should provide a useful tool for the study of nuclear structure and function.

  1. The 2015 super-resolution microscopy roadmap

    NARCIS (Netherlands)

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

    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

  2. Real-time analysis and visualization for single-molecule based super-resolution microscopy.

    Directory of Open Access Journals (Sweden)

    Adel Kechkar

    Full Text Available Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.

  3. Real-time analysis and visualization for single-molecule based super-resolution microscopy.

    Science.gov (United States)

    Kechkar, Adel; Nair, Deepak; Heilemann, Mike; Choquet, Daniel; Sibarita, Jean-Baptiste

    2013-01-01

    Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.

  4. In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster

    Science.gov (United States)

    Schnorrenberg, Sebastian; Grotjohann, Tim; Vorbrüggen, Gerd; Herzig, Alf; Hell, Stefan W; Jakobs, Stefan

    2016-01-01

    Despite remarkable developments in diffraction unlimited super-resolution microscopy, in vivo nanoscopy of tissues and model organisms is still not satisfactorily established and rarely realized. RESOLFT nanoscopy is particularly suited for live cell imaging because it requires relatively low light levels to overcome the diffraction barrier. Previously, we introduced the reversibly switchable fluorescent protein rsEGFP2, which facilitated fast RESOLFT nanoscopy (Grotjohann et al., 2012). In that study, as in most other nanoscopy studies, only cultivated single cells were analyzed. Here, we report on the use of rsEGFP2 for live-cell RESOLFT nanoscopy of sub-cellular structures of intact Drosophila melanogaster larvae and of resected tissues. We generated flies expressing fusion proteins of alpha-tubulin and rsEGFP2 highlighting the microtubule cytoskeleton in all cells. By focusing through the intact larval cuticle, we achieved lateral resolution of <60 nm. RESOLFT nanoscopy enabled time-lapse recordings comprising 40 images and facilitated recordings 40 µm deep within fly tissues. DOI: http://dx.doi.org/10.7554/eLife.15567.001 PMID:27355614

  5. Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy.

    Science.gov (United States)

    Mito, Mari; Kawaguchi, Tetsuya; Hirose, Tetsuro; Nakagawa, Shinichi

    2016-04-01

    A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM).

  6. Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy

    OpenAIRE

    Fitzgibbon, Jessica; Bell,Karen; King, Emma; Oparka, Karl

    2010-01-01

    We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction ("super-resolution") images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specia...

  7. Super-resolution optical microscopy study of telomere structure

    Science.gov (United States)

    Phipps, Mary Lisa; Goodwin, Peter M.; Martinez, Jennifer S.; Goodwin, Edwin H.

    2016-09-01

    Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5‧-TTAGGG-3‧ in humans) repeated more than a thousand times, a short 3‧ single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3‧ overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded "t-loop." Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

  8. Real-Time analysis and visualization for single-molecule based super-resolution microscopy

    OpenAIRE

    Kechkar, Adel; Nair, Deepak; Heilemann, Mike; Choquet, Daniel; Sibarita, Jean-Baptiste

    2013-01-01

    Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct ac...

  9. Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy

    OpenAIRE

    Kechkar, Adel; Nair, Deepak; Heilemann, Mike; Choquet, Daniel; Sibarita, Jean-Baptiste

    2013-01-01

    Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct ac...

  10. SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy

    OpenAIRE

    Graeme Ball; Justin Demmerle; Rainer Kaufmann; Ilan Davis; Dobbie, Ian M.; Lothar Schermelleh

    2015-01-01

    Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities...

  11. Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy.

    Science.gov (United States)

    Barr, Valarie A; Yi, Jason; Samelson, Lawrence E

    2017-01-01

    Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.

  12. Certain uncertainty: using pointwise error estimates in super-resolution microscopy

    CERN Document Server

    Lindén, Martin; Amselem, Elias; Elf, Johan

    2016-01-01

    Point-wise localization of individual fluorophores is a critical step in super-resolution microscopy and single particle tracking. Although the methods are limited by the accuracy in localizing individual flourophores, this point-wise accuracy has so far only been estimated by theoretical best case approximations, disregarding for example motional blur, out of focus broadening of the point spread function and time varying changes in the fluorescence background. Here, we show that pointwise localization uncertainty can be accurately estimated directly from imaging data using a Laplace approximation constrained by simple mircoscope properties. We further demonstrate that the estimated localization uncertainty can be used to improve downstream quantitative analysis, such as estimation of diffusion constants and detection of changes in molecular motion patterns. Most importantly, the accuracy of actual point localizations in live cell super-resolution microscopy can be improved beyond the information theoretic lo...

  13. Super-resolution microscopy reveals compartmentalization of peroxisomal membrane proteins

    DEFF Research Database (Denmark)

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present...... the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins....... Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5...

  14. Super-Resolution Real Imaging in Microsphere-Assisted Microscopy

    Science.gov (United States)

    Wang, Feifei; Li, Yi; Jia, Boliang; Liu, Lianqing; Li, Wen Jung

    2016-01-01

    Microsphere-assisted microscopy has received a lot of attention recently due to its simplicity and its capability to surpass the diffraction limit. However, to date, sub-diffraction-limit features have only been observed in virtual images formed through the microspheres. We show that it is possible to form real, super-resolution images using high-refractive index microspheres. Also, we report on how changes to a microsphere’s refractive index and size affect image formation and planes. The relationship between the focus position and the additional magnification factor is also investigated using experimental and theoretical methods. We demonstrate that such a real imaging mode, combined with the use of larger microspheres, can enlarge sub-diffraction-limit features up to 10 times that of wide-field microscopy’s magnification with a field-of-view diameter of up to 9 μm. PMID:27768774

  15. Wide-field multispectral super-resolution imaging using spin-dependent fluorescence in nanodiamonds.

    Science.gov (United States)

    Chen, Edward H; Gaathon, Ophir; Trusheim, Matthew E; Englund, Dirk

    2013-05-08

    Recent advances in fluorescence microscopy have enabled spatial resolution below the diffraction limit by localizing multiple temporally or spectrally distinguishable fluorophores. Here, we introduce a super-resolution technique that deterministically controls the brightness of uniquely addressable, photostable emitters. We modulate the fluorescence brightness of negatively charged nitrogen-vacancy (NV(-)) centers in nanodiamonds through magnetic resonance techniques. Using a CCD camera, this "deterministic emitter switch microscopy" (DESM) technique enables super-resolution imaging with localization down to 12 nm across a 35 × 35 μm(2) area. DESM is particularly well suited for biological applications such as multispectral particle tracking since fluorescent nanodiamonds are not only cytocompatible but also nonbleaching and bright. We observe fluorescence count rates exceeding 1.5 × 10(6) photons per second from single NV(-) centers at saturation. When combined with emerging NV(-)-based techniques for sensing magnetic and electric fields, DESM opens the door to rapid, super-resolution imaging for tracking and sensing applications in the life and physical sciences.

  16. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  17. SMILE Microscopy : fast and single-plane based super-resolution volume imaging

    CERN Document Server

    Mondal, Partha Pratim

    2016-01-01

    Fast 3D super-resolution imaging is essential for decoding rapidly occurring biological processes. Encoding single molecules to their respective planes enable simultaneous multi-plane super-resolution volume imaging. This saves the data-acquisition time and as a consequence reduce radiation-dose that lead to photobleaching and other undesirable photochemical reactions. Detection and subsequent identification of the locus of individual molecule (both on the focal plane and off-focal planes) holds the key. Experimentally, this is achieved by accurate calibration of system PSF size and its natural spread in off-focal planes using sub-diffraction fluorescent beads. Subsequently the identification and sorting of single molecules that belong to different axial planes is carried out (by setting multiple cut-offs to respective PSFs). Simultaneous Multiplane Imaging based Localization Encoded (SMILE) microscopy technique eliminates the need for multiple z-plane scanning and thereby provides a truly simultaneous multip...

  18. Polarization sensitive localization based super-resolution microscopy with a birefringent wedge

    Science.gov (United States)

    Sinkó, József; Gajdos, Tamás; Czvik, Elvira; Szabó, Gábor; Erdélyi, Miklós

    2017-03-01

    A practical method has been presented for polarization sensitive localization based super-resolution microscopy using a birefringent dual wedge. The measurement of the polarization degree at the single molecule level can reveal the chemical and physical properties of the local environment of the fluorescent dye molecule and can hence provide information about the sub-diffraction sized structure of biological samples. Polarization sensitive STORM imaging of the F-Actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.

  19. Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super-Resolution RESOLFT Microscopy.

    Science.gov (United States)

    Roubinet, Benoît; Bossi, Mariano L; Alt, Philipp; Leutenegger, Marcel; Shojaei, Heydar; Schnorrenberg, Sebastian; Nizamov, Shamil; Irie, Masahiro; Belov, Vladimir N; Hell, Stefan W

    2016-12-05

    Reversibly photoswitchable 1,2-bis(2-ethyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes (EBT) having fluorescent "closed" forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology-related super-resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.

  20. Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy.

    Science.gov (United States)

    Zhang, Zhengyang; Kenny, Samuel J; Hauser, Margaret; Li, Wan; Xu, Ke

    2015-10-01

    By developing a wide-field scheme for spectral measurement and implementing photoswitching, we synchronously obtained the fluorescence spectra and positions of ∼10(6) single molecules in labeled cells in minutes, which consequently enabled spectrally resolved, 'true-color' super-resolution microscopy. The method, called spectrally resolved stochastic optical reconstruction microscopy (SR-STORM), achieved cross-talk-free three-dimensional (3D) imaging for four dyes 10 nm apart in emission spectrum. Excellent resolution was obtained for every channel, and 3D localizations of all molecules were automatically aligned within one imaging path.

  1. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.

    Science.gov (United States)

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-08-12

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

  2. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

    Science.gov (United States)

    Ha, Taekjip; Tinnefeld, Philip

    2012-05-01

    Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own “personalities” regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

  3. Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.

    Science.gov (United States)

    Crossman, David J; Ruygrok, Peter N; Hou, Yu Feng; Soeller, Christian

    2015-03-01

    Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

  4. Spectroscopic super-resolution fluorescence cell imaging using ultra-small Ge quantum dots

    CERN Document Server

    Song, Mingying; Ersoy, Osman; Zhou, Yun; Yang, Yongxin; Zhang, Yuanpeng; Little, William R; Wheeler, Ann P; Sapelkin, Andrei V

    2015-01-01

    In single molecule localisation super-resolution microscopy the need for repeated image capture limits the imaging speed, while the size of fluorescence probes limits the possible theoretical localisation resolution. Here, we demonstrated a spectral imaging based super-resolution approach by separating the overlapping diffraction spots into several detectors during a single scanning period and taking advantage of the size-dependent emission wavelength in nanoparticles. This approach has been tested using off-the-shelf quantum dots (Qdot) and in-house novel ultra-small (~3 nm) Ge QDs. Furthermore, we developed a method-specific Gaussian fitting and maximum likelihood estimation based on a Matlab algorithm for fast QDs localisation. We demonstrate that this methodology results in ~ 40 nm localisation resolution using commercial QDs and ~12 nm localisation resolution using Ge QDs. Using a standard scanning confocal microscope we achieved data acquisition rate of 1.6 seconds/frame. However, we show that this appr...

  5. Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virus.

    Science.gov (United States)

    Müller, Barbara; Heilemann, Mike

    2013-10-01

    For more than 70 years electron microscopy (EM) techniques have played an important role in investigating structures of enveloped viruses. By contrast, use of fluorescence microscopy (FM) methods for this purpose was limited by the fact that the size of virus particles is generally around or below the diffraction limit of light microscopy. Various super-resolution (SR) fluorescence imaging techniques developed over the past two decades bypass the diffraction limit of light microscopy, allowing visualization of subviral details and bridging the gap between conventional FM and EM methods. We summarize here findings on human immunodeficiency virus (HIV-1) obtained using SR-FM techniques. Although the number of published studies is currently limited and some of the pioneering analyses also covered methodological or descriptive aspects, recent publications clearly indicate the potential to approach open questions in HIV-1 replication from a new angle.

  6. 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.

  7. 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 1.6×107 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.

  8. 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.

  9. Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

    Science.gov (United States)

    Johnson, Sam A

    2015-01-01

    Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches.

  10. 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.

  11. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    Science.gov (United States)

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T; Herbert, Alex; Lando, David; Etheridge, Thomas J; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M; Klenerman, David; Lee, Steven F

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

  12. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    Directory of Open Access Journals (Sweden)

    Matthieu Palayret

    Full Text Available Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

  13. Single-Molecule Spectroscopy, Imaging, and Photocontrol: Foundations for Super-Resolution Microscopy (Nobel Lecture).

    Science.gov (United States)

    Moerner, W E William E

    2015-07-06

    The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super-resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.

  14. Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy*

    Science.gov (United States)

    Moerner, W. E. William E.

    2015-10-01

    The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super-resolution microscopy with single molecules. In the room-temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts as a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and selected current developments are summarized.

  15. Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy

    Science.gov (United States)

    Höbartner, Claudia

    2017-01-01

    Continuous improvements in imaging techniques are challenging biologists to search for more accurate methods to label cellular elements. This is particularly relevant for diffraction-unlimited fluorescence imaging, where the perceived resolution is affected by the size of the affinity probes. This is evident when antibodies, which are 10–15 nm in size, are used. Previously it has been suggested that RNA aptamers (~3 nm) can be used to detect cellular proteins under super-resolution imaging. However, a direct comparison between several aptamers and antibodies is needed, to clearly show the advantages and/or disadvantages of the different probes. Here we have conducted such a comparative study, by testing several aptamers and antibodies using stimulated emission depletion microscopy (STED). We have targeted three membrane receptors, EGFR, ErbB2 and Epha2, which are relevant to human health, and recycle between plasma membrane and intracellular organelles. Our results suggest that the aptamers can reveal more epitopes than most antibodies, thus providing a denser labeling of the stained structures. Moreover, this improves the overall quality of the information that can be extracted from the images. We conclude that aptamers could become useful fluorescent labeling tools for light microscopy and super-resolution imaging, and that their development for novel targets is imperative. PMID:28235049

  16. Super-Resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Tool for Alzheimer's Disease Diagnostics.

    Science.gov (United States)

    Zhang, William I; Antonios, Gregory; Rabano, Alberto; Bayer, Thomas A; Schneider, Anja; Rizzoli, Silvio O

    2015-01-01

    Alzheimer's disease (AD) is neuropathologically characterized by aggregates of amyloid-β peptides (Aβ) and tau proteins. The consensus in the AD field is that Aβ and tau should serve as diagnostic biomarkers for AD. However, their aggregates have been difficult to investigate by conventional fluorescence microscopy, since their size is below the diffraction limit (∼200 nm). To solve this, we turned to a super-resolution imaging technique, stimulated emission depletion (STED) microscopy, which has a high enough precision to allow the discrimination of low- and high-molecular weight aggregates prepared in vitro. We used STED to analyze the structural organization of Aβ and tau in cerebrospinal fluid (CSF) from 36 AD patients, 11 patients with mild cognitive impairment (MCI), and 21 controls. We measured the numbers of aggregates in the CSF samples, and the aggregate sizes and intensities. These parameters enabled us to distinguish AD patients from controls with a specificity of ∼87% and a sensitivity of ∼79% . In addition, the aggregate parameters determined with STED microscopy correlated with the severity of cognitive impairment in AD patients. Finally, these parameters may be useful as predictive tools for MCI cases. The STED parameters of two MCI patients who developed AD during the course of the study, as well as of MCI patients whose Aβ ELISA values fall within the accepted range for AD, placed them close to the AD averages. We suggest that super-resolution imaging is a promising tool for AD diagnostics.

  17. Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.

    Science.gov (United States)

    Winter, Peter W; York, Andrew G; Nogare, Damian Dalle; Ingaramo, Maria; Christensen, Ryan; Chitnis, Ajay; Patterson, George H; Shroff, Hari

    2014-09-20

    Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 μm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

  18. CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

    Science.gov (United States)

    Ratz, Michael; Testa, Ilaria; Hell, Stefan W; Jakobs, Stefan

    2015-04-20

    Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

  19. Encoding and decoding spatio-temporal information for super-resolution microscopy.

    Science.gov (United States)

    Lanzanò, Luca; Coto Hernández, Iván; Castello, Marco; Gratton, Enrico; Diaspro, Alberto; Vicidomini, Giuseppe

    2015-04-02

    The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components. Here we present a robust and general method to decode this information using phasor analysis. As an example of the application of this method, we optically generate spatially controlled gradients in the fluorescence lifetime by stimulated emission. Spatial resolution can be increased indefinitely by increasing the number of resolved dynamics components up to a maximum determined by the amount of noise. We demonstrate that the proposed method provides nanoscale imaging of subcellular structures, opening new routes in super-resolution microscopy based on the encoding/decoding of spatial information through manipulation of molecular dynamics.

  20. Fourier ring correlation as a resolution criterion for super-resolution microscopy.

    Science.gov (United States)

    Banterle, Niccolò; Bui, Khanh Huy; Lemke, Edward A; Beck, Martin

    2013-09-01

    Optical nanoscopy techniques using localization based image reconstruction, also termed super-resolution microscopy (SRM), have become a standard tool to bypass the diffraction limit in fluorescence light microscopy. The localization precision measured for the detected fluorophores is commonly used to describe the maximal attainable resolution. However, this measure takes not all experimental factors, which impact onto the finally achieved resolution, into account. Several other methods to measure the resolution of super-resolved images were previously suggested, typically relying on intrinsic standards, such as molecular rulers, or on a priori knowledge about the specimen, e.g. its spatial frequency content. Here we show that Fourier ring correlation provides an easy-to-use, laboratory consistent standard for measuring the resolution of SRM images. We provide a freely available software tool that combines resolution measurement with image reconstruction.

  1. Super-resolution optical microscopy by using dielectric microwires

    Science.gov (United States)

    Darafsheh, Arash; Wu, Gaoxiang; Yang, Shu; Finlay, Jarod C.

    2016-03-01

    We demonstrate that super-resolution imaging of specimens containing sub-diffraction-limited features is feasible by using dielectric microwires fabricated through capillary force lithography followed by photopatterning. As supplementary micron scale cylindrical lenses, we fabricated uniform-sized microwires with and 5 and 10 μm diameters and refractive index ~1.3-1.6. The microwires are placed in contact with the specimen to collect the information of the sub-wavelength features of the specimen and transmit them to the far-field with magnification enabling imaging with two-fold resolution improvement. Potential applications of our imaging technique include biological imaging, microfluidics, and nanophotonics applications.

  2. Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences.

    Science.gov (United States)

    Maglione, Marta; Sigrist, Stephan J

    2013-07-01

    Light microscopy can be applied in vivo and can sample large tissue volumes, features crucial for the study of single neurons and neural circuits. However, light microscopy per se is diffraction-limited in resolution, and the substructure of core signaling compartments of neuronal circuits--axons, presynaptic active zones, postsynaptic densities and dendritic spines-can be only insufficiently characterized by standard light microscopy. Recently, several forms of super-resolution light microscopy breaking the diffraction-imposed resolution limit have started to allow highly resolved, dynamic imaging in the cell-biologically highly relevant 10-100 nanometer range ('mesoscale'). New, sometimes surprising answers concerning how protein mobility and protein architectures shape neuronal communication have already emerged. Here we start by briefly introducing super-resolution microscopy techniques, before we describe their use in the analysis of neuronal compartments. We conclude with long-term prospects for super-resolution light microscopy in the molecular and cellular neurosciences.

  3. Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection.

    Science.gov (United States)

    Zhi, Yanan; Wang, Benquan; Yao, Xincheng

    2015-01-01

    Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches-including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy-have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact-free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina.

  4. Improving spatial resolution of confocal Raman microscopy by super-resolution image restoration.

    Science.gov (United States)

    Cui, Han; Zhao, Weiqian; Wang, Yun; Fan, Ying; Qiu, Lirong; Zhu, Ke

    2016-05-16

    A new super-resolution image restoration confocal Raman microscopy method (SRIR-RAMAN) is proposed for improving the spatial resolution of confocal Raman microscopy. This method can recover the lost high spatial frequency of the confocal Raman microscopy by using Poisson-MAP super-resolution imaging restoration, thereby improving the spatial resolution of confocal Raman microscopy and realizing its super-resolution imaging. Simulation analyses and experimental results indicate that the spatial resolution of SRIR-RAMAN can be improved by 65% to achieve 200 nm with the same confocal Raman microscopy system. This method can provide a new tool for high spatial resolution micro-probe structure detection in physical chemistry, materials science, biomedical science and other areas.

  5. Correlating structure and fluorescence dynamics of quantum dot clusters using super-resolution imaging

    Science.gov (United States)

    Ryan, Duncan P.; Goodwin, Peter M.; Sheehan, Chris J.; Whitcomb, Kevin J.; Gelfand, Martin P.; Van Orden, Alan

    2016-02-01

    Clusters of quantum dots exhibit fluorescent behavior that differs from that of individual particles. Bulk measurements involving a large number of particles obscure these dynamics. Synthesizing clusters with 5-10 particles enables the study of collective behavior where single-molecule fluorescence techniques can be applied. Super-resolution microscopy of these clusters correlated with SEM imaging reveals the influence of geometry and structure on emission dynamics. Signatures of energy transfer can be seen in the form of enhanced blinking. Motion of the emission center of the cluster is tracked, made possible by the independent blinking events of the individual particles. Discrete steps in the localization are observed as random switching between various on/off configurations moves the location of the emission center.

  6. Calibration on the Spot of EMCCD Cameras for Super Resolution Microscopy

    DEFF Research Database (Denmark)

    Mortensen, Kim; Flyvbjerg, Henrik

    2013-01-01

    In single-molecule biophysics and super-resolution microscopy, fluorescent probes are routinely localized with nanometer precision in images taken, e.g., with an EMCCD camera. In such images, an isolated probe images as a diffraction-limited spot of light which was formed by a finite number...... of photons. The probe’s coordinates are estimated from the recorded camera intensities in the spot, and the error on this estimate, the localization error, is given by a mathematical formula that depends on the number of photons in the spot. Translation of measured intensities to photon numbers requires...... a calibration of the camera for the specific setting with which it is used. Here we show how this can be done post festum from just a recorded image. We demonstrate this (i) theoretically, mathematically, (ii) by analyzing images recorded with an EMCCD camera, and (iii) by analyzing simulated EMCCD images...

  7. Follow-up review: recent progress in the development of super-resolution optical microscopy.

    Science.gov (United States)

    Fujita, Katsumasa

    2016-08-01

    The advent of super-resolution microscopy brought a huge impact to various research fields ranging from the fundamental science to medical and industrial applications. The technological development is still ongoing with involving different scientific disciplines and often changing the standard of optical imaging. In this review, I would like to introduce the recent research progress in super-resolution microscopy as a follow-up for the featured issue in Microscopy (Vol. 64, No. 4, 2015) with discussions especially on the current trends and new directions in the technological development.

  8. 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.

  9. Quantum correlation enhanced super-resolution localization microscopy

    CERN Document Server

    Israel, Yonatan; Oron, Dan; Silberberg, Yaron

    2016-01-01

    In standard localization microscopy methods a small number of emitters are sparsely photoswitched, typically not more than one flourophore per diffraction limited spot, limiting the temporal resolution of super-resolved images. Localization of a non-sparse scene requires a precise estimate for the number of active emitters. Quantum correlations in the emitted fluorescence can probe the number of activated emitters, exploiting the fact that a single fluorophore emits a single photon at a time. To obtain this additional information, which is not provided by conventional cameras, we employ a new imaging configuration based on single-photon avalanche detectors (SPAD). Here we demonstrate a 20nm resolution localization and single-particle tracking (SPT) of non-sparsely activated emitters, which may facilitate super-resolved imaging at enhanced temporal resolution.

  10. A microfluidic platform for correlative live-cell and super-resolution microscopy.

    Directory of Open Access Journals (Sweden)

    Johnny Tam

    Full Text Available 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.

  11. 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.

  12. Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.

    Science.gov (United States)

    Jayasinghe, Isuru D; Clowsley, Alexander H; Munro, Michelle; Hou, Yufeng; Crossman, David J; Soeller, Christian

    2015-01-07

    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.

  13. 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.

  14. Super-resolution optical microscopy based on scannable cantilever-combined microsphere.

    Science.gov (United States)

    Wang, Shuying; Zhang, Dongxian; Zhang, Haijun; Han, Xu; Xu, Rui

    2015-12-01

    We report an ingenious method of super-resolution optical microscopy utilizing scannable cantilever-combined microsphere. By scanning the microsphere over the sample surface in a cantilever-combined microsphere-sample contact state, super-resolution images can be acquired at arbitrary sample regions through near-field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub-diffraction-limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy.

  15. Accessing the third dimension in localization-based super-resolution microscopy.

    Science.gov (United States)

    Hajj, Bassam; El Beheiry, Mohamed; Izeddin, Ignacio; Darzacq, Xavier; Dahan, Maxime

    2014-08-21

    Only a few years after its inception, localization-based super-resolution microscopy has become widely employed in biological studies. Yet, it is primarily used in two-dimensional imaging and accessing the organization of cellular structures at the nanoscale in three dimensions (3D) still poses important challenges. Here, we review optical and computational techniques that enable the 3D localization of individual emitters and the reconstruction of 3D super-resolution images. These techniques are grouped into three main categories: PSF engineering, multiple plane imaging and interferometric approaches. We provide an overview of their technical implementation as well as commentary on their applicability. Finally, we discuss future trends in 3D localization-based super-resolution microscopy.

  16. Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy.

    Science.gov (United States)

    Fitzgibbon, Jessica; Bell, Karen; King, Emma; Oparka, Karl

    2010-08-01

    We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction ("super-resolution") images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specialized pore-PD that connect sieve elements (SEs) with their companion cells (CCs) along the tubular sieve element reticulum (SER). The SER was shown to interconnect individual pore-PD at the SE-CC interface. 3D-SIM resolved fine (less than 100 nm) endoplasmic reticulum threads running into individual pore-PD as well as strands that crossed sieve plate pores, structurally linking SEs within a file. Our data reveal that MP entering the SE from the CC may remain associated with the SER. Fluorescence recovery after photobleaching experiments revealed that this MP pool is relatively immobile compared with the membrane probe 3,3'-dihexyloxacarbocyanine iodide, suggesting that MP may become sequestered by the SER once it has entered the SE. The advent of 3D-SIM offers considerable potential in the subdiffraction imaging of plant cells, bridging an important gap between confocal and electron microscopy.

  17. Wavelength scanning achieves pixel super-resolution in holographic on-chip microscopy

    Science.gov (United States)

    Luo, Wei; Göröcs, Zoltan; Zhang, Yibo; Feizi, Alborz; Greenbaum, Alon; Ozcan, Aydogan

    2016-03-01

    Lensfree holographic on-chip imaging is a potent solution for high-resolution and field-portable bright-field imaging over a wide field-of-view. Previous lensfree imaging approaches utilize a pixel super-resolution technique, which relies on sub-pixel lateral displacements between the lensfree diffraction patterns and the image sensor's pixel-array, to achieve sub-micron resolution under unit magnification using state-of-the-art CMOS imager chips, commonly used in e.g., mobile-phones. Here we report, for the first time, a wavelength scanning based pixel super-resolution technique in lensfree holographic imaging. We developed an iterative super-resolution algorithm, which generates high-resolution reconstructions of the specimen from low-resolution (i.e., under-sampled) diffraction patterns recorded at multiple wavelengths within a narrow spectral range (e.g., 10-30 nm). Compared with lateral shift-based pixel super-resolution, this wavelength scanning approach does not require any physical shifts in the imaging setup, and the resolution improvement is uniform in all directions across the sensor-array. Our wavelength scanning super-resolution approach can also be integrated with multi-height and/or multi-angle on-chip imaging techniques to obtain even higher resolution reconstructions. For example, using wavelength scanning together with multi-angle illumination, we achieved a halfpitch resolution of 250 nm, corresponding to a numerical aperture of 1. In addition to pixel super-resolution, the small scanning steps in wavelength also enable us to robustly unwrap phase, revealing the specimen's optical path length in our reconstructed images. We believe that this new wavelength scanning based pixel super-resolution approach can provide competitive microscopy solutions for high-resolution and field-portable imaging needs, potentially impacting tele-pathology applications in resource-limited-settings.

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

    Science.gov (United States)

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

    2017-02-17

    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.

  19. Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

    Science.gov (United States)

    Wang, Yilin; Kanchanawong, Pakorn

    2016-12-01

    Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

  20. DMD-based LED-illumination Super-resolution and optical sectioning microscopy

    OpenAIRE

    Dan, Dan; Ming LEI; 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 ...

  1. Next-generation biomarkers based on 100-parameter functional super-resolution microscopy TIS.

    Science.gov (United States)

    Schubert, Walter; Gieseler, Anne; Krusche, Andreas; Serocka, Peter; Hillert, Reyk

    2012-06-15

    Functional super-resolution (fSR) microscopy is based on the automated toponome imaging system (TIS). fSR-TIS provides insight into the myriad of different cellular functionalities by direct imaging of large subcellular protein networks in morphologically intact cells and tissues, referred to as the toponome. By cyclical fluorescence imaging of at least 100 molecular cell components, fSR-TIS overcomes the spectral limitations of fluorescence microscopy, which is the essential condition for the detection of protein network structures in situ/in vivo. The resulting data sets precisely discriminate between cell types, subcellular structures, cell states and diseases (fSR). With up to 16 bits per protein, the power of combinatorial molecular discrimination (PCMD) is at least 2(100) per subcellular data point. It provides the dimensionality necessary to uncover thousands of distinct protein clusters including their subcellular hierarchies controlling protein network topology and function in the one cell or tissue section. Here we review the technology and findings showing that functional protein networks of the cell surface in different cancers encompass the same hierarchical and spatial coding principle, but express cancer-specific toponome codes within that scheme (referred to as TIS codes). Findings suggest that TIS codes, extracted from large-scale toponome data, have the potential to be next-generation biomarkers because of their cell type and disease specificity. This is functionally substantiated by the observation that blocking toponome-specific lead proteins results in disassembly of molecular networks and loss of function.

  2. Super-resolution scanning laser microscopy through virtually structured detection

    OpenAIRE

    Lu, Rong-Wen; Wang, Ben-Quan; Zhang, Qiu-Xiang; Yao, Xin-Cheng

    2013-01-01

    High resolution microscopy is essential for advanced study of biological structures and accurate diagnosis of medical diseases. The spatial resolution of conventional microscopes is light diffraction limited. Structured illumination has been extensively explored to break the diffraction limit in wide field light microscopy. However, deployable application of the structured illumination in scanning laser microscopy is challenging due to the complexity of the illumination system and possible ph...

  3. STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

    Science.gov (United States)

    Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B Michael; Liersch, Torsten; Jakobs, Stefan

    2014-01-01

    Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

  4. STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

    Directory of Open Access Journals (Sweden)

    Peter Ilgen

    Full Text Available Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

  5. STED microscopy--super-resolution bio-imaging utilizing a stimulated emission depletion.

    Science.gov (United States)

    Otomo, Kohei; Hibi, Terumasa; Kozawa, Yuichi; Nemoto, Tomomi

    2015-08-01

    One of the most popular super-resolution microscopies that breaks the diffraction barrier is stimulated emission depletion (STED) microscopy. As the optical set-up of STED microscopy is based on a laser scanning microscopy (LSM) system, it potentially has several merits of LSM like confocal or two-photon excitation LSM. In this article, we first describe the principles of STED microscopy and then describe the features of our newly developed two-photon excitation STED microscopy. On the basis of our recent results and those of other researchers, we conclude by discussing future research and new technologies in this field.

  6. Nanoscale Spatial Organization of Prokaryotic Cells Studied by Super-Resolution Optical Microscopy

    Science.gov (United States)

    McEvoy, Andrea Lynn

    now see individual proteins inside of large complexes or observe structures with ten times the resolution of conventional imaging. These techniques are known as super-resolution microscopes. In this dissertation, I use super-resolution microscopes to understand how a model microbe, Escherichia coli, assembles complex protein structures. I focus on two spatially organized systems, the chemotaxis network and the cell division machinery. These assembly mechanisms could be general mechanisms for protein assembly in all organisms. I also characterize new fluorescent probes for use in multiple super-resolution imaging modalities and discuss the practicalities of using different super-resolution microscopes. The chemotaxis network in E. coli is the best understood signal transduction network in biology. Chemotaxis receptors cluster into complexes of thousands of proteins located at the cell poles and are used to move bacteria towards favorable stimuli in the environment. In these dense clusters, the receptors can bind each other and communicate to filter out noise and amplify weak signals. It is surprising that chemotaxis receptors are spatially segregated and the mechanism for polar localization of these complexes remains unclear. Using data from PALM images, we develop a model to understand how bacteria organize their receptors into large clusters. The model, stochastic cluster nucleation, is surprising in that is generates micron-scale periodic patterns without the need for accessory proteins to provide scaffolding or active transport. This model may be a general mechanism that cells utilize to organize small and large complexes of proteins. During cell division, E. coli must elongate, replicate its DNA and position its components properly prior to binary fission. Prior to septum formation, a ubiquitous protein called FtsZ, assembles into a ring at mid-cell (Z-ring) which constricts during cell division and recruits the remaining proteins necessary for cytokinesis. Though

  7. Probing nano-organization of astroglia with multi-color super-resolution microscopy.

    Science.gov (United States)

    Heller, Janosch P; Michaluk, Piotr; Sugao, Kohtaroh; Rusakov, Dmitri A

    2017-02-02

    Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca(2+) fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100β and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2016 Wiley Periodicals

  8. Optical far-field super-resolution microscopy using nitrogen vacancy center ensemble in bulk diamond

    Science.gov (United States)

    Li, Shen; Chen, Xiang-dong; Zhao, Bo-Wen; Dong, Yang; Zou, Chong-Wen; Guo, Guang-Can; Sun, Fang-Wen

    2016-09-01

    We demonstrate optical far-field super-resolution microscopy using an array of nitrogen vacancy centers in bulk diamond as near-field optical probes. The local optical field, which transmits through the nanostructures on the diamond surface, is measured by detecting the charge state conversion of the nitrogen vacancy center. Locating the nitrogen vacancy center with a spatial resolution of 6.1 nm is realized with charge state depletion nanoscopy. The nanostructures on the surface of a diamond are then imaged with a resolution below the optical diffraction limit. The results offer an approach to build a general-purpose optical super-resolution microscopy technique and a convenient platform for high spatial resolution quantum sensing with nitrogen vacancy centers.

  9. Optical far-field super-resolution microscopy using nitrogen vacancy center ensemble in bulk diamond

    CERN Document Server

    Li, Shen; Zhao, Bo-Wen; Dong, Yang; Zou, Chong-Wen; Guo, Guang-Can; Sun, Fang-Wen

    2016-01-01

    We demonstrate an optical far-field super-resolution microscopy using array of nitrogen vacancy centers in bulk diamond as near-field optical probes. The local optical field, which transmits through the nanostructures on the diamond surface, is measured by detecting the charge state conversion of nitrogen vacancy center. And the locating of nitrogen vacancy center with spatial resolution of 6.1 nm is realized with the charge state depletion nanoscopy. The nanostructures on the surface of diamond are then imaged with resolution below optical diffraction limit. The results offer an approach to built a general-purpose optical super-resolution microscopy and a convenient platform for high spatial resolution quantum sensing with nitrogen vacancy center.

  10. Super resolution microscopy of lipid bilayer phases and single molecule kinetic studies on merocyanine 540 bound lipid vesicles

    Science.gov (United States)

    Kuo, Chin-Kuei

    Recently, observing biological process and structural details in live cell became feasible after the introduction of super-resolution microscopy. Super-resolution microscopy by single molecule localization is the method that has commonly been used for such purpose. There are mainly three approaches to it: stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and point accumulation in nanoscale topology (PAINT). STORM and PALM rely on external laser control and use of photoactivable fluorescent protein or photoswitchable dyes and are technically challenging. The PAINT method relies on the control of thermal reaction rates to enable the switching between bright and dark states. Therefore, many conventional fluorescent probes can be applied in PAINT method and the images denote different information composed of interactions between the probe and its immediate environment by variations of probe parameters. The existence of lipid rafts has been under debates for decades due to the lack of a tool to directly visualize them in live cells. In the thesis, we combine PAINT with a phase sensitive dye, Merocyanine 540, to enable nanoscale observation of phase separation on supported lipid bilayers of mixed liquid/gel phases. The imaging results are presented in the chapter 3. Given that this is the first example of visualization of nanoscale phase separation of lipid bilayers using an optical microscope, we further looked into the kinetics of MC540 monomer dimer equilibrium in lipid bilayers using single molecule intensity time trajectory analysis and polarization dependent imaging. Our finding confirms that perpendicular monomeric MC540 (to the membrance surface) is the emitting speices in our system and it stays fluorescent for roughly 3 ms before it switches off to dark states. This part of analysis is presented in the chapter 4. All the materials, procedures to carry out experiments and data analysis, methods involved in our

  11. Optical far-field super-resolution microscopy using nitrogen vacancy center ensemble in bulk diamond

    OpenAIRE

    Li, Shen; Chen, Xiang-Dong; Zhao, Bo-Wen; Dong, Yang; Zou, Chong-Wen; Guo, Guang-Can; Sun, Fang-Wen

    2016-01-01

    We demonstrate an optical far-field super-resolution microscopy using array of nitrogen vacancy centers in bulk diamond as near-field optical probes. The local optical field, which transmits through the nanostructures on the diamond surface, is measured by detecting the charge state conversion of nitrogen vacancy center. And the locating of nitrogen vacancy center with spatial resolution of 6.1 nm is realized with the charge state depletion nanoscopy. The nanostructures on the surface of diam...

  12. Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy.

    Science.gov (United States)

    Xu, Jianquan; Tehrani, Kayvan F; Kner, Peter

    2015-03-24

    We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts.

  13. STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.

    Science.gov (United States)

    Jans, Daniel C; Wurm, Christian A; Riedel, Dietmar; Wenzel, Dirk; Stagge, Franziska; Deckers, Markus; Rehling, Peter; Jakobs, Stefan

    2013-05-28

    The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

  14. Second harmonic super-resolution microscopy for quantification of mRNA at single copy sensitivity.

    Science.gov (United States)

    Liu, Jing; Cho, Il-Hoon; Cui, Yi; Irudayaraj, Joseph

    2014-12-23

    Cell-specific information on the quantity and localization of key mRNAs at single copy sensitivity in single cells is critical for evaluating basic cellular process, disease risk, and efficacy of therapy. Quantification of overexpressed mRNAs beyond the diffraction limit is constrained by the optical property of the probes and microscopy techniques. In this report, nanosized barium titanium oxide (BaTiO3, BTO) crystals were utilized as probes for mRNA quantification by a second harmonic super-resolution microscopy (SHaSM). The SHaSM was able to detect a single copy of the human epidermal growth factor receptor 2 (Her2) mRNA at a resolution of 55.6 nm with the ability to resolve multiple mRNA copies in a diffraction-limited spot. Her2 mRNA per cell was counted in SK-BR-3, MCF-7, and HeLa cell lines as 595±79.1, 38.9±8.26, and 1.5±2.8, respectively. Our single-cell quantification results were validated with the fluorescence in situ hybridization studies and quantitative PCR, showing better specificity and selectivity over current single-molecule approaches for transcript detection. The SHaSM is expected to have an upper limit of resolving ∼10(4) transcripts in a single cell with the ability to monitor intracellular transcriptional dynamics at video rate. The developed approach has strong potential in clinical research and in the early diagnosis of life-threatening diseases such as cancer.

  15. Visualizing and Calculating Tip-Substrate Distance in Nanoscale Scanning Electrochemical Microscopy Using 3-Dimensional Super-Resolution Optical Imaging.

    Science.gov (United States)

    Sundaresan, Vignesh; Marchuk, Kyle; Yu, Yun; Titus, Eric J; Wilson, Andrew J; Armstrong, Chadd M; Zhang, Bo; Willets, Katherine A

    2017-01-03

    We report a strategy for the optical determination of tip-substrate distance in nanoscale scanning electrochemical microscopy (SECM) using three-dimensional super-resolution fluorescence imaging. A phase mask is placed in the emission path of our dual SECM/optical microscope, generating a double helix point spread function at the image plane, which allows us to measure the height of emitting objects relative to the focus of the microscope. By exciting both a fluorogenic reaction at the nanoscale electrode tip as well as fluorescent nanoparticles at the substrate, we are able to calculate the tip-substrate distance as the tip approaches the surface with precision better than 25 nm. Attachment of a fluorescent particle to the insulating sheath of the SECM tip extends this technique to nonfluorogenic electrochemical reactions. Correlated electrochemical and optical determination of tip-substrate distance yielded excellent agreement between the two techniques. Not only does super-resolution imaging offer a secondary feedback mechanism for measuring the tip-sample gap during SECM experiments, it also enables facile tip alignment and a strategy for accounting for electrode tilt relative to the substrate.

  16. Fundamental limits of super-resolution microscopy by dielectric microspheres and microfibers

    Science.gov (United States)

    Astratov, V. N.; Maslov, A. V.; Allen, K. W.; Farahi, N.; Li, Y.; Brettin, A.; Limberopoulos, N. I.; Walker, D. E.; Urbas, A. M.; Liberman, V.; Rothschild, M.

    2016-03-01

    In recent years, optical super-resolution by microspheres and microfibers emerged as a new paradigm in nanoscale label-free and fluorescence imaging. However, the mechanisms of such imaging are still not completely understood and the resolution values are debated. In this work, the fundamental limits of super-resolution imaging by high-index barium-titanate microspheres and silica microfibers are studied using nanoplasmonic arrays made from Au and Al. A rigorous resolution analysis is developed based on the object's convolution with the point-spread function that has width well below the conventional (~λ/2) diffraction limit, where λ is the illumination wavelength. A resolution of ~λ/6-λ/7 is demonstrated for imaging nanoplasmonic arrays by microspheres. Similar resolution was demonstrated for microfibers in the direction perpendicular to the fiber axis with hundreds of times larger field-of-view in comparison to microspheres. Using numerical solution of Maxwell's equations, it is shown that extraordinary close point objects can be resolved in the far field, if they oscillate out of phase. Possible super-resolution using resonant excitation of whispering gallery modes is also studied.

  17. Super-resolution microscopy by movable thin-films with embedded microspheres: Resolution analysis

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Kenneth W.; Farahi, Navid; Astratov, Vasily N. [Department of Physics and Optical Science, Center for Optoelectronics and Optical Communications, University of North Carolina at Charlotte, Charlotte, NC, 28223-0001 (United States); Air Force Research Laboratory, Sensors Directorate, Wright-Patterson AFB, OH (United States); Li, Yangcheng [Department of Physics and Optical Science, Center for Optoelectronics and Optical Communications, University of North Carolina at Charlotte, Charlotte, NC, 28223-0001 (United States); Limberopoulos, Nicholaos I.; Walker, Dennis E. Jr. [Air Force Research Laboratory, Sensors Directorate, Wright-Patterson AFB, OH (United States); Urbas, Augustine M. [Air Force Research Laboratory, Materials and Manufacturing Directorate, Wright Patterson AFB, OH (United States); Liberman, Vladimir [Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, Massachusetts 02420 (United States)

    2015-08-15

    Microsphere-assisted imaging has emerged as an extraordinary simple technique of obtaining optical super-resolution. This work addresses two central problems in developing this technology: (i) methodology of the resolution measurements and (ii) limited field-of-view provided by each sphere. It is suggested that a standard method of resolution analysis in far-field microscopy based on convolution with the point-spread function can be extended into the super-resolution area. This allows developing a unified approach to resolution measurements, which can be used for comparing results obtained by different techniques. To develop the surface scanning functionality, the high-index (n ∝ 2) barium titanate glass microspheres were embedded in polydimethylsiloxane (PDMS) thin-films. It is shown that such films adhere to the surface of nanoplasmonic structures so that the tips of embedded spheres experience the objects' optical near-fields. Based on rigorous criteria, the resolution ∝λ/6-λ/7 (where λ is the illumination wavelength) is demonstrated for arrays of Au dimers and bowties. Such films can be translated along the surface of investigated samples after liquid lubrication. It is shown that just after lubrication the resolution is diffraction limited, however the super-resolution gradually recovers as the lubricant evaporates. (copyright 2015 by WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Fabrication of two-color annular hybrid wave plate for three-dimensional super-resolution microscopy

    Science.gov (United States)

    Kumagai, Hiroshi; Iketaki, Yoshinori; Jahn, Kornel; Bokor, Nador

    2016-03-01

    In super-resolution microscopy, we use fluorescence depletion, where an erase beam quenches a molecule in the S1 state generated by a pump beam, and then prevents fluorescence from the S1 state. When a tight doughnut shaped erase beam with is focused on the dyed sample together with a Gaussian pump beam, the remaining fluorescence spot in the focal plane becomes smaller than the diffraction-limited size. Applying destructive interference to the erase beam, erase beam has a minute three-dimensional dark spot surrounded by the light near the focal region. Since this spot introduces fluorescence depletion along the optical axis as in the focal plane, we can achieve three-dimensional super-resolution microscopy. However, to overcome the diffraction limit, an extremely precise optical alignment is required for projecting the focused pump beam into the dark spot of the erase beam. To resolve this technical issue, we fabricated a two-color annular hybrid wave plate (TAHWP) by combining two multi-order wave quartz plates. Although the pump and erase beams co-axially pass through the plate; the pump beam retains its original Gaussian shape, while the erase beam undergoes destructive interference. Inserting the TAHWP into a commercial scanning laser microscope, a three-dimensional spherical fluorescence spot with a volume of (~100 nm)3 can be created. Beside eliminating alignment problems and yielding a compact setup, the TAHWP makes our proposed method very suitable for commercial microscope systems. In this study, we report about detailed fabrication procedure and three-dimensional image properties given by the TAHWP.

  19. VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy

    Science.gov (United States)

    Gray, Robert D. M.; Beerli, Corina; Pereira, Pedro Matos; Scherer, Kathrin Maria; Samolej, Jerzy; Bleck, Christopher Karl Ernst; Mercer, Jason; Henriques, Ricardo

    2016-01-01

    The nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. Their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. The recent development of super-resolution (SR) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. In addition, these techniques provide the added benefit of molecular specific labelling and the capacity to investigate viral structural dynamics using live-cell microscopy. However, there is a lack of robust analytical tools that allow for precise mapping of viral structure within the setting of infection. Here we present an open-source analytical framework that combines super-resolution imaging and naïve single-particle analysis to generate unbiased molecular models. This tool, VirusMapper, is a high-throughput, user-friendly, ImageJ-based software package allowing for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. We demonstrate the usability of VirusMapper by applying it to SIM and STED images of vaccinia virus in isolation and when engaged with host cells. VirusMapper allows for the generation of accurate, high-content, molecular specific virion models and detection of nanoscale changes in viral architecture. PMID:27374400

  20. Limits of single-molecule super-resolution microscopy in thin polymer films

    Science.gov (United States)

    Wang, Muzhou; Davanco, Marcelo; Marr, James M.; Liddle, J. Alexander; Gilman, Jeffrey W.

    Structural characterization by super-resolution microscopy has become increasingly widespread, particularly in the biological community. The technique is powerful because it can produce real-space images with resolutions of tens of nanometers, while sample preparation is relatively non-invasive. Previous studies have applied these techniques to important scientific problems in the life sciences, but relatively little work has explored the attainable limit of resolution using samples of known structure. In this work, we apply photo-activated localization microscopy (PALM) to polymer films that have been nanopatterned using electron-beam lithography. Trace amounts of a rhodamine spiroamide dye are dispersed into nanostructured poly(methyl methacrylate), and UV-induced switching of the fluorophores enables nanoscale localization of single molecules to generate a final composite super-resolution image. Features as small as 50 nm are clearly resolvable. To determine the ultimate resolution limit, we investigate sources of error in the system, particularly from systematic mislocalizations due to the effect of fluorophore orientation on the single-molecule point-spread function.

  1. STED super-resolution microscopy in Drosophila tissue and in mammalian cells

    Science.gov (United States)

    Lau, Lana; Lee, Yin Loon; Matis, Maja; Axelrod, Jeff; Stearns, Tim; Moerner, W. E.

    2011-03-01

    Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

  2. Super-resolution deep imaging with hollow Bessel beam STED microscopy

    CERN Document Server

    Yu, Wentao; Dong, Dashan; Yang, Xusan; Xiao, Yunfeng; Gong, Qihuang; Xi, Peng; Shi, Kebin

    2015-01-01

    Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method beating the diffraction barrier for improved lateral spatial resolution in cellular imaging, lithography, etc. Due to specimen-induced aberrations and scattering distortion, it has been a great challenge for STED to maintain consistent lateral resolution deeply inside the specimens. Here we report on a deep imaging STED microscopy by using Gaussian beam for excitation and hollow Bessel beam for depletion (GB-STED). The proposed scheme shows the improved imaging depth up to ~155{\\mu}m in solid agarose sample, ~115{\\mu}m in PDMS and ~100{\\mu}m in phantom of gray matter in brain tissue with consistent super resolution, while the standard STED microscopy shown a significantly reduced lateral resolution at the same imaging depth. The results indicate the excellent imaging penetration capability of GB-STED, making it a promising tool for deep 3D imaging optical nanoscopy and laser fabrication.

  3. Super-resolution spinning-disk confocal microscopy using optical photon reassignment.

    Science.gov (United States)

    Azuma, Takuya; Kei, Takayuki

    2015-06-01

    Spinning-disk confocal microscopy is a proven technology for investigating 3D structures of biological specimens. Here we report a super-resolution method based on spinning-disk confocal microscopy that optically improves lateral resolution by a factor of 1.37 with a single exposure. Moreover, deconvolution yields twofold improvement over the diffraction limit. With the help of newly modified Nipkow disk which comprises pinholes and micro-lenses on the front and back respectively, emitted photons from specimen can be optically reassigned to the most probable locations they originate from. Consequently, the improvement in resolution is achieved preserving inherent sectioning capabilities of confocal microscopy. This extremely simple implementation will enable reliable observations at super high resolution in biomedical routine research.

  4. 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).

  5. Single-wavelength-controlled in situ dynamic super-resolution fluorescence imaging for block copolymer nanostructures via blue-light-switchable FRAP.

    Science.gov (United States)

    Gong, Wen-Liang; Yan, Jie; Zhao, Ling-Xi; Li, Chong; Huang, Zhen-Li; Tang, Ben Zhong; Zhu, Ming-Qiang

    2016-11-02

    Photoswitchable fluorophores are promising in single-molecule optical devices and super-resolution fluorescence imaging, especially in single-molecule photo-activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). However, the scarcity of current photoswitchable fluorophores stimulates researchers to develop complicated optical systems and processing software, in accordance with the limited photoswitchable fluorescent proteins and organic fluorophores. Previous efforts to develop synthetic photoswitchable fluorophores have exhibited their promising potential in super-resolution fluorescence imaging. Here, we have designed and synthesized a fluorescence molecular switch with reversible green emission, a napthalimide-hexaarylbiimidazole conjugate (NI-N-HABI), which exhibits strong fluorescence in the emissive state, with fast thermal fading of the photochromism and spontaneous fluorescence recovery after photobleaching (FRAP) induced by blue-light. The photoswitchable fluorophore enables the red-edge wavelength of the optical response to red-shift from the initial near-UV region at less than 400 nm, to 500 nm. The relatively fast fading speed of NI-N-HABI and its sensitivity to longer blue-light irradiation (400-500 nm) have allowed simplification of the optical microscopic system from a two-wavelength laser source to a single-wavelength laser. We applied NI-N-HABI in single-wavelength-controlled in situ dynamic super-resolution fluorescence imaging for the self-assembly and solvent annealing of amphiphilic block polymers, with 50 nm of optical resolution. Single-wavelength-controlled dynamic super-resolution fluorescence imaging facilitates nanoscale optical visualization for the dynamic physical and chemical fluctuation processes of stimuli-responsive nanostructures.

  6. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    Science.gov (United States)

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-08-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

  7. Rapid super-resolution line-scanning microscopy through virtually structured detection.

    Science.gov (United States)

    Zhi, Yanan; Lu, Rongwen; Wang, Benquan; Zhang, Qiuxiang; Yao, Xincheng

    2015-04-15

    Virtually structured detection (VSD) has been demonstrated to break the diffraction limit in scanning laser microscopy (SLM). VSD provides an easy, low-cost, and phase-artifact-free strategy to achieve super-resolution imaging. However, practical application of this method is challenging due to a limited image acquisition speed. We report here the combination of VSD and line-scanning microscopy (LSM) to improve the image acquisition speed. A motorized dove prism was used to achieve automatic control of four-angle (i.e., 0°, 45°, 90°, and 135°) scanning, thus ensuring isotropic resolution improvement. Both an optical resolution target and a living frog eyecup were used to verify resolution enhancement.

  8. Super-resolution photoacoustic microscopy using photonic nanojets: a simulation study.

    Science.gov (United States)

    Upputuri, Paul Kumar; Wen, Zhuo-Bin; Wu, Zhe; Pramanik, Manojit

    2014-01-01

    Optical resolution photoacoustic microscopy (ORPAM) is important for various biomedical applications, such as the study of cellular structures, microcirculation systems, and tumor angiogenesis. However, the lateral resolution of a conventional ORPAM is limited by optical diffraction. In this work, we report a simulation study to achieve subdiffraction-limited super-resolution in ORPAM using microspheres. Laser radiation is focused through a microsphere to generate a photonic nanojet, which provides the possibility to break the diffraction limit in ORPAM by reducing the size of the excitation volume. In our simulations using microspheres, we observed improvement in the lateral resolution up to compared to conventional ORPAM. The method is simple, cost effective, and can provide far-field resolution. This approach may provide new opportunities for many biomedical imaging applications that require finer resolution.

  9. Quantum correlation enhanced super-resolution localization microscopy enabled by a fibre bundle camera.

    Science.gov (United States)

    Israel, Yonatan; Tenne, Ron; Oron, Dan; Silberberg, Yaron

    2017-03-13

    Despite advances in low-light-level detection, single-photon methods such as photon correlation have rarely been used in the context of imaging. The few demonstrations, for example of subdiffraction-limited imaging utilizing quantum statistics of photons, have remained in the realm of proof-of-principle demonstrations. This is primarily due to a combination of low values of fill factors, quantum efficiencies, frame rates and signal-to-noise characteristic of most available single-photon sensitive imaging detectors. Here we describe an imaging device based on a fibre bundle coupled to single-photon avalanche detectors that combines a large fill factor, a high quantum efficiency, a low noise and scalable architecture. Our device enables localization-based super-resolution microscopy in a non-sparse non-stationary scene, utilizing information on the number of active emitters, as gathered from non-classical photon statistics.

  10. Quantum correlation enhanced super-resolution localization microscopy enabled by a fibre bundle camera

    Science.gov (United States)

    Israel, Yonatan; Tenne, Ron; Oron, Dan; Silberberg, Yaron

    2017-03-01

    Despite advances in low-light-level detection, single-photon methods such as photon correlation have rarely been used in the context of imaging. The few demonstrations, for example of subdiffraction-limited imaging utilizing quantum statistics of photons, have remained in the realm of proof-of-principle demonstrations. This is primarily due to a combination of low values of fill factors, quantum efficiencies, frame rates and signal-to-noise characteristic of most available single-photon sensitive imaging detectors. Here we describe an imaging device based on a fibre bundle coupled to single-photon avalanche detectors that combines a large fill factor, a high quantum efficiency, a low noise and scalable architecture. Our device enables localization-based super-resolution microscopy in a non-sparse non-stationary scene, utilizing information on the number of active emitters, as gathered from non-classical photon statistics.

  11. 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.

  12. Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

    Science.gov (United States)

    Müller, Marcel; Mönkemöller, Viola; Hennig, Simon; Hübner, Wolfgang; Huser, Thomas

    2016-03-01

    Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses.

  13. Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy.

    Science.gov (United States)

    Bon, Pierre; Bourg, Nicolas; Lécart, Sandrine; Monneret, Serge; Fort, Emmanuel; Wenger, Jérôme; Lévêque-Fort, Sandrine

    2015-07-27

    Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

  14. Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy.

    Science.gov (United States)

    Notelaers, Kristof; Rocha, Susana; Paesen, Rik; Swinnen, Nina; Vangindertael, Jeroen; Meier, Jochen C; Rigo, Jean-Michel; Ameloot, Marcel; Hofkens, Johan

    2014-07-01

    In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.

  15. Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

    Science.gov (United States)

    Mönkemöller, Viola; Øie, Cristina; Hübner, Wolfgang; Huser, Thomas; McCourt, Peter

    2015-11-01

    Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.

  16. Clean localization super-resolution microscopy for 3D biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Mondal, Partha P., E-mail: partha@iap.iisc.ernet.in [Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore 560012 (India); Curthoys, Nikki M.; Hess, Samuel T. [Department of Physics and Astronomy, University of Maine, Orono, Maine 04469 (United States)

    2016-01-15

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  17. Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy.

    Directory of Open Access Journals (Sweden)

    Derek Greenfield

    2009-06-01

    Full Text Available The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

  18. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

    Science.gov (United States)

    Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

    2016-07-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

  19. Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

    Science.gov (United States)

    Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.

    2017-02-01

    Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

  20. Differential localization of SAP102 and PSD-95 is revealed in hippocampal spines using super-resolution light microscopy.

    Science.gov (United States)

    Zheng, Chan-Ying; Wang, Ya-Xia; Kachar, Bechara; Petralia, Ronald S

    2011-01-01

    Synapse-associated protein 102 (SAP102) and postsynaptic density 95 (PSD-95) are two major cytoskeleton proteins in the postsynaptic density (PSD). Both of them belong to the membrane-associated guanylate kinase (MAGUK) family, which clusters and anchors glutamate receptors and other proteins at synapses. In our previous study, we found that SAP102 and PSD-95 have different distributions, using combined light/electron microscopy (LM/EM) methods.1 Here, we double labeled endogenous SAP102 and PSD-95 in mature hippocampal neurons, and then took images by two different kinds of super resolution microscopy-Stimulated Emission Depletion microscopy (STED) and DeltaVision OMX 3D super resolution microscopy. We found that our 2D and 3D super resolution data were consistent with our previous LM/EM data, showing significant differences in the localization of SAP102 and PSD-95 in spines: SAP102 is distributed in both the PSD and cytoplasm of spines, while PSD-95 is concentrated only in the PSD area. These results indicate functional differences between SAP102 and PSD-95 in synaptic organization and plasticity.

  1. Analyzing blinking effects in super resolution localization microscopy with single-photon SPAD imagers

    Science.gov (United States)

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron; Charbon, Edoardo

    2016-02-01

    For many scientific applications, electron multiplying charge coupled devices (EMCCDs) have been the sensor of choice because of their high quantum efficiency and built-in electron amplification. Lately, many researchers introduced scientific complementary metal-oxide semiconductor (sCMOS) imagers in their instrumentation, so as to take advantage of faster readout and the absence of excess noise. Alternatively, single-photon avalanche diode (SPAD) imagers can provide even faster frame rates and zero readout noise. SwissSPAD is a 1-bit 512×128 SPAD imager, one of the largest of its kind, featuring a frame duration of 6.4 μs. Additionally, a gating mechanism enables photosensitive windows as short as 5 ns with a skew better than 150 ps across the entire array. The SwissSPAD photon detection efficiency (PDE) uniformity is very high, thanks on one side to a photon-to-digital conversion and on the other to a reduced fraction of "hot pixels" or "screamers", which would pollute the image with noise. A low native fill factor was recovered to a large extent using a microlens array, leading to a maximum PDE increase of 12×. This enabled us to detect single fluorophores, as required by ground state depletion followed by individual molecule return imaging microscopy (GSDIM). We show the first super resolution results obtained with a SPAD imager, with an estimated localization uncertainty of 30 nm and resolution of 100 nm. The high time resolution of 6.4 μs can be utilized to explore the dye's photophysics or for dye optimization. We also present the methodology for the blinking analysis on experimental data.

  2. Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

    Science.gov (United States)

    Biteen, Julie

    2013-03-01

    Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

  3. Movable thin films with embedded high-index microspheres for super-resolution microscopy

    CERN Document Server

    Allen, Kenneth W; Li, Yangcheng; Limberopoulos, Nicholaos I; Walker, Dennis E; Urbas, Augustine M; Liberman, Vladimir; Astratov, Vasily N

    2015-01-01

    Microsphere-assisted imaging emerged as a surprisingly simple way of achieving optical super-resolution imaging. In this work, we use movable PDMS thin films with embedded high-index barium titanate glass microspheres a sample scanning capability was developed, thus removing the main limitation of this technology based on its small field-of-view.

  4. Introduction to Theories of Several Super-resolution Fluorescence Microscopy Methods and Recent Advance in The Field%几种超分辨率荧光显微技术的原理和近期进展

    Institute of Scientific and Technical Information of China (English)

    吕志坚; 陆敬泽; 吴雅琼; 陈良怡

    2009-01-01

    在生命科学领域,人们常常需要在细胞内精确定位特定的蛋白质以研究其位置与功能的关系.多年来,宽场/共聚焦荧光显微镜的分辨率受限于光的阿贝/瑞利极限,不能分辨出200 nm以下的结构.近年来,随着新的荧光探针和成像理论的出现,研究者开发了多种实现超出普通共聚焦显微镜分辨率的三维超分辨率成像方法.主要介绍这些方法的原理、近期进展和发展趋势.介绍了光源的点扩散函数(point spread function,PSF)的概念和传统分辨率的定义,阐述了提高xy平面分辨率的方法.通过介绍单分子荧光成像技术,引入了单分子成像定位精度的概念,介绍了基于单分子成像的超分辨率显微成像方法,包括光激活定位显微技术(photoactivated localization microscopy,PALM)和随机光学重构显微技术(stochastic optical reconstruction microscopy,STORM).介绍了两大类通过改造光源的点扩散函数米提高成像分辨率的方法,分别是受激发射损耗显微技术(stimulated emission depletion,STED)和饱和结构照明显微技术(saturated structure illumination microscopy,SSIM).比较了不同的z轴提取信息的方法,并阐述了这些方法与xy平面上的超分辨率显微成像技术相结合所得到的各种三维超分辨率显微成像技术的优劣.探讨了目前超分辨率显微成像的发展极限和方向.

  5. Immobilization Techniques of Bacteria for Live Super-resolution Imaging Using Structured Illumination Microscopy.

    Science.gov (United States)

    Bottomley, Amy L; Turnbull, Lynne; Whitchurch, Cynthia B; Harry, Elizabeth J

    2017-01-01

    Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.

  6. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    Science.gov (United States)

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

  7. 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.

  8. 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

    Science.gov (United States)

    Dempwolff, Felix; Schmidt, Felix K.; Hervás, Ana B.; Stroh, Alex; Rösch, Thomas C.; Riese, Cornelius N.; Dersch, Simon; Heimerl, Thomas; Lucena, Daniella; Hülsbusch, Nikola; Stuermer, Claudia A. O.; Takeshita, Norio; Fischer, Reinhard; Graumann, Peter L.

    2016-01-01

    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. PMID:27362352

  9. 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.

    Science.gov (United States)

    Dempwolff, Felix; Schmidt, Felix K; Hervás, Ana B; Stroh, Alex; Rösch, Thomas C; Riese, Cornelius N; Dersch, Simon; Heimerl, Thomas; Lucena, Daniella; Hülsbusch, Nikola; Stuermer, Claudia A O; Takeshita, Norio; Fischer, Reinhard; Eckhardt, Bruno; Graumann, Peter L

    2016-06-01

    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.

  10. Development of a measurement technique for ion distribution in an extended nanochannel by super-resolution-laser-induced fluorescence.

    Science.gov (United States)

    Kazoe, Yutaka; Mawatari, Kazuma; Sugii, Yasuhiko; Kitamori, Takehiko

    2011-11-01

    Ion behavior confined in extended nanospace (10(1)-10(3) nm) is important for nanofluidics and nanochemistry with dominant surface effects. In this paper, we developed a new measurement technique of ion distribution in the nanochannel by super-resolution-laser-induced fluorescence. Stimulated emission depletion microscopy was used to achieve a spatial resolution of 87 nm higher than the diffraction limit. Fluorescein was used for ratiometric measurement of pH with two excitation wavelengths. The pH profile in a 2D nanochannel of 410 nm width and 405 nm depth was successfully measured at an uncertainty of 0.05. The excess protons, showing lower pH than the bulk, nonuniformly distributed in the nanochannel to cancel the negative charge of glass wall, especially when the electric double layer is thick compared to the channel size. The present study first revealed the ion distribution near the surface or in the nanochannel, which is directly related to the electric double layer. In addition, the obtained proton distribution is important to understand the nanoscale water structure between single molecules and continuum phase. This technique will greatly contribute to understanding the basic science in nanoscale and interfacial dynamics, which are strongly required to develop novel miniaturized systems for biochemical analysis and further applications.

  11. Out-of-focus background subtraction for fast structured illumination super-resolution microscopy of optically thick samples.

    Science.gov (United States)

    Vermeulen, P; Zhan, H; Orieux, F; Olivo-Marin, J-C; Lenkei, Z; Loriette, V; Fragola, A

    2015-09-01

    We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

  12. Spectral demixing avoids registration errors and reduces noise in multicolor localization-based super-resolution microscopy

    Science.gov (United States)

    Lampe, André; Tadeus, Georgi; Schmoranzer, Jan

    2015-09-01

    Multicolor single molecule localization-based super-resolution microscopy (SMLM) approaches are challenged by channel crosstalk and errors in multi-channel registration. We recently introduced a spectral demixing-based variant of direct stochastic optical reconstruction microscopy (SD-dSTORM) to perform multicolor SMLM with minimal color crosstalk. Here, we demonstrate that the spectral demixing procedure is inherently free of errors in multicolor registration and therefore does not require multicolor channel alignment. Furthermore, spectral demixing significantly reduces single molecule noise and is applicable to astigmatism-based 3D multicolor imaging achieving 25 nm lateral and 66 nm axial resolution on cellular nanostructures.

  13. Axial super-resolution evanescent wave tomography.

    Science.gov (United States)

    Pendharker, Sarang; Shende, Swapnali; Newman, Ward; Ogg, Stephen; Nazemifard, Neda; Jacob, Zubin

    2016-12-01

    Optical tomographic reconstruction of a three-dimensional (3D) nanoscale specimen is hindered by the axial diffraction limit, which is 2-3 times worse than the focal plane resolution. We propose and experimentally demonstrate an axial super-resolution evanescent wave tomography method that enables the use of regular evanescent wave microscopes like the total internal reflection fluorescence microscope beyond surface imaging and achieve a tomographic reconstruction with axial super-resolution. Our proposed method based on Fourier reconstruction achieves axial super-resolution by extracting information from multiple sets of 3D fluorescence images when the sample is illuminated by an evanescent wave. We propose a procedure to extract super-resolution features from the incremental penetration of an evanescent wave and support our theory by one-dimensional (along the optical axis) and 3D simulations. We validate our claims by experimentally demonstrating tomographic reconstruction of microtubules in HeLa cells with an axial resolution of ∼130  nm. Our method does not require any additional optical components or sample preparation. The proposed method can be combined with focal plane super-resolution techniques like stochastic optical reconstruction microscopy and can also be adapted for THz and microwave near-field tomography.

  14. 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).

  15. 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.

  16. Structured illumination-based super-resolution optical microscopy for hemato- and cyto-pathology applications.

    Science.gov (United States)

    Zhang, Tieqiao; Osborn, Samantha; Brandow, Chloe; Dwyre, Denis; Green, Ralph; Lane, Stephen; Wachsmann-Hogiu, Sebastian

    2013-01-01

    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.

  17. High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers

    Science.gov (United States)

    Siegel, Nisan; Lupashin, Vladimir; Storrie, Brian; Brooker, Gary

    2016-12-01

    Fresnel incoherent correlation holography (FINCH) microscopy is a promising approach for high-resolution biological imaging but has so far been limited to use with low-magnification, low-numerical-aperture configurations. We report the use of in-line incoherent interferometers made from uniaxial birefringent α-barium borate (α-BBO) or calcite crystals that overcome the aberrations and distortions present with previous implementations that employed spatial light modulators or gradient refractive index lenses. FINCH microscopy incorporating these birefringent elements and high-numerical-aperture oil immersion objectives could outperform standard wide-field fluorescence microscopy, with, for example, a 149 nm lateral point spread function at a wavelength of 590 nm. Enhanced resolution was confirmed with sub-resolution fluorescent beads. Taking the Golgi apparatus as a biological example, three different proteins labelled with GFP and two other fluorescent dyes in HeLa cells were resolved with an image quality that is comparable to similar samples captured by structured illumination microscopy.

  18. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    Directory of Open Access Journals (Sweden)

    Alice C N Brown

    2011-09-01

    Full Text Available Natural Killer (NK cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  19. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    Science.gov (United States)

    Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M

    2011-09-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  20. Sectioning and super-resolution using unknown random patterns

    Science.gov (United States)

    Hoffman, Zachary R.; DiMarzio, Charles A.

    2016-03-01

    Random structured illumination patterns are used to demonstrate effective sectioning as well as super-resolution images in conjunction with an incoherent light source. By projecting patterns of varied spatial frequencies and using blind deconvolution of an unknown point spread function, super-resolution is achieved. Random patterns produce more consistent sectioning and super-resolution given an unknown optical transfer function. Further, using a randomly distributed pattern provides a low cost solution to obtaining information similar to that produced in confocal microscopy and other methods of structured illumination, without the requirement of precise projection patterns, coherent light sources, or fluorescence.

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

    CSIR Research Space (South Africa)

    Henriques, R

    2011-05-01

    Full Text Available Live-cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super-resolution imaging systems has pushed light microscopy into the realm...

  2. Origin and compensation of imaging artefacts in localization-based super-resolution microscopy.

    Science.gov (United States)

    Erdélyi, M; Sinkó, J; Kákonyi, R; Kelemen, A; Rees, E; Varga, D; Szabó, G

    2015-10-15

    Interpretation of high resolution images provided by localization-based microscopy techniques is a challenge due to imaging artefacts that can be categorized by their origin. They can be introduced by the optical system, by the studied sample or by the applied algorithms. Some artefacts can be eliminated via precise calibration procedures, others can be reduced only below a certain value. Images studied both theoretically and experimentally are qualified either by pattern specific metrics or by a more general metric based on fluorescence correlation spectroscopy.

  3. Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy.

    Science.gov (United States)

    Winter, Peter W; Chandris, Panagiotis; Fischer, Robert S; Wu, Yicong; Waterman, Clare M; Shroff, Hari

    2015-02-23

    Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.

  4. Super resolution microscopy is poised to reveal new insights into the formation and maturation of dendritic spines [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Cristina M. Robinson

    2016-06-01

    Full Text Available Dendritic spines and synapses are critical for neuronal communication, and they are perturbed in many neurological disorders; however, the study of these structures in living cells has been hindered by their small size. Super resolution microscopy, unlike conventional light microscopy, is diffraction unlimited and thus is well suited for imaging small structures, such as dendritic spines and synapses. Super resolution microscopy has already revealed important new information about spine and synapse morphology, actin remodeling, and nanodomain composition in both healthy cells and diseased states. In this review, we highlight the advancements in probes that make super resolution more amenable to live-cell imaging of spines and synapses. We also discuss recent data obtained by super resolution microscopy that has advanced our knowledge of dendritic spine and synapse structure, organization, and dynamics in both healthy and diseased contexts. Finally, we propose a series of critical questions for understanding spine and synapse formation and maturation that super resolution microscopy is poised to answer.

  5. Direct optical sensing of single unlabeled small proteins and super-resolution microscopy of their binding sites

    CERN Document Server

    Piliarik, Marek

    2013-01-01

    More than twenty years ago, scientists succeeded in pushing the limits of optical detection to single molecules using fluorescence. This breakthrough has revolutionized biophysical measurements, but restrictions in photophysics and labeling protocols have motivated many efforts to achieve fluorescence-free single-molecule sensitivity in biological studies. Although several interesting mechanisms using vibrational spectroscopy, photothermal detection, plasmonics or microcavities have been proposed for biosensing at the single-protein level, no method has succeeded in direct label-free detection of single proteins. Here, we present the first results using interferometric detection of scattering (iSCAT) from single proteins without the need for any label, optical nanostructure or microcavity. Furthermore, we demonstrate super-resolution imaging of protein binding with nanometer localization precision. The ease of iSCAT instrumentation promises a breakthrough for industrial usage as well as fundamental laboratory...

  6. Super resolution imaging of HER2 gene amplification

    Science.gov (United States)

    Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

    2016-02-01

    HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

  7. 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

  8. 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

    2015-11-26

    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

  9. Super-Resolution Microscopy Reveals Altered Desmosomal Protein Organization in Tissue from Patients with Pemphigus Vulgaris.

    Science.gov (United States)

    Stahley, Sara N; Warren, Maxine F; Feldman, Ron J; Swerlick, Robert A; Mattheyses, Alexa L; Kowalczyk, Andrew P

    2016-01-01

    Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease in which autoantibodies (IgG) are directed against the desmosomal cadherin desmoglein 3. To better understand how PV IgG alters desmosome morphology and function in vivo, biopsies from patients with PV were analyzed by structured illumination microscopy, a form of superresolution fluorescence microscopy. In patient tissue, desmosomal proteins were aberrantly clustered and patient IgG colocalized with markers for lipid rafts and endosomes. Additionally, steady-state levels of desmoglein 3 were decreased and desmosomes were reduced in size in patient tissue. Desmosomes at blister sites were occasionally split, with PV IgG decorating the extracellular faces of split desmosomes. Desmosome splitting was recapitulated in vitro by exposing cultured keratinocytes both to PV IgG and to mechanical stress, demonstrating that splitting at the blister interface in patient tissue is due to compromised desmosomal adhesive function. These findings indicate that desmoglein 3 clustering and endocytosis are associated with reduced desmosome size and adhesion defects in tissue of patients with PV. Further, this study reveals that superresolution optical imaging is a powerful approach for studying epidermal adhesion structures in normal and diseased skin.

  10. Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

    OpenAIRE

    Zhi, Yanan; Wang, Benquan; Yao, Xincheng

    2015-01-01

    Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achie...

  11. Measuring Exciton Diffusion in Conjugated Polymer Films with Super-resolution Microscopy

    Science.gov (United States)

    Penwell, Samuel; Ginsberg, Lucas; Noriega Manez, Rodrigo; Ginsberg, Naomi

    2015-03-01

    Conjugated polymers are highly tunable organic semiconductors, which can be solution processed to form thin films, making them prime candidates for organic photovoltaic devices. One of the most important parameters in a conjugated polymer solar cell is the exciton diffusion length, which depends on intermolecular couplings, and is typically on the order of 10 nm. This mean exciton migration can vary dramatically between films and within a single film due to heterogeneities in morphology on length scales of 10's to 100's nm. To study the variability of exciton diffusion and morphology within individual conjugated polymer films, we are adapting stimulated emission depletion microscopy. STED is typically used in biology with well-engineered fluorescent labels or on NV-centers in diamond. I will, however, describe how we have demonstrated STED in conjugated polymer films of MEH-PPV and CN-PPV by taking care to first understand the film's photophysical properties. This new approach provides a way to study exciton diffusion by utilizing subdiffraction optical excitation volumes. In this way, we will obtain a spatiotemporal map of exciton distributions that will help to correlate the energetic landscape to film morphology at the nanoscale. This research is supported in part by the Department of Energy Office of Science Graduate Fellowship Program (DOE SCGF), made possible in part by the American Recovery and Reinvestment Act of 2009, administered by ORISE-ORAU under Contract No. DE-AC05-06.

  12. Fast Super-Resolution Imaging with Ultra-High Labeling Density Achieved by Joint Tagging Super-Resolution Optical Fluctuation Imaging (JT-SOFI)

    CERN Document Server

    Zeng, Zhiping; Wang, Hening; Huang, Ning; Shan, Chunyan; Zhang, Hao; Teng, Junlin; Xi, Peng

    2015-01-01

    Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition....

  13. Super-resolution fluorescence imaging and correlation spectroscopy: Principles and examples of application

    Directory of Open Access Journals (Sweden)

    Jovanović-Talisman Tijana

    2013-01-01

    Full Text Available Self-organization of cell-surface receptors in structurally distinct domains in the plasma membrane is of vital interest for correct cellular signaling. However, this dynamic process is difficult to study in cells with sufficiently high temporal and spatial resolution. We present here two quantitative high-resolution methods with single-molecule sensitivity, Fluorescence Correlation Spectroscopy (FCS and pair-correlation Photoactivated Localization Microscopy (pcPALM, which enable nondestructive study of receptor diffusion and lateral organization at the nanoscale level. We introduce here the methods and review their application in studies of lateral organization of G Protein-Coupled Receptors (GPCRs. Examples from our own work on opioid receptor lateral organization are presented in order to illustrate the most recent advances in the field. [Projekat Ministarstva nauke Republike Srbije, br. 172015 i br. 45001

  14. Unidirectional Living Growth of Self-Assembled Protein Nanofibrils Revealed by Super-resolution Microscopy

    NARCIS (Netherlands)

    Beun, Lennart H.; Albertazzi, Lorenzo; Zwaag, van der Daan; Vries, de Renko; Cohen Stuart, Martien A.

    2016-01-01

    Protein-based nanofibrils are emerging as a promising class of materials that provide unique properties for applications such as biomedical and food engineering. Here, we use atomic force microscopy and stochastic optical reconstruction microscopy imaging to elucidate the growth dynamics, exchang

  15. Cellular cartography : mapping the neuronal microtubule network using super-resolution microscopy

    NARCIS (Netherlands)

    Cloin, B.M.C.

    2016-01-01

    Described in this thesis are the development and use of novel single molecule localization microscopy technologies to gain new insights into (neuronal) microtubule organization. The image quality of single molecule localization microscopy (SMLM) depends on a sound optical setup. Aberrations introduc

  16. Optimized localization analysis for single-molecule tracking and super-resolution microscopy

    DEFF Research Database (Denmark)

    Mortensen, Kim; Churchman, L. S.; Spudich, J. A.;

    2010-01-01

    We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitt......We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least...

  17. Note on the classification of super-resolution in far-field microscopy and information theory

    CERN Document Server

    Passon, Oliver

    2016-01-01

    In recent years several far-field microscopy techniques have been developed which manage to overcome the diffraction limit of resolution. A unifying classification scheme for them is clearly desirable. We argue that existing schemes based on the information capacity of the optical system can not easily be extended to cover e.g., STED microscopy or techniques based on single molecule imaging. We suggest a classification based on a reconstruction of the Abbe limit.

  18. Note on the classification of super-resolution in far-field microscopy and information theory

    Science.gov (United States)

    Passon, Oliver; Grebe-Ellis, Johannes

    2016-07-01

    In recent years several far-field microscopy techniques have been developed which manage to overcome the diffraction limit of resolution. A unifying classification scheme for them is clearly desirable. We argue that existing schemes based on the information capacity of the optical system can not easily be extended to cover e.g., STED microscopy or techniques based on single molecule imaging. We suggest a classification based on a reconstruction of the Abbe limit.

  19. DURIP: Super-Resolution Module for Confocal Microscopy of Reconfigurable Matter

    Science.gov (United States)

    2014-09-28

    Proceeding publications (other than abstracts): (d) Manuscripts Received Paper TOTAL: Received Paper TOTAL: Received Paper TOTAL: Received Book TOTAL: Patents ...Submitted Patents Awarded Awards Graduate Students No Patents Submitted No Patents Awarded No Honors and Awards to Report Names of Post Doctorates...anisotropic particles, which explains our interest in this microscopy method. Materials and Methods. 200 and 310 nm diameter polystyrene (PS) beads

  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. Clustering and Functional Coupling of Diverse Ion Channels and Signaling Proteins Revealed by Super-resolution STORM Microscopy in Neurons.

    Science.gov (United States)

    Zhang, Jie; Carver, Chase M; Choveau, Frank S; Shapiro, Mark S

    2016-10-19

    The fidelity of neuronal signaling requires organization of signaling molecules into macromolecular complexes, whose components are in intimate proximity. The intrinsic diffraction limit of light makes visualization of individual signaling complexes using visible light extremely difficult. However, using super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K(+), L-type Ca(2+), or TRPV1 channels) and G protein-coupled receptors coupled by the scaffolding protein A-kinase-anchoring protein (AKAP)79/150. Some channels assembled as multi-channel supercomplexes. Surprisingly, we identified novel layers of interplay within macromolecular complexes containing diverse channel types at the single-complex level in sensory neurons, dependent on AKAP79/150. Electrophysiological studies revealed that such ion channels are functionally coupled as well. Our findings illustrate the novel role of AKAP79/150 as a molecular coupler of different channels that conveys crosstalk between channel activities within single microdomains in tuning the physiological response of neurons.

  2. Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.

    Energy Technology Data Exchange (ETDEWEB)

    Carson, Bryan D.; Aaron, Jesse S.; Timlin, Jerilyn Ann

    2010-10-01

    Over the past decade optical approaches were introduced that effectively break the diffraction barrier. Of particular note were introductions of Stimulated Emission/Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), and the closely related Stochastic Optical Reconstruction Microscopy (STORM). STORM represents an attractive method for researchers, as it does not require highly specialized optical setups, can be implemented using commercially available dyes, and is more easily amenable to multicolor imaging. We implemented a simultaneous dual-color, direct-STORM imaging system through the use of an objective-based TIRF microscope and filter-based image splitter. This system allows for excitation and detection of two fluorophors simultaneously, via projection of each fluorophor's signal onto separate regions of a detector. We imaged the sub-resolution organization of the TLR4 receptor, a key mediator of innate immune response, after challenge with lipopolysaccharide (LPS), a bacteria-specific antigen. While distinct forms of LPS have evolved among various bacteria, only some LPS variations (such as that derived from E. coli) typically result in significant cellular immune response. Others (such as from the plague bacteria Y. pestis) do not, despite affinity to TLR4. We will show that challenge with LPS antigens produces a statistically significant increase in TLR4 receptor clusters on the cell membrane, presumably due to recruitment of receptors to lipid rafts. These changes, however, are only detectable below the diffraction limit and are not evident using conventional imaging methods. Furthermore, we will compare the spatiotemporal behavior of TLR4 receptors in response to different LPS chemotypes in order to elucidate possible routes by which pathogens such as Y. pestis are able to circumvent the innate immune system. Finally, we will exploit the dual-color STORM capabilities to simultaneously image LPS and TLR4 receptors in the

  3. Lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution.

    Science.gov (United States)

    Isikman, Serhan O; Bishara, Waheb; Ozcan, Aydogan

    2012-08-16

    Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos. Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of <1 μm x <1 μm x <3 μm in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm(3), and can be particularly useful for lab-on-a-chip platforms.

  4. Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm

    Science.gov (United States)

    El Khatib, Mariam; Martins, Alexandre; Bourgeois, Dominique; Colletier, Jacques-Philippe; Adam, Virgile

    2016-01-01

    Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments. PMID:26732634

  5. Super-Resolution Molecular and Functional imaging of Nanoscale Architectures in Life and Materials Science

    Directory of Open Access Journals (Sweden)

    Satoshi eHabuchi

    2014-06-01

    Full Text Available Super-resolution 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 super-resolution fluorescence microscopy techniques along with the latest developments of fluorophores and labeling for the super-resolution microscopy. I discuss the applications of super-resolution 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.

  6. 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.

  7. Super-resolution microscopy reveals structural diversity in molecular exchange among peptide amphiphile nanofibres

    Science.gov (United States)

    da Silva, Ricardo M. P.; van der Zwaag, Daan; Albertazzi, Lorenzo; Lee, Sungsoo S.; Meijer, E. W.; Stupp, Samuel I.

    2016-05-01

    The dynamic behaviour of supramolecular systems is an important dimension of their potential functions. Here, we report on the use of stochastic optical reconstruction microscopy to study the molecular exchange of peptide amphiphile nanofibres, supramolecular systems known to have important biomedical functions. Solutions of nanofibres labelled with different dyes (Cy3 and Cy5) were mixed, and the distribution of dyes inserting into initially single-colour nanofibres was quantified using correlative image analysis. Our observations are consistent with an exchange mechanism involving monomers or small clusters of molecules inserting randomly into a fibre. Different exchange rates are observed within the same fibre, suggesting that local cohesive structures exist on the basis of β-sheet discontinuous domains. The results reported here show that peptide amphiphile supramolecular systems can be dynamic and that their intermolecular interactions affect exchange patterns. This information can be used to generate useful aggregate morphologies for improved biomedical function.

  8. Novel organic dyes for multicolor localization-based super-resolution microscopy.

    Science.gov (United States)

    Lehmann, Martin; Lichtner, Gregor; Klenz, Haider; Schmoranzer, Jan

    2016-01-01

    Precise multicolor single molecule localization-based microscopy (SMLM) requires bright probes with compatible photo-chemical and spectral properties to resolve distinct molecular species at the nanoscale. The accuracy of multicolor SMLM is further challenged by color channel crosstalk and chromatic alignment errors. These constrains limit the applicability of known reversibly switchable organic dyes for optimized multicolor SMLM. Here, we tested 28 commercially available dyes for their suitability to super-resolve a known cellular nanostructure. We identified eight novel dyes in different spectral regimes that enable high quality dSTORM imaging. Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing-based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3-color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.

  9. Super-resolution microscopy reveals γ-secretase at both sides of the neuronal synapse.

    Science.gov (United States)

    Schedin-Weiss, Sophia; Caesar, Ina; Winblad, Bengt; Blom, Hans; Tjernberg, Lars O

    2016-03-31

    The transmembrane protein assembly γ-secretase is a key protease in regulated intramembrane processing (RIP) of around 100 type-1 transmembrane proteins. Importantly, it has a pathological role in Alzheimer disease (AD) as it generates the neurotoxic amyloid β-peptide from the amyloid precursor protein (APP). Studies on γ-secretase location are therefore crucial both from a biological and a therapeutic perspective. Despite several years of efforts in many laboratories, it is not clear where in the neuron γ-secretase exerts it's activities. Technical challenges include the fact that the active enzyme contains four protein components and that most subcellular compartments cannot be spatially resolved by traditional light microscopy. Here, we have used a powerful combination of the two nanoscopy techniques STORM and STED microscopy to visualize the location of γ-secretase in neurons using an active-site specific probe, with a focus on the synapse. We show that γ-secretase is present in both the pre-and postsynaptic compartments. We further show that the enzyme is enriched very close to the synaptic cleft in the postsynaptic membrane, as well as to NMDA receptors, demonstrating that γ-secretase is present in the postsynaptic plasma membrane. Importantly, the expression of γ-secretase increased in the pre- and postsynaptic compartments with the size of the synapse, suggesting a correlation between γ-secretase activity and synapse maturation. Thus, our data shows the synaptic location with high precision in three dimensions and settles the long-lasting debate on the synaptic location of γ-secretase.

  10. Trade-offs between spatial and temporal resolutions in stochastic super-resolution microscopy techniques

    CERN Document Server

    Rupprecht, Jean-Francois; Tessier, Gilles

    2016-01-01

    Widefield stochastic microscopy techniques such as PALM or STORM rely on the progressive accumulation of a large number of frames, each containing a scarce number of super-resolved point images. We justify that the redundancy in the localization of detected events imposes a specific limit on the temporal resolution. Based on a theoretical model, we derive analytical predictions for the minimal time required to obtain a reliable image at a given spatial resolution, called image completion time. In contrast to standard assumptions, we find that the image completion time scales logarithmically with the ratio of the image size by the spatial resolution volume. We justify that this non-linear relation is the hallmark of a random coverage problem. We propose a method to estimate the risk that the image reconstruction is not complete, which we apply to an experimental data set. Our results provide a theoretical framework to quantify the pattern detection efficiency and to optimize the trade-off between image coverag...

  11. 光学超分辨荧光显微成像--2014年诺贝尔化学奖解析%Super-resolution fluorescent microscopy:A brief introduction to the Nobel Prize in Chemistry 2014

    Institute of Scientific and Technical Information of China (English)

    纪伟; 徐涛; 刘贝

    2014-01-01

    Super-resolution fluorescent microscopy becomes a powerful tool for biomedical research, and extent the application of fluorescent microscopy to a brand new level. The Royal Swedish Academy of Sciences decided to award Erik Betzig, Stefan W. Hell and W. E. Moerner the Nobel Prize in Chemistry 2014 for the development of super-resolution fluorescence microscopy. Their award proved the importance of this multidisciplinary field consist of chemistry, biology and physics. In this article, we briefly introduced the historical background of super-resolution imaging, and dissect the born and development of each techniques. Finally, the current problems and the challenges for future research were presented.%超分辨成像显微镜的出现为现代生物医学研究提供了新的强有力的工具,将荧光显微镜的应用推到了新的高度。2014年诺贝尔化学奖授予了Eric Betzig、Stefan Hell以及William Moerner三位科学家,以表彰他们在“发展超高分辨荧光显微镜”上的贡献。他们的获奖肯定了化学生物物理多学科交叉对于当今前沿科技发展的重要性。本文主要介绍了超分辨荧光显微成像诞生的历史背景,以及各成像技术的发生发展过程,最后对此技术的未来发展做了展望。

  12. Marker-Less Stage Drift Correction in Super-Resolution Microscopy Using the Single-Cluster PHD Filter

    NARCIS (Netherlands)

    Schlangen, I. (Isabel); Franco, J. (José); Houssineau, J. (Jérémie); Pitkeathly, W.T.E. (William T.E.); Clark, D. (Daniel); I. Smal (Ihor); Rickman, C. (Colin)

    2016-01-01

    textabstractFluorescence microscopy is a technique which allows the imaging of cellular and intracellular dynamics through the activation of fluorescent molecules attached to them. It is a very important technique because it can be used to analyze the behavior of intracellular processes in vivo in c

  13. Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

    Science.gov (United States)

    Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

    2016-01-01

    Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

  14. 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.

  15. In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy.

    Science.gov (United States)

    Buss, Jackson; Coltharp, Carla; Huang, Tao; Pohlmeyer, Chris; Wang, Shih-Chin; Hatem, Christine; Xiao, Jie

    2013-09-01

    In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule-based super-resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments. © 2013 John Wiley & Sons Ltd.

  16. Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm

    Science.gov (United States)

    Zhang, Peng; Kim, Kyungsoo; Lee, Seungah; Chakkarapani, Suresh Kumar; Fang, Ning; Kang, Seong Ho

    2016-09-01

    Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D.

  17. 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.

  18. Fast super-resolution imaging with ultra-high labeling density achieved by joint tagging super-resolution optical fluctuation imaging.

    Science.gov (United States)

    Zeng, Zhiping; Chen, Xuanze; Wang, Hening; Huang, Ning; Shan, Chunyan; Zhang, Hao; Teng, Junlin; Xi, Peng

    2015-02-10

    Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, thereby bring it closer to single-molecule state, we can faithfully reconstruct the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods.

  19. Fast Super-Resolution Imaging with Ultra-High Labeling Density Achieved by Joint Tagging Super-Resolution Optical Fluctuation Imaging

    Science.gov (United States)

    Zeng, Zhiping; Chen, Xuanze; Wang, Hening; Huang, Ning; Shan, Chunyan; Zhang, Hao; Teng, Junlin; Xi, Peng

    2015-01-01

    Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, thereby bring it closer to single-molecule state, we can faithfully reconstruct the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods. PMID:25665878

  20. 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.

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

    Science.gov (United States)

    Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U; Westerling, Katherine M; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L

    2015-01-01

    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.

  2. Axial Super-resolution Evanescent Wave Tomography

    CERN Document Server

    Pendharker, Sarang; Newman, Ward; Ogg, Stephen; Nazemifard, Neda; Jacob, Zubin

    2016-01-01

    Optical tomographic reconstruction of a 3D nanoscale specimen is hindered by the axial diffraction limit, which is 2-3 times worse than the focal plane resolution. We propose and experimentally demonstrate an axial super-resolution evanescent wave tomography (AxSET) method that enables the use of regular evanescent wave microscopes like Total Internal Reflection Fluorescence Microscope (TIRF) beyond surface imaging, and achieve tomographic reconstruction with axial super-resolution. Our proposed method based on Fourier reconstruction achieves axial super-resolution by extracting information from multiple sets of three-dimensional fluorescence images when the sample is illuminated by an evanescent wave. We propose a procedure to extract super-resolution features from the incremental penetration of an evanescent wave and support our theory by 1D (along the optical axis) and 3D simulations. We validate our claims by experimentally demonstrating tomographic reconstruction of microtubules in HeLa cells with an axi...

  3. Super-resolution

    DEFF Research Database (Denmark)

    Nasrollahi, Kamal; Moeslund, Thomas B.

    2014-01-01

    and aerial imaging to medical image processing, to facial image analysis, text image analysis, sign and number plates reading, and biometrics recognition, to name a few. This has resulted in many research papers, each developing a new super-resolution algorithm for a specific purpose. The current......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...... the contributions of different authors to the basic concepts of each group. Furthermore, common issues in super-resolution algorithms, such as imaging models and registration algorithms, optimization of the cost functions employed, dealing with color information, improvement factors, assessment of super...

  4. 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...... the contributions of different authors to the basic concepts of each group. Furthermore, common issues in super-resolution algorithms, such as imaging models and registration algorithms, optimization of the cost functions employed, dealing with color information, improvement factors, assessment of super...

  5. Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

    Science.gov (United States)

    Chizhik, Anna M; Stein, Simon; Dekaliuk, Mariia O; Battle, Christopher; Li, Weixing; Huss, Anja; Platen, Mitja; Schaap, Iwan A T; Gregor, Ingo; Demchenko, Alexander P; Schmidt, Christoph F; Enderlein, Jörg; Chizhik, Alexey I

    2016-01-13

    Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

  6. Axial super-resolution evanescent wave tomography

    Science.gov (United States)

    Pendharker, Sarang; Shende, Swapnali; Newman, Ward; Ogg, Stephen; Nazemifard, Neda; Jacob, Zubin

    2016-12-01

    Optical tomographic reconstruction of a 3D nanoscale specimen is hindered by the axial diffraction limit, which is 2-3 times worse than the focal plane resolution. We propose and experimentally demonstrate an axial super-resolution evanescent wave tomography (AxSET) method that enables the use of regular evanescent wave microscopes like Total Internal Reflection Fluorescence Microscope (TIRF) beyond surface imaging, and achieve tomographic reconstruction with axial super-resolution. Our proposed method based on Fourier reconstruction achieves axial super-resolution by extracting information from multiple sets of three-dimensional fluorescence images when the sample is illuminated by an evanescent wave. We propose a procedure to extract super-resolution features from the incremental penetration of an evanescent wave and support our theory by 1D (along the optical axis) and 3D simulations. We validate our claims by experimentally demonstrating tomographic reconstruction of microtubules in HeLa cells with an axial resolution of $\\sim$130 nm. Our method does not require any additional optical components or sample preparation. The proposed method can be combined with focal plane super-resolution techniques like STORM and can also be adapted for THz and microwave near-field tomography.

  7. 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.

  8. 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.

  9. A theoretical analysis of the super-resolution capacity of imagers using speckle illuminations

    CERN Document Server

    Idier, Jérôme; Liu, Penghuan; Allain, Marc; Bourguignon, Sébastien; Sentenac, Anne

    2015-01-01

    Speckle based imaging consists in forming a super-resolved reconstruction of an unknown object from low-resolution images obtained under random inhomogeneous illuminations (speckles). However, the origin of this super-resolution is unclear. In this work, we demonstrate that, under physically realistic conditions, the correlation of the data have a super-resolution power corresponding to the squaring of the imager point spread function. This theoretical result is important for many practical imaging systems such as acoustic and electromagnetic tomographies, fluorescence and photoacoustic microscopies or synthetic aperture radar imaging.

  10. Fluorescence antibunching microscopy

    CERN Document Server

    Schwartz, Osip

    2011-01-01

    Breaking the diffraction limit in microscopy by utilizing quantum properties of light has been the goal of intense research in the recent years. We propose a quantum superresolution technique based on non-classical emission statistics of fluorescent markers, routinely used as contrast labels for bio-imaging. The technique can be readily implemented using standard fluorescence microscopy equipment.

  11. LEDs for fluorescence microscopy

    NARCIS (Netherlands)

    Young, I.T.; Garini, Y.; Dietrich, H.R.C.; Van Oel, W.; Liqui Lung, G.

    2004-01-01

    Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light

  12. Assessing resolution in super-resolution imaging.

    Science.gov (United States)

    Demmerle, Justin; Wegel, Eva; Schermelleh, Lothar; Dobbie, Ian M

    2015-10-15

    Resolution is a central concept in all imaging fields, and particularly in optical microscopy, but it can be easily misinterpreted. The mathematical definition of optical resolution was codified by Abbe, and practically defined by the Rayleigh Criterion in the late 19th century. The limit of conventional resolution was also achieved in this period, and it was thought that fundamental constraints of physics prevented further increases in resolution. With the recent development of a range of super-resolution techniques, it is necessary to revisit the concept of optical resolution. Fundamental differences in super-resolution modalities mean that resolution is not a directly transferrable metric between techniques. This article considers the issues in resolution raised by these new technologies, and presents approaches for comparing resolution between different super-resolution methods.

  13. High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization.

    Science.gov (United States)

    Holden, Seamus J; Pengo, Thomas; Meibom, Karin L; Fernandez Fernandez, Carmen; Collier, Justine; Manley, Suliana

    2014-03-25

    We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

  14. Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

    Science.gov (United States)

    Bongiovanni, Marie N.; Godet, Julien; Horrocks, Mathew H.; Tosatto, Laura; Carr, Alexander R.; Wirthensohn, David C.; Ranasinghe, Rohan T.; Lee, Ji-Eun; Ponjavic, Aleks; Fritz, Joelle V.; Dobson, Christopher M.; Klenerman, David; Lee, Steven F.

    2016-12-01

    Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

  15. Away from resolution, assessing the information content of super-resolution images

    CERN Document Server

    Pengo, Thomas; Manley, Suliana

    2015-01-01

    Super-resolution microscopy has revolutionized optical fluorescence imaging by improving 3D resolution by 1-2 orders of magnitude. While different methods can successfully increase the resolution, all methods share significant differences with standard imaging methods, making the usual measures of resolution inapplicable. In particular image quality and information content are spatially heterogeneous with variabilities that can be comparable to their mean values, limiting the use of the average resolution as a predictor for local information. A common use of super-resolution data is to test or establish structural models, and in these cases it would be valuable to assess the capacity of the data to validate a model. We focus here on single-molecule localization methods and present a new way of assessing the quality and reliability of super-resolution data.

  16. Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

    OpenAIRE

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

    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 ima...

  17. 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.

  18. 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.

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

    Science.gov (United States)

    2016-05-25

    Typically, a “north” and “east” direction marking is sufficient to locate cells of interest (Box 2). ?TROUBLESHOOTING ii) Seed tissue culture...lysine solution for 20 minutes. ii) Adherent cultured cells should be seeded onto the coverslip allowing room for growth to reach a final confluency... shadowing of the coating will aid in more complete coating of cellular structures and minimize charging effects during SEM imaging. ix) Image by SEM

  20. Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super-Resolution Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chien, Miao-Ping; Carlini, Andrea S.; Hu, Dehong; Barback, Christopher V.; Rush, Anthony M.; Hall, David J.; Orr, Galya; Gianneschi, Nathan C.

    2013-12-18

    Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block, and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo, and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micron-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

  1. Super-resolved fluorescence microscopy: Nobel Prize in Chemistry 2014 for Eric Betzig, Stefan Hell, and William E. Moerner.

    Science.gov (United States)

    Möckl, Leonhard; Lamb, Don C; Bräuchle, Christoph

    2014-12-15

    A big honor for small objects: The Nobel Prize in Chemistry 2014 was jointly awarded to Eric Betzig, Stefan Hell, and William E. Moerner "for the development of super-resolved fluorescence microscopy". This Highlight describes how the field of super-resolution microscopy developed from the first detection of a single molecule in 1989 to the sophisticated techniques of today.

  2. Super-resolution imaging strategies for cell biologists using a spinning disk microscope.

    Science.gov (United States)

    Hosny, Neveen A; Song, Mingying; Connelly, John T; Ameer-Beg, Simon; Knight, Martin M; Wheeler, Ann P

    2013-01-01

    In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.

  3. Super-resolution imaging strategies for cell biologists using a spinning disk microscope.

    Directory of Open Access Journals (Sweden)

    Neveen A Hosny

    Full Text Available In this study we use a spinning disk confocal microscope (SD to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM/Stochastic Optical Reconstruction Microscopy (STORM methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.

  4. Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

    Science.gov (United States)

    Hosny, Neveen A.; Song, Mingying; Connelly, John T.; Ameer-Beg, Simon; Knight, Martin M.; Wheeler, Ann P.

    2013-01-01

    In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging. PMID:24130668

  5. 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...

  6. Super-resolution optical telescopes with local light diffraction shrinkage

    OpenAIRE

    Changtao Wang; Dongliang Tang; Yanqin Wang; Zeyu Zhao; Jiong Wang; Mingbo Pu; Yudong Zhang; Wei Yan; Ping Gao; Xiangang Luo

    2015-01-01

    Suffering from giant size of objective lenses and infeasible manipulations of distant targets, telescopes could not seek helps from present super-resolution imaging, such as scanning near-field optical microscopy, perfect lens and stimulated emission depletion microscopy. In this paper, local light diffraction shrinkage associated with optical super-oscillatory phenomenon is proposed for real-time and optically restoring super-resolution imaging information in a telescope system. It is found ...

  7. Single cell genomic quantification by non-fluorescence nonlinear microscopy

    Science.gov (United States)

    Kota, Divya; Liu, Jing

    2017-02-01

    Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.

  8. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).

    Science.gov (United States)

    Dertinger, T; Colyer, R; Iyer, G; Weiss, S; Enderlein, J

    2009-12-29

    Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

  9. Super-resolution imaging in glycoscience: New developments and challenges

    Directory of Open Access Journals (Sweden)

    Junling Chen

    2016-05-01

    Full Text Available Carbohydrates on cell surfaces play a crucial role in a wide variety of biological processes, including cell adhesion, recognition and signaling, viral and bacterial infection, inflammation and metastasis. However, owing to the large diversity and complexity of carbohydrate structure and nongenetically synthesis, glycoscience is the least understood field compared with genomics and proteomics. Although the structures and functions of carbohydrates have been investigated by various conventional analysis methods, the distribution and role of carbohydrates in cell membranes remain elusive. This review focuses on the developments and challenges of super-resolution imaging in glycoscience through introduction of imaging principle and the available fluorescent probes for super-resolution imaging, the labeling strategies of carbohydrates, and the recent applications of super-resolution imaging in glycoscience, which will promote the super-resolution imaging technology as a promising tool to provide new insights into the study of glycoscience.

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

    CERN Document Server

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

    2013-01-01

    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 F\\"orster 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-...

  11. Optical Super-Resolution Imaging of β-Amyloid Aggregation In Vitro and In Vivo: Method and Techniques.

    Science.gov (United States)

    Pinotsi, Dorothea; Kaminski Schierle, Gabriele S; Kaminski, Clemens F

    2016-01-01

    Super-resolution microscopy has emerged as a powerful and non-invasive tool for the study of molecular processes both in vitro and in live cells. In particular, super-resolution microscopy has proven valuable for research studies in protein aggregation. In this chapter we present details of recent advances in this method and the specific techniques, enabling the study of amyloid beta aggregation optically, both in vitro and in cells. First, we show that variants of optical super-resolution microscopy provide a capability to visualize oligomeric and fibrillar structures directly, providing detailed information on species morphology in vitro and even in situ, in the cellular environment. We focus on direct Stochastic Optical Reconstruction Microscopy, dSTORM, which provides morphological detail on spatial scales below 20 nm, and provide detailed protocols for its implementation in the context of amyloid beta research. Secondly, we present a range of optical techniques that offer super-resolution indirectly, which we call multi-parametric microscopy. The latter offers molecular scale information on self-assembly reactions via changes in protein or fluorophore spectral signatures. These techniques are empowered by our recent discovery that disease related amyloid proteins adopt intrinsic energy states upon fibrilisation. We show that fluorescence lifetime imaging provides a particularly sensitive readout to report on the aggregation state, which is robustly quantifiable for experiments performed either in vitro or in vivo.

  12. Subsurface Super-resolution Imaging of Unstained Polymer Nanostructures

    Science.gov (United States)

    Urban, Ben E.; Dong, Biqin; Nguyen, The-Quyen; Backman, Vadim; Sun, Cheng; Zhang, Hao F.

    2016-06-01

    Optical imaging has offered unique advantages in material researches, such as spectroscopy and lifetime measurements of deeply embedded materials, which cannot be matched using electron or scanning-probe microscopy. Unfortunately, conventional optical imaging cannot provide the spatial resolutions necessary for many nanoscopic studies. Despite recent rapid progress, super-resolution optical imaging has yet to be widely applied to non-biological materials. Herein we describe a method for nanoscopic optical imaging of buried polymer nanostructures without the need for extrinsic staining. We observed intrinsic stochastic fluorescence emission or blinking from unstained polymers and performed spatial-temporal spectral analysis to investigate its origin. We further applied photon localization super-resolution imaging reconstruction to the detected stochastic blinking, and achieved a spatial resolution of at least 100 nm, which corresponds to a six-fold increase over the optical diffraction limit. This work demonstrates the potential for studying the static heterogeneities of intrinsic polymer molecular-specific properties at sub-diffraction-limited optical resolutions.

  13. Microsphere Super-resolution Imaging

    CERN Document Server

    Wang, Zengbo

    2015-01-01

    Recently, it was discovered that microsphere can generate super-resolution focusing beyond diffraction limit. This has led to the development of an exciting super-resolution imaging technique -microsphere nanoscopy- that features a record resolution of 50 nm under white lights. Different samples have been directly imaged in high resolution and real time without labelling, including both non-biological (nano devices, structures and materials) and biological (subcellular details, viruses) samples. This chapter reviews the technique, which covers its background, fundamentals, experiments, mechanisms as well as the future outlook.

  14. 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

  15. Multimodal super-resolution optical microscopy using a transition metal-based probe provides unprecedented capabilities for imaging both nucle-ar chromatin and mitochondria.

    Science.gov (United States)

    Sreedharan, Sreejesh; Gill, Martin; Garcia, Esther; Saeed, Hiwa K; Robinson, Darren; Byrne, Aisling; Cadby, Ashley James; Keyes, Tia E; Smythe, Carl G W; Pellett, Patrina; Bernardino de la Serna, Jorge; Thomas, Jim Antony

    2017-10-04

    Detailed studies on the live cell uptake properties of a dinuclear membrane permeable permeable RuII cell probe show that, at low concentrations, the complex localizes and images mitochondria. At concentrations above ~20 μM the complex images nuclear DNA. Since the complex is extremely photostable, has a large Stokes shift, and displays intrinsic subcellular targeting, its compatibility with super-resolution techniques was investigated. It was found to be very well suited to image mitochondria and nuclear chromatin in two col-our, 2C-SIM; and STED and 3D-STED both in fixed and live cell. In particular, due to its vastly improved photostability compared to conventional SR probes, it can provide images of nuclear DNA at unprecedented resolution.

  16. Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent

    Directory of Open Access Journals (Sweden)

    Meng-Tsen Ke

    2016-03-01

    Full Text Available Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2. SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518, thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of >100 μm, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels.

  17. 超分辨显微,至极至美:2014年诺贝尔化学奖述评%Beyond the limit:super-resolution microscopy earned the Nobel Prize in Chemistry 2014

    Institute of Scientific and Technical Information of China (English)

    李明

    2014-01-01

    Three physicists, Eric Betzig, Stefan Hell and William E. Moerner were award-ed the Nobel Prize in Chemistry 2014 for developing super-resolution optical microscopy. They pushed the techniques of their time to extremes to image single molecules, discovered the on/off switching behaviors of fluorescent molecules, and applied the well-known stimulated emission phe-nomenon to bypass a presumed scientific limitation stipulating that an optical microscope can nev-er yield a resolution better than 200 nm. The new techniques will lead to a revolution in life sci-ence. Using them, scientists can now monitor the interplay between individual molecules inside cells and track cell division at the nano-level, to name but a few.%三个物理学家,因为对生命科学的贡献,赢得2014年的诺贝尔化学奖。他们做了什么重大贡献?恩斯特·阿贝为常规光学显微镜的分辨率设定了一个限制——半波长极限。贝齐格、赫尔和莫纳将已知的技术推至极限,最早探测到凝聚态体系中的单个荧光分子,利用荧光分子的开关效应,加上物理教科书上的受激辐射原理和数据分析中常用的拟合定位方法,绕开了这个似乎不能突破的极限。他们将光学显微技术带入到纳米尺度,引发了常温下活体生物学研究的又一场革命。他们对科学的追求堪称至极至美。这样的典范将来还会有,尤其是在物理学与生命科学的交叉领域。

  18. 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.

  19. SOFI Simulation Tool: A Software Package for Simulating and Testing Super-Resolution Optical Fluctuation Imaging.

    Science.gov (United States)

    Girsault, Arik; Lukes, Tomas; Sharipov, Azat; Geissbuehler, Stefan; Leutenegger, Marcel; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Lasser, Theo

    2016-01-01

    Super-resolution optical fluctuation imaging (SOFI) allows one to perform sub-diffraction fluorescence microscopy of living cells. By analyzing the acquired image sequence with an advanced correlation method, i.e. a high-order cross-cumulant analysis, super-resolution in all three spatial dimensions can be achieved. Here we introduce a software tool for a simple qualitative comparison of SOFI images under simulated conditions considering parameters of the microscope setup and essential properties of the biological sample. This tool incorporates SOFI and STORM algorithms, displays and describes the SOFI image processing steps in a tutorial-like fashion. Fast testing of various parameters simplifies the parameter optimization prior to experimental work. The performance of the simulation tool is demonstrated by comparing simulated results with experimentally acquired data.

  20. Super-resolution 2-photon microscopy reveals that the morphology of each dendritic spine correlates with diffusive but not synaptic properties

    Directory of Open Access Journals (Sweden)

    Kevin eTakasaki

    2014-05-01

    Full Text Available The structure of dendritic spines suggests a specialized function in compartmentalizing synaptic signals near active synapses. Indeed, theoretical and experimental analyses indicate that the diffusive resistance of the spine neck is sufficient to effectively compartmentalize some signaling molecules in a spine for the duration of their activated lifetime. Here we describe the application of 2-photon microscopy combined with stimulated emission depletion (STED-2P to the biophysical study of the relationship between synaptic signals and spine morphology, demonstrating the utility of combining STED-2P with modern optical and electrophysiological techniques. Morphological determinants of fluorescence recovery time were identified and evaluated within the context of a simple compartmental model describing diffusive transfer between spine and dendrite. Correlations between the neck geometry and the amplitude of synapse potentials and calcium transients evoked by 2-photon glutamate uncaging were also investigated.

  1. Bridging fluorescence microscopy and electron microscopy

    NARCIS (Netherlands)

    Giepmans, Ben N. G.

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

  2. Bridging fluorescence microscopy and electron microscopy

    NARCIS (Netherlands)

    Giepmans, Ben N. G.

    2008-01-01

    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 breakth

  3. Common fluorescent proteins for single-molecule localization microscopy

    Science.gov (United States)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  4. Color-Coded Super-Resolution Small-Molecule Imaging.

    Science.gov (United States)

    Beuzer, Paolo; La Clair, James J; Cang, Hu

    2016-06-02

    Although the development of super-resolution microscopy dates back to 1994, its applications have been primarily focused on visualizing cellular structures and targets, including proteins, DNA and sugars. We now report on a system that allows both monitoring of the localization of exogenous small molecules in live cells at low resolution and subsequent super-resolution imaging by using stochastic optical reconstruction microscopy (STORM) on fixed cells. This represents a powerful new tool to understand the dynamics of subcellular trafficking associated with the mode and mechanism of action of exogenous small molecules.

  5. 3D multicolor super-resolution imaging offers improved accuracy in neuron tracing.

    Directory of Open Access Journals (Sweden)

    Melike Lakadamyali

    Full Text Available The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D, multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM, for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.

  6. 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.

  7. 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.

  8. 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.

  9. Fundamentals of fluorescence and fluorescence microscopy.

    Science.gov (United States)

    Wolf, David E

    2013-01-01

    This chapter discusses the fundamental physics of fluorescence. The application of fluorescence to microscopy represents an important transition in the development of microscopy, particularly as it applies to biology. It enables quantitating the amounts of specific molecules within a cell, determining whether molecules are complexing on a molecular level, measuring changes in ionic concentrations within cells and organelles, and measuring molecular dynamics. This chapter also discusses the issues important to quantitative measurement of fluorescence and focuses on four of quantitative measurements of fluorescence--boxcar-gated detection, streak cameras, photon correlation, and phase modulation. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. This chapter also examines how fluorescence is measured in the steady state and time domain and how fluorescence is applied in the modern epifluorescence microscope.

  10. Correlative stochastic optical reconstruction microscopy and electron microscopy.

    Directory of Open Access Journals (Sweden)

    Doory Kim

    Full Text Available 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.

  11. Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing.

    Science.gov (United States)

    Cho, Won-Ki; Jayanth, Namrata; Mullen, Susan; Tan, Tzer Han; Jung, Yoon J; Cissé, Ibrahim I

    2016-10-26

    Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging.

  12. Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing

    Science.gov (United States)

    Cho, Won-Ki; Jayanth, Namrata; Mullen, Susan; Tan, Tzer Han; Jung, Yoon J.; Cissé, Ibrahim I.

    2016-01-01

    Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging. PMID:27782203

  13. Membranes and Fluorescence microscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2009-01-01

    be provided by microscopy-related techniques. In this chapter, I will attempt to summarize representative examples concerning how microscopy (which provides information on membrane lateral organization by direct visualization) and spectroscopy techniques (which provides information about molecular interaction...

  14. 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.

  15. Exploiting speckle correlations to improve the resolution of wide-field fluorescence microscopy

    CERN Document Server

    Yilmaz, Hasan; Bertolotti, Jacopo; Lagendijk, Ad; Vos, Willem L; Mosk, Allard P

    2014-01-01

    Fluorescence microscopy is indispensable in nanoscience and biological sciences. The versatility of labeling target structures with fluorescent dyes permits to visualize structure and function at a subcellular resolution with a wide field of view. Due to the diffraction limit, conventional optical microscopes are limited to resolving structures larger than 200 nm. The resolution can be enhanced by near-field and far-field super-resolution microscopy methods. Near-field methods typically have a limited field of view and far-field methods are limited by the involved conventional optics. Here, we introduce a combined high-resolution and wide-field fluorescence microscopy method that improves the resolution of a conventional optical microscope by exploiting correlations in speckle illumination through a randomly scattering high-index medium: Speckle correlation resolution enhancement (SCORE). As a test, we collect two-dimensional fluorescence images of 100-nm diameter dye-doped nanospheres. We demonstrate a decon...

  16. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.

    Science.gov (United States)

    Unnersjö-Jess, David; Scott, Lena; Blom, Hans; Brismar, Hjalmar

    2016-01-01

    The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.

  17. Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Xin; Szymanski, Craig J.; Wang, Zhaoying; Zhou, Yufan; Ma, Xiang; Yu, Jiachao; Evans, James E.; Orr, Galya; Liu, Songqin; Zhu, Zihua; Yu, Xiao-Ying

    2016-05-15

    Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics at the molecular level.

  18. Super-resolution quantum sensing using NV centers based on rotating linear polarized light and Monte-Carlo method

    CERN Document Server

    Zhang, Hua-Yu; Guo, Guang-Can; Sun, Fang-Wen

    2016-01-01

    The nitrogen vacancy (NV) center in diamond has been widely applied for quantum information and sensing in last decade. Based on the laser polarization dependent excitation of fluorescence emission, we propose a super-resolution microscopy of NV center. A series of wide field images of NV centers are taken with different polarizations of the linear polarized excitation laser. The fluorescence intensity of NV center is changed with the relative angle between excitation laser polarization and the orientation of NV center dipole. The images pumped by different excitation laser polarizations are analyzed with Monte Carlo method. Then the symmetry axis and position of NV center are obtained with sub-diffraction resolution.

  19. Correlative fluorescence and electron microscopy.

    Science.gov (United States)

    Schirra, Randall T; Zhang, Peijun

    2014-10-01

    Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation--integration.

  20. Combining fluorescence and bioluminescence microscopy.

    Science.gov (United States)

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  1. Super-Resolution Imaging at Mid-Infrared Waveband in Graphene-nanocavity formed on meta-surface

    Science.gov (United States)

    Yang, Jingzhong; Wang, Taisheng; Chen, Zuolong; Hu, Bingliang; Yu, Weixing

    2016-11-01

    Plasmonic structured illumination microscopy (PSIM) is one of the promising wide filed optical imaging methods, which takes advantage of the surface plasmons to break the optical diffraction limit and thus to achieve a super-resolution optical image. To further improve the imaging resolution of PSIM, we propose in this work a so called graphene nanocavity on meta-surface structure (GNMS) to excite graphene surface plasmons with a deep sub-wavelength at mid-infrared waveband. It is found that surface plasmonic interference pattern with a period of around 52 nm can be achieved in graphene nanocavity formed on structured meta-surface for a 7 μm wavelength incident light. Moreover, the periodic plasmonic interference pattern can be tuned by simply changing the nanostructures fabricated on meta-surface for different application purposes. At last, the proposed GNMS structure is applied for super-resolution imaging in PSIM and it is found that an imaging resolution of 26 nm can be achieved, which is nearly 100 folds higher than that can be achieved by conventional epi-fluorescence microscopy. In comparison with visible waveband, mid-infrared is more gently and safe to biological cells and thus this work opens the new possibility for optical super-resolution imaging at mid-infrared waveband for biological research field.

  2. 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.

  3. Super-resolution optical telescopes with local light diffraction shrinkage

    Science.gov (United States)

    Wang, Changtao; Tang, Dongliang; Wang, Yanqin; Zhao, Zeyu; Wang, Jiong; Pu, Mingbo; Zhang, Yudong; Yan, Wei; Gao, Ping; Luo, Xiangang

    2015-12-01

    Suffering from giant size of objective lenses and infeasible manipulations of distant targets, telescopes could not seek helps from present super-resolution imaging, such as scanning near-field optical microscopy, perfect lens and stimulated emission depletion microscopy. In this paper, local light diffraction shrinkage associated with optical super-oscillatory phenomenon is proposed for real-time and optically restoring super-resolution imaging information in a telescope system. It is found that fine target features concealed in diffraction-limited optical images of a telescope could be observed in a small local field of view, benefiting from a relayed metasurface-based super-oscillatory imaging optics in which some local Fourier components beyond the cut-off frequency of telescope could be restored. As experimental examples, a minimal resolution to 0.55 of Rayleigh criterion is obtained, and imaging complex targets and large targets by superimposing multiple local fields of views are demonstrated as well. This investigation provides an access for real-time, incoherent and super-resolution telescopes without the manipulation of distant targets. More importantly, it gives counterintuitive evidence to the common knowledge that relayed optics could not deliver more imaging details than objective systems.

  4. New approach for super-resolution imaging of NV-nanodiamonds

    Science.gov (United States)

    Arai, Keigo; Le Sage, David; Bar-Gill, Nir; Belthangady, Chinmay; Glenn, David; Linh Pham, My; Zhang, Huiliang; Walsworth, Ronald

    2012-06-01

    We describe a new approach for super-resolution imaging of nanodiamonds (NDs) containing NV centers. The random orientation of NDs in a static magnetic field allow each ND to be distinguished by the NV ESR Zeeman shift and spin-state-dependent fluorescence rate. We exploit this behavior as a photo-switch such that adjacent NDs emit fluorescence sequentially in time. Post-analysis of a series of images at each ESR resonance frequency can localize individual NDs with sub-wavelength resolution. This technique has the advantage of being compatible with CCD-based wide-field microscopy, and involves significantly less laser intensity and experimental complexity than STED-based approaches.

  5. 基于时间相关单光子计数的离线式g-STED超分辨显微术%Super Resolution Microscopy of Offline g-STED Microscopy Based on Time-Correlated Single Photon Counting

    Institute of Scientific and Technical Information of China (English)

    郝翔; 匡翠方; 顾兆泰; 李帅; 刘旭

    2013-01-01

    提出了一种离线式基于时间门的荧光受激发射损耗(g-STED)显微方法.基于在强光照条件下荧光寿命缩短的理论模型,在常规STED架构基础上,使用时间相关单光子记数(TCSPC)算法获取图像的荧光寿命信息,离线设置合理的时间门阈值,丢弃短寿命信号数据,对荧光信号有效点扩展函数(PSF)进行压缩,达到超分辨显微的目的.与传统STED显微术相比,此方法所需光功率大幅度降低,减少了荧光漂白及光毒性;离线式处理则同时增加了时间门设置的灵活性.在实验中,使用45 rnW的连续STED光,最终获取了约80 nm的图像空间分辨率.进一步对时间门的设置对获取图像信号的分辨率和信噪比的影响进行了讨论.%The offline time-gated stimulated emission depletion (g-STED) microscopy, which is based on time-correlated single photon counting (TCSPC) algorithm, is proposed. As STED beam can eliminate the ratio of spontaneous fluorescent emission while reducing the fluorescence lifetime, the lifetime of fluorescent signals in the center of excitation focal spot and that in the surrounding doughnut area which are overlap by the STED focal spot are significant different. Based on this principle, in a general continuous wave STED (CW-STED), the fluorescent lifetimes of the whole imaging region are calculated by TCSPC, and the signals with shorter lifetime are discarded after all data recorded. The effective point spread function (PSF) of each fluorescent labels are shrinked in order to enhance the resolution. Compared with traditional ones, this offline g-STED not only decreases the incident intensity of laser to avoid the risk of fluorescence photobleaching and optical toxicity, but also increases the flexibility of time-gate manipulation. A spatial resolution of 80 nm is obtained in the experiment when only 45 mW STED beam is introduced. The potential influences of time-gate selection to the resolution and signal-to-noise ratio

  6. Highlights of the optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, G H

    2011-07-01

    The development of super-resolution microscopy techniques using molecular localization, such as photoactivated localization microscopy, fluorescence photoactivated localization microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy with independent running acquisition and many others, has heightened interest in molecules that will be grouped here into a category referred to as 'optical highlighter' fluorescent proteins. This review will survey many of the advances in development of fluorescent proteins for optically highlighting sub-populations of fluorescently labelled molecules.

  7. Exploring the formation of focal adhesions on patterned surfaces using super-resolution imaging.

    Science.gov (United States)

    Chien, Fan-Ching; Kuo, Chiung Wen; Yang, Zong-Han; Chueh, Di-Yen; Chen, Peilin

    2011-10-17

    The formation of focal adhesions on various sizes of fibronectin patterns, ranging from 200 μm to 250 nm, was systematically investigated by total internal reflection fluorescence microscopy and super-resolution imaging. It was found that cells adhered to and spread on these micro/nanopatterns, forming focal adhesions. On a micrometer scale the shape of the focal adhesions was elongated. However, on the nanometer scale, the shape of focal adhesions became dotlike. To further explore the distribution of focal adhesion proteins formed on surfaces, a localization-based super-resolution imaging technique was employed in order to determine the position and density of vinculin proteins. A characteristic distance of 50 nm was found between vinculin molecules in the focal adhesions, which did not depend on the size of the fibronectin nanopatterns. This distance was found to be crucial for the formation of focal adhesions. In addition, the density of vinculin at the focal adhesions formed on the nanopatterns increased as the pattern size decreased. The density of the protein was found to be 425 ± 247, 584 ± 302, and 703 ± 305 proteins μm(-2) on the 600, 400, and 250 nm fibronectin patterns respectively. Whereas 226 ± 77 proteins μm(-2) was measured for the matured focal adhesions on homogeneous fibronectin coated substrates. The increase in vinculin density implies that an increase in mechanical load was applied to the focal adhesions formed on the smaller nanopatterns.

  8. Super resolution of images and video

    CERN Document Server

    Katsaggelos, Aggelos K

    2007-01-01

    This book focuses on the super resolution of images and video. The authors' use of the term super resolution (SR) is used to describe the process of obtaining a high resolution (HR) image, or a sequence of HR images, from a set of low resolution (LR) observations. This process has also been referred to in the literature as resolution enhancement (RE). SR has been applied primarily to spatial and temporal RE, but also to hyperspectral image enhancement. This book concentrates on motion based spatial RE, although the authors also describe motion free and hyperspectral image SR problems. Also exa

  9. 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.

  10. 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. Limita

  11. Super-resolution near field imaging device

    DEFF Research Database (Denmark)

    2014-01-01

    Super-resolution imaging device comprising at least a first and a second elongated coupling element, each having a first transverse dimension at a first end and a second transverse dimension at a second end and being adapted for guiding light between their respective first and second ends, each...

  12. Multi-pulse pumping for far-field super-resolution imaging

    Science.gov (United States)

    Requena, Sebastian; Raut, Sangram; Doan, Hung; Kimball, Joe; Fudala, Rafal; Borejdo, Julian; Gryczynski, Ignacy; Strzhemechny, Yuri; Gryczynski, Zygmunt

    2016-02-01

    Recently, far-field optical imaging with a resolution significantly beyond diffraction limit has attracted tremendous attention allowing for high resolution imaging in living objects. Various methods have been proposed that are divided in to two basic approaches; deterministic super-resolution like STED or RESOLFT and stochastic super-resolution like PALM or STORM. We propose to achieve super-resolution in far-field fluorescence imaging by the use of controllable (on-demand) bursts of pulses that can change the fluorescence signal of long-lived component over one order of magnitude. We demonstrate that two beads, one labeled with a long-lived dye and another with a short-lived dye, separated by a distance lower than 100 nm can be easily resolved in a single experiment. The proposed method can be used to separate two biological structures in a cell by targeting them with two antibodies labeled with long-lived and short-lived fluorophores.

  13. Robust super-resolution without regularization

    Energy Technology Data Exchange (ETDEWEB)

    Pham, T Q [Canon Information Systems Research Australia, 1 Thomas Holt drive, North Ryde, NSW 2113 (Australia); Vliet, L J v [Quantitative Imaging Group, Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft (Netherlands); Schutte, K [Electro-Optics Group, TNO Defence, Security and Safety, PO Box 96864, 2509 JG The Hague (Netherlands)

    2008-07-15

    Super-resolution restoration is the problem of restoring a high-resolution scene from multiple degraded low-resolution images under motion. Due to imaging blur and noise, this problem is ill-posed. Additional constraints such as smoothness of the solution (i.e. regularization) is often required to obtain a stable solution. While regularizing the cost function is a standard practice in image restoration, we propose a restoration algorithm that does not require this extra regularization term. The robustness of the algorithm is achieved by a robust error norm that does not response to intensity outliers. With the outliers suppressed, our solution behaves similarly to a maximum-likelihood solution under the presence of Gaussian noise. The effectiveness of our algorithm is demonstrated with super-resolution restoration of real infrared image sequences under severe aliasing and intensity outliers.

  14. Penrose Pixels for Super-Resolution.

    Science.gov (United States)

    Ben-Ezra, M; Lin, Zhouchen; Wilburn, Bennett; Zhang, Wei

    2011-07-01

    We present a novel approach to reconstruction-based super-resolution that uses aperiodic pixel tilings, such as a Penrose tiling or a biological retina, for improved performance. To this aim, we develop a new variant of the well-known error back projection super-resolution algorithm that makes use of the exact detector model in its back projection operator for better accuracy. Pixels in our model can vary in shape and size, and there may be gaps between adjacent pixels. The algorithm applies equally well to periodic or aperiodic pixel tilings. We present analysis and extensive tests using synthetic and real images to show that our approach using aperiodic layouts substantially outperforms existing reconstruction-based algorithms for regular pixel arrays. We close with a discussion of the feasibility of manufacturing CMOS or CCD chips with pixels arranged in Penrose tilings.

  15. Camera simulation engine enables efficient system optimization for super-resolution imaging

    Science.gov (United States)

    Fullerton, Stephanie; Bennett, Keith; Toda, Eiji; Takahashi, Teruo

    2012-02-01

    Quantitative fluorescent imaging requires optimization of the complete optical system, from the sample to the detector. Such considerations are especially true for precision localization microscopy such as PALM and (d)STORM where the precision of the result is limited by the noise in both the optical and detection systems. Here, we present a Camera Simulation Engine (CSE) that allows comparison of imaging results from CCD, CMOS and EM-CCD cameras under various sample conditions and can accurately validate the quality of precision localization algorithms and camera performance. To achieve these results, the CSE incorporates the following parameters: 1) Sample conditions including optical intensity, wavelength, optical signal shot noise, and optical background shot noise; 2) Camera specifications including QE, pixel size, dark current, read noise, EM-CCD excess noise; 3) Camera operating conditions such as exposure, binning and gain. A key feature of the CSE is that, from a single image (either real or simulated "ideal") we generate a stack of statistically realistic images. We have used the CSE to validate experimental data showing that certain current scientific CMOS technology outperforms EM-CCD in most super-resolution scenarios. Our results support using the CSE to efficiently and methodically select cameras for quantitative imaging applications. Furthermore, the CSE can be used to robustly compare and evaluate new algorithms for data analysis and image reconstruction. These uses of the CSE are particularly relevant to super-resolution precision localization microscopy and provide a faster, simpler and more cost effective means of system optimization, especially camera selection.

  16. Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting.

    Directory of Open Access Journals (Sweden)

    Sarah L Veatch

    Full Text Available We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM and scanning electron microscopy (SEM. We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by

  17. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    Science.gov (United States)

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  18. Toward Super-Resolution Imaging at Green Wavelengths Employing Stratified Metal-Insulator Metamaterials

    Directory of Open Access Journals (Sweden)

    Masanobu Iwanaga

    2015-05-01

    Full Text Available Metamaterials (MMs are subwavelength-structured materials that have been rapidly developed in this century and have various potentials to realize novel phenomena, such as negative refraction, cloaking and super-resolution. Theoretical proposals for super-resolution image transfer using metallic thin films were experimentally demonstrated at ultraviolet and violet wavelengths from 365 to 405 nm. However, the most preferred wavelengths of optical imaging are green wavelengths around 500 nm, because optical microscopy is most extensively exploited in the area of biotechnology. In order to make the super-resolution techniques using MMs more practical, we propose the design of a stratified metal-insulator MM that has super-resolution image transfer modes at green wavelengths, which we here call hyper modes. The design assumed only Ag and SiO2 as constituent materials and was found employing Bloch-state analysis, which is based on a rigorous transfer-matrix method for the metal-insulator MMs. It is numerically substantiated that the designed stratified metal-insulator metamaterial (SMIM is capable of forming super-resolution images at the green wavelengths, and optical loss reduction is also studied. We discuss the results derived by the Bloch-state analysis and by effective medium models usually used for the metal-insulator MMs and show that the Bloch-state analysis is more suitable to reproduce the experimental data.

  19. Super-Resolution Imaging on Microfluidic Super-Resolution Near-Field Structure

    Institute of Scientific and Technical Information of China (English)

    WANG Pei; TANG Lin; ZHANG Dou-Guo; LU Yong-Hua; JIAO Xiao-Jin; XIE Jian-Ping; MING Hai

    2005-01-01

    @@ We present a new concept of the microfluidic super-resolution near-field structure (MSRENS) based on a microfluidic structure and a super-resolution near-field structure. The near-field distance control, "nano-probe"and scanning can be realized simultaneously using the MSRENS, which is similar to a near-field scanning optical microscope. The design and simulation results are presented. Numerical simulation has demonstrated that the MSRENS with spatial resolution beyond the diffraction limit could be applicable in chemistry, biologics, and many other fields.

  20. Fluorescence microscopy: A tool to study autophagy

    Science.gov (United States)

    Rai, Shashank; Manjithaya, Ravi

    2015-08-01

    Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

  1. Super-resolution imaging of plasma membrane proteins with click chemistry

    Directory of Open Access Journals (Sweden)

    Pablo Mateos-Gil

    2016-09-01

    Full Text Available Besides its function as a passive cell wall, the plasma membrane (PM serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM with single-molecule sensitivity.

  2. Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry

    Science.gov (United States)

    Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Sören; Sauer, Markus

    2016-01-01

    Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size, and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity. PMID:27668214

  3. STED microscopy of living cells--new frontiers in membrane and neurobiology.

    Science.gov (United States)

    Eggeling, Christian; Willig, Katrin I; Barrantes, Francisco J

    2013-07-01

    Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.

  4. Convolutional Neural Network Based dem Super Resolution

    Science.gov (United States)

    Chen, Zixuan; Wang, Xuewen; Xu, Zekai; Hou, Wenguang

    2016-06-01

    DEM super resolution is proposed in our previous publication to improve the resolution for a DEM on basis of some learning examples. Meanwhile, the nonlocal algorithm is introduced to deal with it and lots of experiments show that the strategy is feasible. In our publication, the learning examples are defined as the partial original DEM and their related high measurements due to this way can avoid the incompatibility between the data to be processed and the learning examples. To further extent the applications of this new strategy, the learning examples should be diverse and easy to obtain. Yet, it may cause the problem of incompatibility and unrobustness. To overcome it, we intend to investigate a convolutional neural network based method. The input of the convolutional neural network is a low resolution DEM and the output is expected to be its high resolution one. A three layers model will be adopted. The first layer is used to detect some features from the input, the second integrates the detected features to some compressed ones and the final step transforms the compressed features as a new DEM. According to this designed structure, some learning DEMs will be taken to train it. Specifically, the designed network will be optimized by minimizing the error of the output and its expected high resolution DEM. In practical applications, a testing DEM will be input to the convolutional neural network and a super resolution will be obtained. Many experiments show that the CNN based method can obtain better reconstructions than many classic interpolation methods.

  5. SuReSim: simulating localization microscopy experiments from ground truth models.

    Science.gov (United States)

    Venkataramani, Varun; Herrmannsdörfer, Frank; Heilemann, Mike; Kuner, Thomas

    2016-04-01

    Super-resolution fluorescence microscopy has become a widely used tool in many areas of research. However, designing and validating super-resolution experiments to address a research question in a technically feasible and scientifically rigorous manner remains a fundamental challenge. We developed SuReSim, a software tool that simulates localization data of arbitrary three-dimensional structures represented by ground truth models, allowing users to systematically explore how changing experimental parameters can affect potential imaging outcomes.

  6. Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy.

    Science.gov (United States)

    Liv, Nalan; van Oosten Slingeland, Daan S B; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W; Hoogenboom, Jacob P

    2016-01-26

    We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization.

  7. 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...... 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...... confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata. Conclusions Super-resolution light microscopy of PFS-stained cellulose fibrils is possible...

  8. Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

    Science.gov (United States)

    Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian

    2016-10-01

    Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

  9. 远场超分辨随机光重建显微镜(STORM)研究进展%Progress in far-field super-resolution stochastic optical reconstruction microscopy(STORM)

    Institute of Scientific and Technical Information of China (English)

    王成; 马俊领; 魏勋斌

    2011-01-01

    Understanding intracellular molecule-scale characteristic of dynamics and structures is urgently demanded to solve issues in today's life science. In order to solve this problem, a far field optical imaging obtained nanometer or sub-nanometer scale 3D resolution will be demanded. The far-field fluorescence microscopy, which broken diffraction barrier, Stochastic Optical Restructure Microscopy (STORM) is introduced. The STORM can be achieved resolution of 20 nm laterally and 50 nm axially. In theory, the STORM can be achieved single molecule location precision. Imaging foundational principle, progress of 3D and multi-color imaging, recently faced challenge as well as the direction of development about the STORM is talked in detailed.%了解细胞内分子尺度的动态和结构的特征是生命科学迫切需要解决的问题,要求远场光学成像要求纳米或亚纳米量级的空间分辨率.介绍了一种实现打破衍射极限的远场荧光显微成像技术--随机光重建显微术(STORM),其分辨率可以达到横向分辨率20 nm,轴向分辨率50 nm,理论上这种方法的空间分辨率可以达到单分子定位的精度.具体介绍了其成像的基本原理,在三维、多色成像方面的进展,和目前面临的问题及今后的发展方向.

  10. A NOVEL SUPER-RESOLUTION BEAMFORMING ALGORITHM

    Institute of Scientific and Technical Information of China (English)

    Guo Li; Guo Yan; Li Ning

    2007-01-01

    A novel simply-structured hybrid smart antenna system suitable to be used in ad-hoc network terminals is proposed in this letter.The super-resolution beamforming algorithm is also presented based on the system using DOA estimation results.The algorithm can switch the beamforming to the direction of the expected signal and get the best transmitting performance after the pre-beamforming of the Butler matrix.The shifting value formulas are presented to obtain the best SNR when there is no interfering signal and to acquire the highest Signal to Interference Ratio(SIR)as there is one interfering signal.When there are more than one interfering signals,the pre-beamforming feature of the Butler matrix Can also suppress the interfering signals.Simulation results verified the algorithm.

  11. A Super-Resolution Laser Altimetry Concept

    Science.gov (United States)

    Lu, Xiaomei; Hu, Yongxiang; Trepte, Charles; Liu, Zhaoyan

    2014-01-01

    A super-resolution laser altimetry technique has been proposed to provide improved lidar altimetry from Cloud Aerosol Lidar and Infrared Pathfinder Satellite Observation (CALIPSO) lidar data, and it is applicable to other similar atmospheric profiling lidar with low-pass filters. To achieve high altimetry resolution, the new technique relies on an empirical relationship between the peak signal ratio and the distance between land surface and the peak signal range bin center, which is directly derived from the CALIPSO lidar measurements and does not require the CALIPSO's transient response. The CALIPSO surface elevation results in Northern America retrieved by the new technique agree with the National Elevation Database high resolution elevation maps, and the comparisons suggest that the precision of the technique is much better than 1.4 m. The preliminary data product of land surface elevation retrieved by the new technique from CALIPSO lidar measurements is available to the altimetry community for evaluation.

  12. Temporal super resolution using variational methods

    DEFF Research Database (Denmark)

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

    2010-01-01

    and intensities are calculated simultaneously in a multiresolution setting. A frame doubling version of our algorithm is implemented and in testing it, we focus on making the motion of high contrast edges to seem smooth and thus reestablish the illusion of motion pictures.......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...... conversion (where also lower frame rates than recorded is sometimes required). We discuss and detail the requirements imposed by the human visual system (HVS) on TSR algorithms, of which the need for (apparent) fluid motion, also known as the phi-effect, is the principal one. This problem is typically...

  13. Solid-State Camera System for Fluorescence Lifetime Microscopy

    NARCIS (Netherlands)

    Zhao, Q.

    2014-01-01

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

  14. Super-resolution photoacoustic imaging of single gold nanoparticles

    Science.gov (United States)

    Lee, Seunghyun; Kwon, Owoong; Jeon, Mansik; Song, Jaejung; Jo, Minguk; Kim, Sungjee; Son, Junwoo; Kim, Yunseok; Kim, Chulhong

    2016-03-01

    Photoacoustic imaging (PAI) is an emerging hybrid imaging modality that can provide a strong optical absorption contrast using the photoacoustic (PA) effect, and breaks through the fundamental imaging depth limit of existing optical microscopy such as optical coherence tomography (OCT), confocal or two-photon microscopy. In PAI, a short-pulsed laser is illuminated to the tissue, and the PA waves are generated by thermoelastic expansion. Despite the high lateral resolution of optical-resolution photoacoustic microscopy (OR-PAM) thanks to the tight optical focus, the lateral resolution of OR-PAM is limited to the optical diffraction limit, which is approximately a half of the excitation wavelength. Here, we demonstrate a new super-resolution photoacoustic microscopy (SR-PAM) system by breaking the optical diffraction limit. The conventional microscopes with nanoscale resolutions such as a scanning electron microscope (SEM) and transmission electron microscope (TEM) are typically used to image the structures of nanomaterials, but these systems should work in a high vacuum environment and cannot provide the optical properties of the materials. Our newly developed SR-PAM system provides the optical properties with a nanoscale resolution in a normal atmosphere. We have photoacoustically imaged single gold nanoparticles with an average size of 80 nm in diameter and shown their PA expansion properties individually. The lateral resolution of this system was approximately 20 nm. Therefore, this tool will provide an unprecedented optical absorption property with an accurate nanoscale resolution and greatly impact on materials science and nanotechnology field.

  15. Novel optical super-resolution pattern with upright edges diffracted by a tiny thin aperture.

    Science.gov (United States)

    Wu, Jiu Hui; Zhou, Kejiang

    2015-08-24

    In the past decade numerous efforts have been concentrated to achieve optical imaging resolution beyond the diffraction limit. In this letter a thin microcavity theory of near-field optics is proposed by using the power flow theorem firstly. According to this theory, the near-field optical diffraction from a tiny aperture whose diameter is less than one-tenth incident wavelength embedded in a thin conducting film is investigated by considering this tiny aperture as a thin nanocavity. It is very surprising that there exists a kind of novel super-resolution diffraction patterns showing resolution better than λ/80 (λ is the incident wavelength), which is revealed for the first time to our knowledge in this letter. The mechanism that has allowed the imaging with this kind of super-resolution patterns is due to the interaction between the incident wave and the thin nanocavity with a complex wavenumber. More precisely, these super-resolution patterns with discontinuous upright peaks are formed by one or three items of the integration series about the cylindrical waves according to our simulation results. This novel optical super-resolution with upright edges by using the thin microcavity theory presented in the study could have potential applications in the future semiconductor lithography process, nano-size laser-drilling technology, microscopy, optical storage, optical switch, and optical information processing.

  16. 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.

  17. 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.

  18. 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.

  19. Three dimensional super-resolution in metamaterial slab lenses

    CERN Document Server

    Mesa, F; Freire, M; Baena, J D

    2005-01-01

    This letter presents a theoretical and experimental study on the viability of obtaining three dimensional super-resolution (i.e. resolution overcoming the diffraction limit for all directions in space) by means of metamaterial slab lenses. Although the source field cannot be actually reproduced at the back side of the lens with super-resolution in all space directions, the matching capabilities of metamaterial slabs does make it possible the detection of images with three-dimensional super-resolution. This imaging takes place because of the coupling between the evanescent space harmonic components of the field generated at both the source and the detector.

  20. Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles

    Directory of Open Access Journals (Sweden)

    Taro eIchimura

    2014-07-01

    Full Text Available Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometr scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies.

  1. Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

    CERN Document Server

    Deschout, Hendrik; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

    2016-01-01

    Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of m...

  2. Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

    2016-12-01

    Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

  3. Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes.

    Science.gov (United States)

    Rossi, Andrea; Moritz, Tobias J; Ratelade, Julien; Verkman, A S

    2012-09-15

    Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ~20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm(2) with decreasing M1:M23 ratio from 1:1 to 1:3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs with an area of 2142±829 nm(2), which increased to 5137±1119 nm(2) with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ~10(-12) cm(2)/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.

  4. Super-Resolution Genome Mapping in Silicon Nanochannels.

    Science.gov (United States)

    Jeffet, Jonathan; Kobo, Asaf; Su, Tianxiang; Grunwald, Assaf; Green, Ori; Nilsson, Adam N; Eisenberg, Eli; Ambjörnsson, Tobias; Westerlund, Fredrik; Weinhold, Elmar; Shabat, Doron; Purohit, Prashant K; Ebenstein, Yuval

    2016-11-22

    Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value scores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition.

  5. 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.

  6. Photobleaching correction in fluorescence microscopy images

    Energy Technology Data Exchange (ETDEWEB)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H [Microscopy Laboratory, School of Engineering - Bioengineering, National University of Entre Rios (UNER), Ruta 11, Km 10 (3101), Oro Verde, Entre Rios (Argentina)

    2007-11-15

    Fluorophores are used to detect molecular expression by highly specific antigen-antibody reactions in fluorescence microscopy techniques. A portion of the fluorophore emits fluorescence when irradiated with electromagnetic waves of particular wavelengths, enabling its detection. Photobleaching irreversibly destroys fluorophores stimulated by radiation within the excitation spectrum, thus eliminating potentially useful information. Since this process may not be completely prevented, techniques have been developed to slow it down or to correct resulting alterations (mainly, the decrease in fluorescent signal). In the present work, the correction by photobleaching curve was studied using E-cadherin (a cell-cell adhesion molecule) expression in Bufo arenarum embryos. Significant improvements were observed when applying this simple, inexpensive and fast technique.

  7. Least-squares based iterative multipath super-resolution technique

    CERN Document Server

    Nam, Wooseok

    2011-01-01

    In this paper, we study the problem of multipath channel estimation for direct sequence spread spectrum signals. To resolve multipath components arriving within a short interval, we propose a new algorithm called the least-squares based iterative multipath super-resolution (LIMS). Compared to conventional super-resolution techniques, such as the multiple signal classification (MUSIC) and the estimation of signal parameters via rotation invariance techniques (ESPRIT), our algorithm has several appealing features. In particular, even in critical situations where the conventional super-resolution techniques are not very powerful due to limited data or the correlation between path coefficients, the LIMS algorithm can produce successful results. In addition, due to its iterative nature, the LIMS algorithm is suitable for recursive multipath tracking, whereas the conventional super-resolution techniques may not be. Through numerical simulations, we show that the LIMS algorithm can resolve the first arrival path amo...

  8. SIMToolbox: a MATLAB toolbox for structured illumination fluorescence microscopy.

    Science.gov (United States)

    Křížek, Pavel; Lukeš, Tomáš; Ovesný, Martin; Fliegel, Karel; Hagen, Guy M

    2016-01-15

    SIMToolbox is an open-source, modular set of functions for MATLAB equipped with a user-friendly graphical interface and designed for processing two-dimensional and three-dimensional data acquired by structured illumination microscopy (SIM). Both optical sectioning and super-resolution applications are supported. The software is also capable of maximum a posteriori probability image estimation (MAP-SIM), an alternative method for reconstruction of structured illumination images. MAP-SIM can potentially reduce reconstruction artifacts, which commonly occur due to refractive index mismatch within the sample and to imperfections in the illumination. SIMToolbox, example data and the online documentation are freely accessible at http://mmtg.fel.cvut.cz/SIMToolbox. ghagen@uccs.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

    CSIR Research Space (South Africa)

    Mabaso, M

    2012-10-01

    Full Text Available Detection of messenger Ribonucleic Acid (mRNA) spots in fluorescence microscopy images is of great importance for biologists seeking better understanding of cell functionality. Fluorescence microscopy and specific staining methods make biological...

  10. Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy

    Science.gov (United States)

    Liu, Yujia; Lu, Yiqing; Yang, Xusan; Zheng, Xianlin; Wen, Shihui; Wang, Fan; Vidal, Xavier; Zhao, Jiangbo; Liu, Deming; Zhou, Zhiguang; Ma, Chenshuo; Zhou, Jiajia; Piper, James A.; Xi, Peng; Jin, Dayong

    2017-02-01

    Lanthanide-doped glasses and crystals are attractive for laser applications because the metastable energy levels of the trivalent lanthanide ions facilitate the establishment of population inversion and amplified stimulated emission at relatively low pump power. At the nanometre scale, lanthanide-doped upconversion nanoparticles (UCNPs) can now be made with precisely controlled phase, dimension and doping level. When excited in the near-infrared, these UCNPs emit stable, bright visible luminescence at a variety of selectable wavelengths, with single-nanoparticle sensitivity, which makes them suitable for advanced luminescence microscopy applications. Here we show that UCNPs doped with high concentrations of thulium ions (Tm3+), excited at a wavelength of 980 nanometres, can readily establish a population inversion on their intermediate metastable 3H4 level: the reduced inter-emitter distance at high Tm3+ doping concentration leads to intense cross-relaxation, inducing a photon-avalanche-like effect that rapidly populates the metastable 3H4 level, resulting in population inversion relative to the 3H6 ground level within a single nanoparticle. As a result, illumination by a laser at 808 nanometres, matching the upconversion band of the 3H4 → 3H6 transition, can trigger amplified stimulated emission to discharge the 3H4 intermediate level, so that the upconversion pathway to generate blue luminescence can be optically inhibited. We harness these properties to realize low-power super-resolution stimulated emission depletion (STED) microscopy and achieve nanometre-scale optical resolution (nanoscopy), imaging single UCNPs; the resolution is 28 nanometres, that is, 1/36th of the wavelength. These engineered nanocrystals offer saturation intensity two orders of magnitude lower than those of fluorescent probes currently employed in stimulated emission depletion microscopy, suggesting a new way of alleviating the square-root law that typically limits the

  11. Biological applications of confocal fluorescence polarization microscopy

    Science.gov (United States)

    Bigelow, Chad E.

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

  12. Gradient Permittivity Meta-Structure model for Wide-field Super-resolution imaging with a sub-45 nm resolution.

    Science.gov (United States)

    Cao, Shun; Wang, Taisheng; Xu, Wenbin; Liu, Hua; Zhang, Hongxin; Hu, Bingliang; Yu, Weixing

    2016-03-21

    A gradient permittivity meta-structure (GPMS) model and its application in super-resolution imaging were proposed and discussed in this work. The proposed GPMS consists of alternate metallic and dielectric films with a gradient permittivity which can support surface plasmons (SPs) standing wave interference patterns with a super resolution. By employing the rigorous numerical FDTD simulation method, the GPMS was carefully simulated to find that the period of the SPs interference pattern is only 84 nm for a 532 nm incident light. Furthermore, the potential application of the GPMS for wide-field super-resolution imaging was also discussed and the simulation results show that an imaging resolution of sub-45 nm can be achieved based on the plasmonic structure illumination microscopic method, which means a 5.3-fold improvement on resolution has been achieved in comparison with conventional epifluorescence microscopy. Moreover, besides the super-resolution imaging application, the proposed GPMS model can also be applied for nanolithography and other areas where super resolution patterns are needed.

  13. From single-molecule spectroscopy to super-resolution imaging of the neuron: a review

    Science.gov (United States)

    Laine, Romain F.; Kaminski Schierle, Gabriele S.; van de Linde, Sebastian; Kaminski, Clemens F.

    2016-06-01

    For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research.

  14. Super-resolution mbPAINT for optical localization of single-stranded DNA.

    Science.gov (United States)

    Chen, Jixin; Bremauntz, Alberto; Kisley, Lydia; Shuang, Bo; Landes, Christy F

    2013-10-09

    We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as "motion blur" point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.

  15. Non-radiative excitation fluorescence microscopy

    Science.gov (United States)

    Riachy, Lina; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-03-01

    Non-radiative Excitation Fluorescence Microscopy (NEFM) constitutes a new way to observe biological samples beyond the diffraction limit. Non-radiative excitation of the samples is achieved by coating the substrate with donor species, such as quantum dots (QDs). Thus the dyes are not excited directly by the laser source, as in common fluorescence microscopy, but through a non-radiative energy transfer. To prevent dewetting of the donor film, we have recently implemented a silanization process to covalently bond the QDs on the substrate. An homogeneous monolayer of QDs was then deposited on only one side of the coverslips. Atomic force microscopy was then used to characterize the QD layer. We highlight the potential of our method through the study of Giant Unilamellar Vesicles (GUVs) labeled with DiD as acceptor, in interaction with surface functionalized with poly-L-lysine. In the presence of GUVs, we observed a quenching of QDs emission, together with an emission of DiD located in the membrane, which clearly indicated that non-radiative energy transfer from QDs to DiD occurs.

  16. SLAP: Small Labeling Pair for Single-Molecule Super-Resolution Imaging.

    Science.gov (United States)

    Wieneke, Ralph; Raulf, Anika; Kollmannsperger, Alina; Heilemann, Mike; Tampé, Robert

    2015-08-24

    Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).

  17. 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.

  18. Read-only memory disk with AgOx super-resolution mask layer

    Institute of Scientific and Technical Information of China (English)

    Feng Zhang; Yang Wang; Wendong Xu; Hongren Shi; Fuxi Gan

    2005-01-01

    @@ A novel read-only memory (ROM) disk with an AgOx mask layer was proposed and studied in this letter.The AgOx films sputtered on the premastered substrates, with pits depth of 50 nm and pits length of 380 nm, were studied by an atomic force microscopy. The transmittances of these AgOx films were also measured by a spectrophotometer. Disk measurement was carried out by a dynamic setup with a laser wavelength of 632.8 nm and a lens numerical aperture (NA) of 0.40. The readout resolution limit of this setup was λ/(4NA) (400 nm). Results showed that the super-resolution readout happened only when the oxygen flow ratios were at suitable values for these disks. The best super-resolution performance was achieved at the oxygen flow ratio of 0.5 with the smoothest film surface. The super-resolution readout mechanism of these ROM disks was analyzed as well.

  19. Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

    Science.gov (United States)

    Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.

    2016-01-01

    The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP

  20. Gibbs artifact reduction for POCS super-resolution image reconstruction

    Institute of Scientific and Technical Information of China (English)

    Chuangbai XIAO; Jing YU; Kaina SU

    2008-01-01

    The topic of super-resolution image reconstruc-tion has recently received considerable attention among the research community. Super-resolution image reconstruc-tion methods attempt to create a single high-resolution image from a number of low-resolution images (or a video sequence). The method of projections onto convex sets (POCS) for super-resolution image reconstruction attracts many researchers' attention. In this paper, we propose an improvement to reduce the amount of Gibbs artifacts pre-senting on the edges of the high-resolution image recon-structed by the POCS method. The proposed method weights the blur PSF centered at an edge pixel with an exponential function, and consequently decreases the coef-ficients of the PSF in the direction orthogonal to the edge. Experiment results show that the modification reduces effectively the visibility of Gibbs artifacts on edges and improves obviously the quality of the reconstructed high-resolution image.

  1. 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.

  2. GF-4 Images Super Resolution Reconstruction Based on POCS

    Directory of Open Access Journals (Sweden)

    XU Lina

    2017-08-01

    Full Text Available The super resolution reconstruction of GF-4 is made by projection on convex sets (POCS. Papoulis-Gerchberg is used to construct reference frame which can reduce iteration and improve algorithm efficiency.Vandewalle is used to estimate motion parameter which is benefit to block process. Tested and analyzed by real GF-4 series images, it shows that sharpness of super resolution result is positive correlatie to frame amount, and signal to noise ratio (SNR is negative correlate to frame amount. After processing by 5 frames, information entropy (IE changes little; sharpness (average gradient increases from 7.803 to 14.386; SNR reduces a little, from 3.411 to 3.336. The experiment shows that after super resolution reconstruction, sharpness and detail information of results can be greatly improved.

  3. OBJECT-BASED SUPER RESOLUTION FOR INTELLIGENT VISUAL SURVEILLANCE VIDEO

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Construction of high resolution images from low resolution sequences is often important in surveillance applications. In this letter, an affine based multi-scale block-matching image registration algorithm is first proposed. The images to be registered are divided into overlapped blocks of different size according to its motions. The Least Square (LS) image registration algorithm is extended to match the blocks. Then an object based Super Resolution (SR) scheme is designed, the Maximum A Priori (MAP) super resolution algorithm is extended to enhance the resolution of the interest objects. Experimental results show that the proposed multi-scale registration method provides more accurate registration between frames. Further more, the object based super resolution scheme shows an enhanced performance compared with the traditional MAP method.

  4. Light Microscopy: An ongoing contemporary revolution

    CERN Document Server

    Weisenburger, Siegfried

    2014-01-01

    Optical microscopy is one of the oldest scientific instruments that is still used in forefront research. Ernst Abbe's nineteenth century formulation of the resolution limit in microscopy let generations of scientists believe that optical studies of individual molecules and resolving sub-wavelength structures were not feasible. The Nobel Prize in 2014 for super-resolution fluorescence microscopy marks a clear recognition that the old beliefs have to be revisited. In this article, we present a critical overview of various recent developments in optical microscopy. In addition to the popular super-resolution fluorescence methods, we discuss the prospects of various other techniques and imaging contrasts and consider some of the fundamental and practical challenges that lie ahead.

  5. Robust Microbubble Tracking for Super Resolution Imaging in Ultrasound

    DEFF Research Database (Denmark)

    Hansen, kristoffer B; Villagómez-Hoyos, Carlos A; 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...

  6. 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...... for super-resolved sequences. Computing super-resolved flows has to our knowledge not been done before. Most advanced super-resolution (SR) methods found in literature cannot be applied to general video with arbitrary scene content and/or arbitrary optical flows, as it is possible with our simultaneous VSR...

  7. Benchmarking Compressed Sensing, Super-Resolution, and Filter Diagonalization

    CERN Document Server

    Markovich, Thomas; Sanders, Jacob N; Aspuru-Guzik, Alan

    2015-01-01

    Signal processing techniques have been developed that use different strategies to bypass the Nyquist sampling theorem in order to recover more information than a traditional discrete Fourier transform. Here we examine three such methods: filter diagonalization, compressed sensing, and super-resolution. We apply them to a broad range of signal forms commonly found in science and engineering in order to discover when and how each method can be used most profitably. We find that filter diagonalization provides the best results for Lorentzian signals, while compressed sensing and super-resolution perform better for arbitrary signals.

  8. Modulated CMOS camera for fluorescence lifetime microscopy.

    Science.gov (United States)

    Chen, Hongtao; Holst, Gerhard; Gratton, Enrico

    2015-12-01

    Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition.

  9. Super-resolution Restoration of Remote-sensing Images

    Institute of Scientific and Technical Information of China (English)

    LIU Yang-yang; JIN Wei-qi; SU Bing-hua; CHEN Hua; ZHANG Nan

    2006-01-01

    A novel image restoration scheme, which is super-resolution image restoration algorithm Poisson-maximum-afterword-probability based on Markvo constraint (MPMAP) combined with evaluating image detail parameter D, has been proposed. The advantage of super-resolution algorithm MPMAP incorporated with parameter D lies in the fact that super-resolution algorithm MPMAP model is discrete, which is in accordance with remote-sensing imaging model, and the algorithm MPMAP is proved applicable to linear and non-linear imaging models with a unique solution when noise is not severe. According to simulation experiments for practical images, super-resolution algorithm MPMAP can retain image details better than most of traditional restoration methods; at the same time, the proposed parameter D can help to identify real point spread function (PSF) value of degradation process. Processing result of practical remote-sensing images by MPMAP combined with parameter D are given, it illustrates that MPMAP restoration scheme combined PSF estimation has a better restoration result than that of Photoshop processing, based on the same original images. It is proved that the proposed scheme is helpful to offset the lack of resolution of the original remote-sensing images and has its extensive application foreground.

  10. Generation of super-resolution stills from video

    CSIR Research Space (South Africa)

    Duvenhage, B

    2014-11-01

    Full Text Available The real-time super-resolution technique discussed in this paper increases the effective pixel density of an image sensor by combining consecutive image frames from a video. In surveillance, the higher pixel density lowers the Nyquist rate...

  11. Bioaerosol Analysis by Online Fluorescence Detection and Fluorescence Microscopy

    Science.gov (United States)

    Huffman, Alex; Pöhlker, Christopher; Treutlein, Bärbel; Pöschl, Ulrich

    2010-05-01

    substantial proportion of coarse aerosol particle number and mass in continental boundary layer air. Moreover, they suggest that the number concentration of viable bioparticles is dominated by fungal spores or agglomerated bacteria with aerodynamic diameters around 3 μm rather than single bacterial cells with diameters around 1 μm. Filter samples were later collected at the same sampling location and analyzed with a fluorescence microscope. By observing collected particles both with transmitted white light and with fluorescent emission from near-UV excitation, the technique provides information about whether individual particles are biological and regarding their viability. Characteristic images of FBAPs are shown. Further goals are to correlate size distributions from the UV-APS with size information gained from microscopy, and also to constrain uncertainties that arise from non-biological particles that also exhibit fluorescence. [1] Huffman et al. (2009) Atmos. Chem. Phys. Discuss., 9, 17705 - 17751.

  12. State space approach to single molecule localization in fluorescence microscopy.

    Science.gov (United States)

    Vahid, Milad R; Chao, Jerry; Kim, Dongyoung; Ward, E Sally; Ober, Raimund J

    2017-03-01

    Single molecule super-resolution microscopy enables imaging at sub-diffraction-limit resolution by producing images of subsets of stochastically photoactivated fluorophores over a sequence of frames. In each frame of the sequence, the fluorophores are accurately localized, and the estimated locations are used to construct a high-resolution image of the cellular structures labeled by the fluorophores. Many methods have been developed for localizing fluorophores from the images. The majority of these methods comprise two separate steps: detection and estimation. In the detection step, fluorophores are identified. In the estimation step, the locations of the identified fluorophores are estimated through an iterative approach. Here, we propose a non-iterative state space-based localization method which combines the detection and estimation steps. We demonstrate that the estimated locations obtained from the proposed method can be used as initial conditions in an estimation routine to potentially obtain improved location estimates. The proposed method models the given image as the frequency response of a multi-order system obtained with a balanced state space realization algorithm based on the singular value decomposition of a Hankel matrix. The locations of the poles of the resulting system determine the peak locations in the frequency domain, and the locations of the most significant peaks correspond to the single molecule locations in the original image. The performance of the method is validated using both simulated and experimental data.

  13. Super-resolution and nonlinear absorption with metallodielectric stacks

    Science.gov (United States)

    Katte, Nkorni

    We investigate sub-wavelength imaging, i.e. super-resolution, in metal-dielectric film systems, which are simply referred to as metallodielectrics. Our simulations incorporate experimentally derived material dielectric dispersion properties across the visible region. For demonstration purposes we designed metallodielectric stacks for super-resolution containing GaP and TiO2, dielectric films, and either Ag or Au as the metallic materials. Using the known optical properties of the constituent materials found designs that could be good candidates for super-resolution. We did not have the resources to fabricate these samples; however, based on our computer simulations we are confident that the designed samples would produce super-resolution approaching one-twentieth of a wavelength in air. We examined for the first time the broad bandwidth of the super-resolution phenomenon in metallodielectrics. We validate the results using the finite element method (FEM) and the transfer matrix method (TMM). We also show that the measurement of super-resolution is highly dependent on the distance of the probe from the exit surface; high resolution at the exit plane can quickly decay with a few tens of nanometers when high resolution is sought. Secondly we numerically studied the nonlinear optical transmission of an optical beam through heterogeneous metallodielectric stacks under the action of nonlinear absorption. One film layer is a metal and the other layer is a dielectric; the heterogeneous material is called a metallodielectric stack (MDS). In these studies we also used applied FEM with two-dimensional transverse effects and TMM simulation techniques. Our samples consisted of Ag/ZnS, Ag/SiO 2 and Cu/ZnS. We numerically simulate using two transverse dimensions in our FEM codes, Z-scan experiments for two different MDS designs and draw general observations from these cases. We experimentally examined the nonlinear absorption effect in samples of Ag/SiO2 when irradiated by a

  14. 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.

  15. Super-Resolution for Traditional and Omnidirectional Image Sequences

    Directory of Open Access Journals (Sweden)

    Attila Nagy

    2009-03-01

    Full Text Available This article presents a simple method on how to implement a super-resolutionbased video enhancement technique in .NET using the functions of the OpenCV library.First, we outline the goal of this project and after that, a short review of the steps of superresolutiontechnique is given. As a part of the discussion about the implementation itself,the general design aspects are detailed in short. Then, the different optical flow algorithmsare analyzed and the super-resolution calculation of omnidirectional image sequences isdiscussed. After all that, the achieved results can be seen and finally, a short generalconclusion can be read. This paper is a revision of our previous work [1]. In this edition,we focus on the super-resolution of omnidirectional image sequences rather than thetechnological issues that were discussed in our previous article. Further information aboutthe implementation and wrapper development can be found in [1 and 12].

  16. Super-resolution for scanning light stimulation systems

    Science.gov (United States)

    Bitzer, L. A.; Neumann, K.; Benson, N.; Schmechel, R.

    2016-09-01

    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.

  17. Super-resolution by pupil plane phase filtering

    Indian Academy of Sciences (India)

    L N Hazra; N Reza

    2010-11-01

    Resolution capability of any optical imaging system is limited by residual aberrations as well as diffraction effects. Overcoming this fundamental limit is called super-resolution. Several new paradigms for super-resolution in optical systems use ‘a posteriori’ digital image processing. In these ventures the three-dimensional point spread function (PSF) of the lens plays a key role in image acquisition. A straightforward tailoring of the PSF can be performed by appropriate pupil plane filtering. With a brief review of the state-of-art in this research area, this paper dwells upon the inverse problem of global optimization of the pupil function by phase filtering in accordance with the desired PSF.

  18. Fluorescence Microscopy of Nanoscale Silver Oxide Thin Films

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-Yu; JIANG Hong-Bing; LIU Chun-Ling; GONG Qi-Huang; ZHANG Xi-Yao; ZHANG Qi-Feng; XU Bei-Xue; WU Jin-Lei

    2003-01-01

    The experimental conditions for photoactivated intermittent fluorescence from nanoscale silver oxide were studied with fluorescence microscopy. Strong fluorescence was observed from the Ag?O particles with size of 10-20nm excited with both blue and green light. We observed the saturation of photoexcitation with blue light and explained the experimental results using the model of agglomeration of silver atoms to form small clusters and the fluorescence of Ag2 and Ags clusters.

  19. On the Adaptability of Neural Network Image Super-Resolution

    OpenAIRE

    Chua, Kah Keong; Tay, Yong Haur

    2012-01-01

    In this paper, we described and developed a framework for Multilayer Perceptron (MLP) to work on low level image processing, where MLP will be used to perform image super-resolution. Meanwhile, MLP are trained with different types of images from various categories, hence analyse the behaviour and performance of the neural network. The tests are carried out using qualitative test, in which Mean Squared Error (MSE), Peak Signal-to-Noise Ratio (PSNR) and Structural Similarity Index (SSIM). The r...

  20. Super resolution WiFi indoor localization and tracking

    OpenAIRE

    Salman, N.; Alsindi, N; Mihaylova, L.; Kemp, AH

    2014-01-01

    In this paper, we present a complete framework for accurate indoor positioning and tracking using the 802.11a WiFi network. Channel frequency response is first estimated via the least squares (LS) method using an orthogonal frequency division multiplexing (OFDM) pilot symbol. For accurate time of arrival (ToA) distance estimates in multipath environments, super resolution technique i.e. Multiple Signal Classification (MUSIC) is used which capitalizes on the autocorrelation matrix of the estim...

  1. New resolving power for light microscopy: applications to neurobiology.

    Science.gov (United States)

    Dani, Adish; Huang, Bo

    2010-10-01

    The recent invention of super-resolution fluorescence microscopy brings more than an order of magnitude gain in the spatial resolution of light microscopy. New opportunities keep emerging with the multicolor, three-dimensional, and live imaging functionalities gained in the past three years. The power of this technology has been demonstrated by imaging the organization of organelles and molecular complexes, with recent applications increasingly showing its potential in neurobiology. These developments are exemplified by the visualization of components inside dendritic spines to fine morphologies of neurons. In combination with correlative electron microscopy, functional imaging, and electrical/optogenetic stimulation tools, super-resolution fluorescence microscopy has the potential to provide further insights ranging from the molecular details of neurons up to the functional mechanisms of neuronal circuits.

  2. Super-Resolution Recording by an Organic Photochromic Mask Layer

    Institute of Scientific and Technical Information of China (English)

    SHI Ming; ZHAO Sheng-Min; YI Jia-Xiang; ZHAO Fu-Qun; NIU Li-Hong; LI Zhong-Yu; ZHANG Fu-Shi

    2007-01-01

    By using the super-resolution near-field structure(super-RENS)method,the super-resolution recording marks are obtained practically by an organic photochromic diarylethene mask layer,under much lower recording laser Dower of 0.45mW.The size of recording marks is decreased by 60% (from 1.6μm to 0.7μm) for a diarylethene (photo-mode)recording layer by the optical detection method(limited by optical diffraction),or decreased by 97%(from 1600nm to 50nm)for a heptaoxyl copper phthalocyanine(thermo-optical)recording layer,the latter is much smaller than the limitation of optical diffraction.In order to obtain a desirable result,a proper extent or Dhotochemistry reaction in the mask layer is needed.Thus,the super-resolution recording marks can be obtained by adjusting the concentration of diarylethene in the mask layer,the recording laser power,and the moving speed of the sample disc.

  3. Multiframe Blind Super Resolution Imaging Based on Blind Deconvolution

    Institute of Scientific and Technical Information of China (English)

    元伟; 张立毅

    2016-01-01

    As an ill-posed problem, multiframe blind super resolution imaging recovers a high resolution image from a group of low resolution images with some degradations when the information of blur kernel is limited. Note that the quality of the recovered image is influenced more by the accuracy of blur estimation than an advanced regularization. We study the traditional model of the multiframe super resolution and modify it for blind deblurring. Based on the analysis, we proposed two algorithms. The first one is based on the total variation blind deconvolution algorithm and formulated as a functional for optimization with the regularization of blur. Based on the alternating minimization and the gradient descent algorithm, the high resolution image and the unknown blur kernel are esti-mated iteratively. By using the median shift and add operator, the second algorithm is more robust to the outlier influence. The MSAA initialization simplifies the interpolation process to reconstruct the blurred high resolution image for blind deblurring and improves the accuracy of blind super resolution imaging. The experimental results demonstrate the superiority and accuracy of our novel algorithms.

  4. 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

  5. Imaging and Intracellular Tracking of Cancer-Derived Exosomes Using Single-Molecule Localization-Based Super-Resolution Microscope.

    Science.gov (United States)

    Chen, Chen; Zong, Shenfei; Wang, Zhuyuan; Lu, Ju; Zhu, Dan; Zhang, Yizhi; Cui, Yiping

    2016-10-05

    Exosomes are small membrane vesicles secreted by cells and enriched with plenty of proteins. Considering their significant roles in different physical activities and potential value for diagnostic drug delivery, researchers have put great efforts in in vitro tracking and content analysis of exosomes. Recently, the emergence of different kinds of super-resolution microscopy provides powerful tools for exosome study. Here, we demonstrate the application of single-molecule localization based super-resolution imaging technique in the imaging and tracking of cancer-derived exosomes. In the experiment, first, cancer-derived exosomes are extracted from the culture media of tumor cells. Then the exosome membrane receptors are labeled with photoswitchable probes, which allow super-resolution imaging of these membrane receptors via photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). By using human breast cancer cell-derived exosomes, we demonstrated simultaneous dual-color PALM/STORM imaging of two kinds of membrane receptors on the exosome membrane. Moreover, the successful labeling and imaging of exosomes make it possible to observe the interaction between cancer-derived exosomes and normal cells. Meanwhile, we realized the colocalization of cancer-derived exosomes and lysosomes in recipient cells with PALM/STORM imaging. Since exosomes play a vital role in intercellular communications, we anticipate that the presented PALM/STORM-based imaging and tracking of exosomes holds a great potential in the investigation of the mechanism of exosome-mediated cancer metastasis.

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

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

    Full Text Available of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies, Rome, Italy. 21-23 February, 2016 A Framework for Creating Realistic Synthetic Fluorescence Microscopy Image Sequences Matsilele Mabaso1, Daniel Withey1...

  7. In vivo acoustic super-resolution and super-resolved velocity mapping using microbubbles.

    Science.gov (United States)

    Christensen-Jeffries, Kirsten; Browning, Richard J; Tang, Meng-Xing; Dunsby, Christopher; Eckersley, Robert J

    2015-02-01

    The structure of microvasculature cannot be resolved using standard clinical ultrasound (US) imaging frequencies due to the fundamental diffraction limit of US waves. In this work, we use a standard clinical US system to perform in vivo sub-diffraction imaging on a CD1, female mouse aged eight weeks by localizing isolated US signals from microbubbles flowing within the ear microvasculature, and compare our results to optical microscopy. Furthermore, we develop a new technique to map blood velocity at super-resolution by tracking individual bubbles through the vasculature. Resolution is improved from a measured lateral and axial resolution of 112 μm and 94 μ m respectively in original US data, to super-resolved images of microvasculature where vessel features as fine as 19 μm are clearly visualized. Velocity maps clearly distinguish opposing flow direction and separated speed distributions in adjacent vessels, thereby enabling further differentiation between vessels otherwise not spatially separated in the image. This technique overcomes the diffraction limit to provide a noninvasive means of imaging the microvasculature at super-resolution, to depths of many centimeters. In the future, this method could noninvasively image pathological or therapeutic changes in the microvasculature at centimeter depths in vivo.

  8. Gradient Permittivity Meta-Structure model for Wide-field Super-resolution imaging with a sub-45 nm resolution

    Science.gov (United States)

    Cao, Shun; Wang, Taisheng; Xu, Wenbin; Liu, Hua; Zhang, Hongxin; Hu, Bingliang; Yu, Weixing

    2016-03-01

    A gradient permittivity meta-structure (GPMS) model and its application in super-resolution imaging were proposed and discussed in this work. The proposed GPMS consists of alternate metallic and dielectric films with a gradient permittivity which can support surface plasmons (SPs) standing wave interference patterns with a super resolution. By employing the rigorous numerical FDTD simulation method, the GPMS was carefully simulated to find that the period of the SPs interference pattern is only 84 nm for a 532 nm incident light. Furthermore, the potential application of the GPMS for wide-field super-resolution imaging was also discussed and the simulation results show that an imaging resolution of sub‑45 nm can be achieved based on the plasmonic structure illumination microscopic method, which means a 5.3-fold improvement on resolution has been achieved in comparison with conventional epifluorescence microscopy. Moreover, besides the super-resolution imaging application, the proposed GPMS model can also be applied for nanolithography and other areas where super resolution patterns are needed.

  9. The internal architecture of dendritic spines revealed by super-resolution imaging: What did we learn so far?

    Energy Technology Data Exchange (ETDEWEB)

    MacGillavry, Harold D., E-mail: h.d.macgillavry@uu.nl; Hoogenraad, Casper C., E-mail: c.hoogenraad@uu.nl

    2015-07-15

    The molecular architecture of dendritic spines defines the efficiency of signal transmission across excitatory synapses. It is therefore critical to understand the mechanisms that control the dynamic localization of the molecular constituents within spines. However, because of the small scale at which most processes within spines take place, conventional light microscopy techniques are not adequate to provide the necessary level of resolution. Recently, super-resolution imaging techniques have overcome the classical barrier imposed by the diffraction of light, and can now resolve the localization and dynamic behavior of proteins within small compartments with nanometer precision, revolutionizing the study of dendritic spine architecture. Here, we highlight exciting new findings from recent super-resolution studies on neuronal spines, and discuss how these studies revealed important new insights into how protein complexes are assembled and how their dynamic behavior shapes the efficiency of synaptic transmission.

  10. Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states.

    Science.gov (United States)

    Ehmann, Nadine; van de Linde, Sebastian; Alon, Amit; Ljaschenko, Dmitrij; Keung, Xi Zhen; Holm, Thorge; Rings, Annika; DiAntonio, Aaron; Hallermann, Stefan; Ashery, Uri; Heckmann, Manfred; Sauer, Markus; Kittel, Robert J

    2014-08-18

    The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

  11. 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

  12. Super-resolution of facial images in forensics scenarios

    DEFF Research Database (Denmark)

    Satiro, Joao; Nasrollahi, Kamal; Correia, Paulo

    2015-01-01

    Forensics facial images are usually provided by surveillance cameras and are therefore of poor quality and resolution. Simple upsampling algorithms can not produce artifact-free higher resolution images from such low-resolution (LR) images. To deal with that, reconstruction-based super-resolution......Forensics facial images are usually provided by surveillance cameras and are therefore of poor quality and resolution. Simple upsampling algorithms can not produce artifact-free higher resolution images from such low-resolution (LR) images. To deal with that, reconstruction-based super...

  13. Super-resolution photoacoustic fluctuation imaging with multiple speckle illumination

    CERN Document Server

    Chaigne, Thomas; Allain, Marc; Katz, Ori; Gigan, Sylvain; Sentenac, Anne; Bossy, Emmanuel

    2015-01-01

    In deep tissue photoacoustic imaging, the spatial resolution is inherently limited by acoustic diffraction. Moreover, as the ultrasound attenuation increases with frequency, resolution is often traded-off for penetration depth. Here we report on super-resolution photoacoustic imaging by use of multiple speckle illumination. Specifically, we show that the analysis of second-order fluctuations of the photoacoustic images combined with image deconvolution enables resolving optically absorbing structures beyond the acoustic diffraction limit. A resolution increase of almost a factor 2 is demonstrated experimentally. Our method introduces a new framework that could potentially lead to deep tissue photoacoustic imaging with sub-acoustic resolution.

  14. Image super-resolution using windowed ordinary Kriging interpolation

    Science.gov (United States)

    Zhang, Qianying; Wu, Jitao

    2015-02-01

    This paper presents a novel interpolation approach for single image super-resolution based on ordinary Kriging interpolation, which has been widely used in geostatistics. The proposed method simultaneously considers the intensity distances and geometry of the pixel data. We employ a new intensity distance definition and local windows surrounding each unknown high-resolution pixel to implement the algorithm. The proposed approach is able to produce adaptive weights and edge preservation is achieved. Our experimental results show the efficiency of the proposed approach compared to conventional interpolation methods in terms of the peak signal-to-noise (PNSR) and visual perception.

  15. 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.

  16. Single image super-resolution based on image patch classification

    Science.gov (United States)

    Xia, Ping; Yan, Hua; Li, Jing; Sun, Jiande

    2017-06-01

    This paper proposed a single image super-resolution algorithm based on image patch classification and sparse representation where gradient information is used to classify image patches into three different classes in order to reflect the difference between the different types of image patches. Compared with other classification algorithms, gradient information based algorithm is simpler and more effective. In this paper, each class is learned to get a corresponding sub-dictionary. High-resolution image patch can be reconstructed by the dictionary and sparse representation coefficients of corresponding class of image patches. The result of the experiments demonstrated that the proposed algorithm has a better effect compared with the other algorithms.

  17. From single molecules to life: microscopy at the nanoscale.

    Science.gov (United States)

    Turkowyd, Bartosz; Virant, David; Endesfelder, Ulrike

    2016-10-01

    Super-resolution microscopy is the term commonly given to fluorescence microscopy techniques with resolutions that are not limited by the diffraction of light. Since their conception a little over a decade ago, these techniques have quickly become the method of choice for many biologists studying structures and processes of single cells at the nanoscale. In this review, we present the three main approaches used to tackle the diffraction barrier of ∼200 nm: stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). We first present a theoretical overview of the techniques and underlying physics, followed by a practical guide to all of the facets involved in designing a super-resolution experiment, including an approachable explanation of the photochemistry involved, labeling methods available, and sample preparation procedures. Finally, we highlight some of the most exciting recent applications of and developments in these techniques, and discuss the outlook for this field. Graphical Abstract Super-resolution microscopy techniques. Working principles of the common approaches stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM).

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

    Science.gov (United States)

    Greenbaum, Alon; Akbari, Najva; Feizi, Alborz; Luo, Wei; Ozcan, Aydogan

    2013-01-01

    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.

  19. Towards a Mathematical Theory of Super-Resolution

    CERN Document Server

    Candes, Emmanuel

    2012-01-01

    This paper develops a mathematical theory of super-resolution. Broadly speaking, super-resolution is the problem of recovering the fine details of an object---the high end of its spectrum---from coarse scale information only---from samples at the low end of the spectrum. Suppose we have many point sources at unknown locations in $[0,1]$ and with unknown complex-valued amplitudes. We only observe Fourier samples of this object up until a frequency cut-off $f_c$. We show that one can super-resolve these point sources with infinite precision---i.e. recover the exact locations and amplitudes---by solving a simple convex program. This holds provided that the distance between sources is at least $2/f_c$. This result extends to higher dimensions and other models. In one dimension for instance, it is possible to recover a piecewise smooth function by resolving the discontinuity points with infinite precision as well. We also show that the theory and methods are robust to noise. In particular, we develop some theoreti...

  20. 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.

  1. 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.

  2. 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.

  3. Super Resolution from Hyperview Image Stack by Spatial Multiplexing

    Science.gov (United States)

    Grasnick, Armin

    2016-09-01

    An image stack for a hyperview representation could contain millions of different perspective views with extreme image similarity. The recording of all views from a computational 3d model implicates a lateral displacement of the virtual camera. Because of the huge number of views, the offset in between two adjoining camera positions can be very minor. If such a virtual setup reproduces a real hyperview screen setup, the offset can be below the wavelength of the visible light. But even with such small changes, there is an intrinsic probability for a measurable difference in between two neighbour images. Such image dissimilarity can be proofed successfully also in very basic 3d scenes. By using a quantity of juxtapositional images from the hyperview image stack, the resolution of the rendered images can be considerably improved, which is commonly known as super resolution. The utilisation of super resolution images in hyperview could cut the necessity of full frame computing and will reduce the effective render time.

  4. 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.

  5. Decoupling absorption and emission processes in super-resolution localization of emitters in a plasmonic hotspot

    Science.gov (United States)

    Mack, David L.; Cortés, Emiliano; Giannini, Vincenzo; Török, Peter; Roschuk, Tyler; Maier, Stefan A.

    2017-02-01

    The absorption process of an emitter close to a plasmonic antenna is enhanced due to strong local electromagnetic (EM) fields. The emission, if resonant with the plasmonic system, re-radiates to the far-field by coupling with the antenna via plasmonic states, whose presence increases the local density of states. Far-field collection of the emission of single molecules close to plasmonic antennas, therefore, provides mixed information of both the local EM field strength and the local density of states. Moreover, super-resolution localizations from these emission-coupled events do not report the real position of the molecules. Here we propose using a fluorescent molecule with a large Stokes shift in order to spectrally decouple the emission from the plasmonic system, leaving the absorption strongly resonant with the antenna's enhanced EM fields. We demonstrate that this technique provides an effective way of mapping the EM field or the local density of states with nanometre spatial resolution.

  6. Confocal microscopy via multimode fibers: fluorescence bandwidth

    Science.gov (United States)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2016-03-01

    We recently described a method for confocal reflection imaging through fibers, as a way to increase contrast when imaging unstained biological specimens. Using a transmission matrix, focused spots can be created at the distal end of a fiber. The backscattered field coming back from the sample can be filtered using optical correlation to obtain spatial selectivity in the detection. In this proceedings article, we briefly review the working principle of this method, and we discuss how the scheme could be adapted to confocal fluorescence imaging. In particular, we show simulations of the achievable detection bandwidth when using step-index multimode fibers as imaging devices.

  7. Super-Resolution Imaging of a Dielectric Microsphere Is Governed by the Waist of Its Photonic Nanojet.

    Science.gov (United States)

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

    2016-08-10

    Dielectric microspheres with appropriate refractive index can image objects with super-resolution, that is, with a precision well beyond the classical diffraction limit. A microsphere is also known to generate upon illumination a photonic nanojet, which is a scattered beam of light with a high-intensity main lobe and very narrow waist. Here, we report a systematic study of the imaging of water-immersed nanostructures by barium titanate glass microspheres of different size. A numerical study of the light propagation through a microsphere points out the light focusing capability of microspheres of different size and the waist of their photonic nanojet. The former correlates to the magnification factor of the virtual images obtained from linear test nanostructures, the biggest magnification being obtained with microspheres of ∼6-7 μm in size. Analyzing the light intensity distribution of microscopy images allows determining analytically the point spread function of the optical system and thereby quantifies its resolution. We find that the super-resolution imaging of a microsphere is dependent on the waist of its photonic nanojet, the best resolution being obtained with a 6 μm Ø microsphere, which generates the nanojet with the minimum waist. This comparison allows elucidating the super-resolution imaging mechanism.

  8. Static recording characteristics of new type super-resolution near-field structure

    Institute of Scientific and Technical Information of China (English)

    Feng Zhang(张锋); Wendong Xu(徐文东); Yang Wang(王阳); Jinsong Wei(魏劲松); Fei Zhou(周飞); Xiumin Gao(高秀敏); Fuxi Gan(干福熹)

    2004-01-01

    A novel super-resolution near-field optical structure (super-RENS) with bismuth (Bi) mask layer is proposed in this paper. Static optical recording tests with and without super-RENS are carried out using a 650-nm semiconductor laser at recording powers of 14 and 7 mW with pulse duration of 100 ns. The recording marks are observed by high-resolution optical microscopy with a charge-coupled device (CCD)camera. The results show that the Bi mask layer can also concentrate energy into the center of a laser beam at low laser power similar to the traditional Sb mask layer. The results above are further confirmed by another Ar+ laser system. The third-order nonlinear response induced by the plasma oscillation at the Bi/SiN interface during laser irradiation can be used to explain the phenomenon. The calculation results are basically consistent with our experimental results.

  9. Recovering a stochastic process from super-resolution noisy ensembles of single-particle trajectories.

    Science.gov (United States)

    Hoze, N; Holcman, D

    2015-11-01

    Recovering a stochastic process from noisy ensembles of single-particle trajectories is resolved here using the coarse-grained Langevin equation as a model. The massive redundancy contained in single-particle tracking data allows recovering local parameters of the underlying physical model. We use several parametric and nonparametric estimators to compute the first and second moments of the process, to recover the local drift, its derivative, and the diffusion tensor, and to deconvolve the instrumental from the physical noise. We use numerical simulations to also explore the range of validity for these estimators. The present analysis allows defining what can exactly be recovered from statistics of super-resolution microscopy trajectories used for characterizing molecular trafficking underlying cellular functions.

  10. Partial internal reflections on total internal reflection fluorescent microscopy.

    Science.gov (United States)

    Simon, Sanford M

    2009-11-01

    Microscopy, especially fluorescence microscopy, has proven to be a powerful method for studying biological processes. Unfortunately, some of the same features that make biological membranes powerful (for example, all of the action taking place across a narrow 4nm film) also make it difficult to visualize by fluorescence. Over the past 30 years, numerous tricks have been developed to narrow the plane over which data is collected. One approach, total internal reflection (TIR) fluorescence microscopy, is particularly well suited for studying membrane events. A key issue to address when using TIR to tackle a new biological problem is: how can one judge whether the signals being observed are actually the biological phenomena that one wishes to study?

  11. Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy

    Science.gov (United States)

    Ito, Takashi

    2008-01-01

    This article describes experiments for an undergraduate instrumental analysis laboratory that aim to observe individual double-stranded DNA (dsDNA) molecules using fluorescence microscopy and atomic force microscopy (AFM). dsDNA molecules are observed under several different conditions to discuss their chemical and physical properties. In…

  12. Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy

    Science.gov (United States)

    Ito, Takashi

    2008-01-01

    This article describes experiments for an undergraduate instrumental analysis laboratory that aim to observe individual double-stranded DNA (dsDNA) molecules using fluorescence microscopy and atomic force microscopy (AFM). dsDNA molecules are observed under several different conditions to discuss their chemical and physical properties. In…

  13. X-ray fluorescence microscopy of olfactory receptor neurons

    Energy Technology Data Exchange (ETDEWEB)

    Ducic, T; Herbst, J; Novakova, E; Salditt, T [Institute for X-ray Physics, Georg-August-University, Friedrich-Hund-Pl. 1, 37077 Goettingen (Germany); Breunig, E; Schild, D [Department of Molecular Neurophysiology, Georg-August University Goettingen (Germany); Susini, J; Tucoulu, R, E-mail: tducic@gwdg.d [European Synchrotron Radiation Facility ESRF, 6 rue Jules Horowitz, 38043 Grenoble (France)

    2009-09-01

    We report a x-ray fluorescence microscopy study of cells and tissues from the olfactory system of Xenopus laevis. In this experiment we focus on sample preparation and experimental issues, and present first results of fluorescence maps of the elemental distribution of Cl, K, Ca, P, S and Na both in individual isolated neural cells and in cross-sections of the same tissue.

  14. Automated quantification of synapses by fluorescence microscopy.

    Science.gov (United States)

    Schätzle, Philipp; Wuttke, René; Ziegler, Urs; Sonderegger, Peter

    2012-02-15

    The quantification of synapses in neuronal cultures is essential in studies of the molecular mechanisms underlying synaptogenesis and synaptic plasticity. Conventional counting of synapses based on morphological or immunocytochemical criteria is extremely work-intensive. We developed a fully automated method which quantifies synaptic elements and complete synapses based on immunocytochemistry. Pre- and postsynaptic elements are detected by their corresponding fluorescence signals and their proximity to dendrites. Synapses are defined as the combination of a pre- and postsynaptic element within a given distance. The analysis is performed in three dimensions and all parameters required for quantification can be easily adjusted by a graphical user interface. The integrated batch processing enables the analysis of large datasets without any further user interaction and is therefore efficient and timesaving. The potential of this method was demonstrated by an extensive quantification of synapses in neuronal cultures from DIV 7 to DIV 21. The method can be applied to all datasets containing a pre- and postsynaptic labeling plus a dendritic or cell surface marker.

  15. 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

  16. Fluorescence cryo-microscopy: current challenges and prospects

    OpenAIRE

    Kaufmann, Rainer; Hagen, Christoph; Grünewald, Kay

    2014-01-01

    Studying biological structures with fine details does not only require a microscope with high resolution, but also a sample preparation process that preserves the structures in a near-native state. Live-cell imaging is restricted mostly to the field of light microscopy. For studies requiring much higher resolution, fast freezing techniques (vitrification) are successfully used to immobilize the sample in a near-native state for imaging with electron and X-ray cryo-microscopy. Fluorescence cry...

  17. 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.

  18. Image Super-Resolution Using Deep Convolutional Networks.

    Science.gov (United States)

    Dong, Chao; Loy, Chen Change; He, Kaiming; Tang, Xiaoou

    2016-02-01

    We propose a deep learning method for single image super-resolution (SR). Our method directly learns an end-to-end mapping between the low/high-resolution images. The mapping is represented as a deep convolutional neural network (CNN) that takes the low-resolution image as the input and outputs the high-resolution one. We further show that traditional sparse-coding-based SR methods can also be viewed as a deep convolutional network. But unlike traditional methods that handle each component separately, our method jointly optimizes all layers. Our deep CNN has a lightweight structure, yet demonstrates state-of-the-art restoration quality, and achieves fast speed for practical on-line usage. We explore different network structures and parameter settings to achieve trade-offs between performance and speed. Moreover, we extend our network to cope with three color channels simultaneously, and show better overall reconstruction quality.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    Action recognition systems mostly work with videos of proper quality and resolution. Even most challenging bench- mark databases for action recognition, hardly include videos of low-resolution from, e.g., surveillance cameras. In videos recorded by such cameras, due to the distance between people...... and cameras, people are pictured very small and hence challenge action recognition algorithms. Simple upsampling methods, like bicubic interpolation, cannot retrieve all the detailed information that can help the recognition. To deal with this problem, in this paper we combine results of bicubic interpolation...... 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...

  20. A Fast Super-Resolution Reconstruction from Image Sequence

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Based on the mechanism of imagery, a novel method called the delaminating combining template method, used for the problem of super-resolution reconstruction from image sequence, is described in this paper. The combining template method contains two steps: a delaminating strategy and a combining template algorithm. The delaminating strategy divides the original problem into several sub-problems;each of them is only connected to one degrading factor. The combining template algorithm is suggested to resolve each sub-problem. In addition, to verify the valid of the method, a new index called oriental entropy is presented. The results from the theoretical analysis and experiments illustrate that this method to be promising and efficient.

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

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; Berg, van den 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

  2. Photon budget analysis for fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Zhao, Q.; Young, I.T.; De Jong, J.G.S.

    2011-01-01

    We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon “budget.” These

  3. Blind fluorescence structured illumination microscopy: A new reconstruction strategy

    CERN Document Server

    Labouesse, S; Idier, J; Bourguignon, S; Liu, P; Sentenac, A

    2016-01-01

    In this communication, a fast reconstruction algorithm is proposed for fluorescence \\textit{blind} structured illumination microscopy (SIM) under the sample positivity constraint. This new algorithm is by far simpler and faster than existing solutions, paving the way to 3D and/or real-time 2D reconstruction.

  4. Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

    NARCIS (Netherlands)

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

    2009-01-01

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

  5. 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 g

  6. Stage-dependent changes of the nuclear architecture, envelope and lamina during mammalian early embryonic development studied with a novel 3D structured illumination microscopy protocol

    OpenAIRE

    Popken, Jens

    2016-01-01

    Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over conventional microscopy and is well established on flat, adherent cells. However, blastomeres in mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to non-adherent embryos moving during scanning and a large distance to the cover glass. The biggest challenge an...

  7. Super-resolution chemical imaging with dynamic placement of plasmonic hotspots

    Science.gov (United States)

    Olson, Aeli P.; Ertsgaard, Christopher T.; McKoskey, Rachel M.; Rich, Isabel S.; Lindquist, Nathan C.

    2015-08-01

    We demonstrate dynamic placement of plasmonic "hotspots" for super-resolution chemical imaging via Surface Enhanced Raman Spectroscopy (SERS). A silver nanohole array surface was coated with biological samples and illuminated with a laser. Due to the large plasmonic field enhancements, blinking behavior of the SERS hotspots was observed and processed using a Stochastic Optical Reconstruction Microscopy (STORM) algorithm enabling localization to within 10 nm. However, illumination of the sample with a single static laser beam (i.e., a slightly defocused Gaussian beam) only produced SERS hotspots in fixed locations on the surface, leaving noticeable gaps in any final image. But, by using a spatial light modulator (SLM), the illumination profile of the beam could be altered, shifting any hotspots across the nanohole array surface in sub-wavelength steps. Therefore, by properly structuring an illuminating light field with the SLM, we show the possibility of positioning plasmonic hotspots over a metallic nanohole surface on-the-fly. Using this and our SERS-STORM imaging technique, we show potential for high-resolution chemical imaging without the noticeable gaps that were present with static laser illumination. Interestingly, even illuminating the surface with randomly shifting SLM phase profiles was sufficient to completely fill in a wide field of view for super-resolution SERS imaging of a single strand of 100-nm thick collagen protein fibrils. Images were then compared to those obtained with a scanning electron microscope (SEM). Additionally, we explored alternative methods of phase shifting other than holographic illumination through the SLM to create localization of hotspots necessary for SERS-STORM imaging.

  8. 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.

  9. 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.

  10. Nanoscale resolution for fluorescence microscopy via adiabatic passage

    CERN Document Server

    Rubio, Juan Luis; Ahufinger, Verònica; Mompart, Jordi

    2015-01-01

    We propose the use of the subwavelength localization via adiabatic passage technique for fluorescence microscopy with nanoscale resolution in the far field. This technique uses a {\\Lambda}-type medium coherently coupled to two laser pulses: the pump, with a node in its spatial profile, and the Stokes. The population of the {\\Lambda} system is adiabatically transferred from one ground state to the other except at the node position, yielding a narrow population peak. This coherent localization allows fluorescence imaging with nanometer lateral resolution. We derive an analytical expression to asses the resolution and perform a comparison with the coherent population trapping and the stimulated-emission-depletion techniques.

  11. Mueller matrix signature in advanced fluorescence microscopy imaging

    Science.gov (United States)

    Mazumder, Nirmal; Qiu, Jianjun; Kao, Fu-Jen; Diaspro, Alberto

    2017-02-01

    We have demonstrated the measurement and characterization of the polarization properties of a fluorescence signal using four-channel photon counting based Stokes-Mueller polarization microscopy. Thus, Lu-Chipman decomposition was applied to extract the critical polarization properties such as depolarization, linear retardance and the optical rotation of collagen type I fiber. We observed the spatial distribution of anisotropic and helical molecules of collagen from the reconstructed 2D Mueller images based on the fluorescence signal in a pixel-by-pixel manner.

  12. Fluorescent microscopy approaches of quantitative soil microbial analysis

    Science.gov (United States)

    Ivanov, Konstantin; Polyanskaya, Lubov

    2015-04-01

    Classical fluorescent microscopy method was used during the last decades in various microbiological studies of terrestrial ecosystems. The method provides representative results and simple application which is allow to use it both as routine part of amplitudinous research and in small-scaled laboratories. Furthermore, depending on research targets a lot of modifications of fluorescent microscopy method were established. Combination and comparison of several approaches is an opportunity of quantitative estimation of microbial community in soil. The first analytical part of the study was dedicated to soil bacterial density estimation by fluorescent microscopy in dynamic of several 30-days experiments. The purpose of research was estimation of changes in soil bacterial community on the different soil horizons under aerobic and anaerobic conditions with adding nutrients in two experimental sets: cellulose and chitin. Was modified the nalidixic acid method for inhibition of DNA division of gram-negative bacteria, and the method provides the quantification of this bacterial group by fluorescent microscopy. Established approach allowed to estimate 3-4 times more cells of gram-negative bacteria in soil. The functions of actinomyces in soil polymer destruction are traditionally considered as dominant in comparison to gram-negative bacterial group. However, quantification of gram-negative bacteria in chernozem and peatland provides underestimation of classical notion for this bacterial group. Chitin introduction had no positive effect to gram-negative bacterial population density changes in chernozem but concurrently this nutrient provided the fast growing dynamics at the first 3 days of experiment both under aerobic and anaerobic conditions. This is confirming chitinolytic activity of gram-negative bacteria in soil organic matter decomposition. At the next part of research modified method for soil gram-negative bacteria quantification was compared to fluorescent in situ

  13. Super-resolution imaging in optical scanning holography using structured illumination

    Science.gov (United States)

    Ren, Zhenbo; Lam, Edmund Y.

    2016-10-01

    As a specific digital holographic microscopy system, optical scanning holography (OSH) is an appealing technique that makes use of the advantages of holography in the application of optical microscopy. In OSH system, a three-dimensional object is scanned with a Fresnel zone plate in a raster fashion, and the electrical signals are demodulated into a complex hologram by heterodyne detection. Then the recorded light wavefront information contained in the hologram allows one to digitally reconstruct the specimen for multiple purposes such as optical sectioning, extended focused imaging as well as three-dimensional imaging. According to Abbe criterion, however, akin to those conventional microscopic imaging systems, OSH suffers from limited resolving power due to the finite sizes of the objective lens and the aperture, i.e., low numerical aperture. To bypass the diffraction barrier in light microscopy, various super-resolution imaging techniques have been proposed. Among those methods, structured illumination is an ensemble imaging concept that modulates the spatial frequency by projecting additional well-defined patterns with different orientation and phase shift onto the specimen. Computational algorithms are then applied to remove the effect of the structure and to reconstruct a super-resolved image beyond the diffraction-limit. In this paper, we introduce this technique in OSH system to scale down the spatial resolution beyond the diffraction limit. The performance of the proposed method is validated by simulation and experimental results.

  14. 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.

  15. Image Super-Resolution via Adaptive Regularization and Sparse Representation.

    Science.gov (United States)

    Cao, Feilong; Cai, Miaomiao; Tan, Yuanpeng; Zhao, Jianwei

    2016-07-01

    Previous studies have shown that image patches can be well represented as a sparse linear combination of elements from an appropriately selected over-complete dictionary. Recently, single-image super-resolution (SISR) via sparse representation using blurred and downsampled low-resolution images has attracted increasing interest, where the aim is to obtain the coefficients for sparse representation by solving an l0 or l1 norm optimization problem. The l0 optimization is a nonconvex and NP-hard problem, while the l1 optimization usually requires many more measurements and presents new challenges even when the image is the usual size, so we propose a new approach for SISR recovery based on regularization nonconvex optimization. The proposed approach is potentially a powerful method for recovering SISR via sparse representations, and it can yield a sparser solution than the l1 regularization method. We also consider the best choice for lp regularization with all p in (0, 1), where we propose a scheme that adaptively selects the norm value for each image patch. In addition, we provide a method for estimating the best value of the regularization parameter λ adaptively, and we discuss an alternate iteration method for selecting p and λ . We perform experiments, which demonstrates that the proposed regularization nonconvex optimization method can outperform the convex optimization method and generate higher quality images.

  16. Coupled Deep Autoencoder for Single Image Super-Resolution.

    Science.gov (United States)

    Zeng, Kun; Yu, Jun; Wang, Ruxin; Li, Cuihua; Tao, Dacheng

    2017-01-01

    Sparse coding has been widely applied to learning-based single image super-resolution (SR) and has obtained promising performance by jointly learning effective representations for low-resolution (LR) and high-resolution (HR) image patch pairs. However, the resulting HR images often suffer from ringing, jaggy, and blurring artifacts due to the strong yet ad hoc assumptions that the LR image patch representation is equal to, is linear with, lies on a manifold similar to, or has the same support set as the corresponding HR image patch representation. Motivated by the success of deep learning, we develop a data-driven model coupled deep autoencoder (CDA) for single image SR. CDA is based on a new deep architecture and has high representational capability. CDA simultaneously learns the intrinsic representations of LR and HR image patches and a big-data-driven function that precisely maps these LR representations to their corresponding HR representations. Extensive experimentation demonstrates the superior effectiveness and efficiency of CDA for single image SR compared to other state-of-the-art methods on Set5 and Set14 datasets.

  17. 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.

  18. 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.

  19. 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...

  20. 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.

  1. 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.

  2. Structured light sheet fluorescence microscopy based on four beam interference.

    Science.gov (United States)

    Lei, Ming; Zumbusch, Andreas

    2010-08-30

    A 3D structured light sheet microscope using a four-faceted symmetric pyramid is presented. The sample is illuminated by the resulting four beam interference field. This approach combines advantages of standing wave and structured illumination microscopy. Examples of micrographs of fluorescently labeled Chinese hamster ovary (CHO) cells as well as of the compound eyes of drosophila are shown and the optical sectioning ability of our system is demonstrated. The capabilities and the limitations of the scheme are discussed.

  3. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging.

    Science.gov (United States)

    Geissbuehler, Stefan; Sharipov, Azat; Godinat, Aurélien; Bocchio, Noelia L; Sandoz, Patrick A; Huss, Anja; Jensen, Nickels A; Jakobs, Stefan; Enderlein, Jörg; Gisou van der Goot, F; Dubikovskaya, Elena A; Lasser, Theo; Leutenegger, Marcel

    2014-12-18

    Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm(3) without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.

  4. 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.

  5. Compressive Fluorescence Microscopy for Biological and Hyperspectral Imaging

    CERN Document Server

    Studer, Vincent; Chahid, Makhlad; Moussavi, Hamed; Candes, Emmanuel; Dahan, Maxime

    2012-01-01

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

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

    Science.gov (United States)

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

    2008-06-01

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

  7. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  8. Coordinate-targeted fluorescence nanoscopy with multiple off states

    Science.gov (United States)

    Danzl, Johann G.; Sidenstein, Sven C.; Gregor, Carola; Urban, Nicolai T.; Ilgen, Peter; Jakobs, Stefan; Hell, Stefan W.

    2016-02-01

    Far-field super-resolution fluorescence microscopy discerns fluorophores residing closer than the diffraction barrier by briefly transferring them in different (typically ON and OFF) states before detection. In coordinate-targeted super-resolution variants, such as stimulated emission depletion (STED) microscopy, this state difference is created by the intensity minima and maxima of an optical pattern, causing all fluorophores to assume the off state, for instance, except at the minima. Although strong spatial confinement of the on state enables high resolution, it also subjects the fluorophores to excess intensities and state cycles at the maxima. Here, we address these issues by driving the fluorophores into a second off state that is inert to the excess light. By using reversibly switchable fluorescent proteins as labels, our approach reduces bleaching and enhances resolution and contrast in live-cell STED microscopy. Using two or more transitions to off states is a useful strategy for augmenting the power of coordinate-targeted super-resolution microscopy.

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

    Directory of Open Access Journals (Sweden)

    Sean C Warren

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

  10. Single image super-resolution with multiscale similarity learning.

    Science.gov (United States)

    Zhang, Kaibing; Gao, Xinbo; Tao, Dacheng; Li, Xuelong

    2013-10-01

    Example learning-based image super-resolution (SR) is recognized as an effective way to produce a high-resolution (HR) image with the help of an external training set. The effectiveness of learning-based SR methods, however, depends highly upon the consistency between the supporting training set and low-resolution (LR) images to be handled. To reduce the adverse effect brought by incompatible high-frequency details in the training set, we propose a single image SR approach by learning multiscale self-similarities from an LR image itself. The proposed SR approach is based upon an observation that small patches in natural images tend to redundantly repeat themselves many times both within the same scale and across different scales. To synthesize the missing details, we establish the HR-LR patch pairs using the initial LR input and its down-sampled version to capture the similarities across different scales and utilize the neighbor embedding algorithm to estimate the relationship between the LR and HR image pairs. To fully exploit the similarities across various scales inside the input LR image, we accumulate the previous resultant images as training examples for the subsequent reconstruction processes and adopt a gradual magnification scheme to upscale the LR input to the desired size step by step. In addition, to preserve sharper edges and suppress aliasing artifacts, we further apply the nonlocal means method to learn the similarity within the same scale and formulate a nonlocal prior regularization term to well pose SR estimation under a reconstruction-based SR framework. Experimental results demonstrate that the proposed method can produce compelling SR recovery both quantitatively and perceptually in comparison with other state-of-the-art baselines.

  11. Multiphoton excitation fluorescence microscopy in planar membrane systems.

    Science.gov (United States)

    Brewer, Jonathan; Bernardino de la Serna, Jorge; Wagner, Kerstin; Bagatolli, Luis A

    2010-07-01

    The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

  12. Three-dimensional multimodal sub-diffraction imaging with spinning-disk confocal microscopy using blinking/ fluctuating probes

    Institute of Scientific and Technical Information of China (English)

    Xuanze Chen[2; Zhiping zeng[2; Hening Wang[1; Peng Xi[1

    2015-01-01

    Three-dimensional imaging cannot be achieved easily using previously developed localization super-resolution techniques. Here, we present a three-dimensional multimodal sub-diffraction imaging technique with spinning-disk (SD) confocal microscopy called 3D-MUSIC, which not only has all the advantages of SD confocal microscopy such as fast imaging speed, high signal-to-noise ratio, and optical-sectioning capability, but also extends its spatial resolution limit along all three dimensions. Both axial and lateral resolution can be improved simul- taneously by virtue of the blinking/fluctuating nature of modified fluorescent probes, exemplified with the quantum dots. Further, super-resolution images with dual modality can be obtained through super-resolution optical fluctuation imaging (SOFI) and bleaching/blinking-assisted localization microscopy (BALM). Therefore, fast super-resolution imaging can be achieved with SD-SOFI by capturing only 100 frames while SD-BaLM yields high-resolution imaging.

  13. 超分辨率荧光显微技术——解析2014年诺贝尔化学奖%Super-resolution fluorescent microscopy: commentary on the 2014 Nobel Prize in Chemistry

    Institute of Scientific and Technical Information of China (English)

    席鹏; 孙育杰

    2015-01-01

    2014年诺贝尔化学奖授予Eric Betzig,Stefan W.Hell和William E.Moerner3位科学家,以表彰他们在超分辨率荧光显微成像技术方面的重大贡献.本文从显微镜分辨率的起因入手,对超分辨荧光显微技术进行了深入阐述.此外,对光学显微技术的发展前景进行展望.

  14. Near-Infrared Super Resolution Imaging with Metallic Nanoshell Particle Chain Array

    CERN Document Server

    Kong, Weijie; Cao, Penfei; Cheng, Lin; Gong, Li; Zhao, Xining; Yang, Lili

    2012-01-01

    We propose a near-infrared super resolution imaging system without a lens or a mirror but with an array of metallic nanoshell particle chain. The imaging array can plasmonically transfer the near-field components of dipole sources in the incoherent and coherent manners and the super resolution images can be reconstructed in the output plane. By tunning the parameters of the metallic nanoshell particle, the plasmon resonance band of the isolate nanoshell particle red-shifts to the near-infrared region. The near-infrared super resolution images are obtained subsequently. We calculate the field intensity distribution at the different planes of imaging process using the finite element method and find that the array has super resolution imaging capability at near-infrared wavelengths. We also show that the image formation highly depends on the coherence of the dipole sources and the image-array distance.

  15. 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.

  16. In vivo multiphoton fluorescence microscopy of epithelial precancer

    Science.gov (United States)

    Zheng, Wei; Li, Dong; Zeng, Yan; Qu, Jianan Y.

    2011-03-01

    Most human cancers arise from epithelium, the superficial layer covering the exterior of body or lining the internal body cavities. Endogenous fluorophores such as aromatic amino acids, reduced nicotinamide adenine dinucleotide (NADH), flavoprotein (FAD), keratin, collagen, and elastin can provide abundant information to reveal the changes in biochemistry, metabolism, and morphology of living tissues. Thus, autofluorescence spectroscopy and microscopy have been recognized as potential tools for discrimination of cancer from normal tissues. However, current fluorescence diagnostic studies mostly rely on spectral analysis or morphological differentiation. It is challenged since the emission spectra of endogenous fluorophores are broad and usually overlapping with each other and the fluorescence intensity could be affected by many factors. In this study, we instrumented a nonlinear optical microscopy system to characterize the morphologic and biochemical features in the epithelial precancer in vivo. The 7,12-dimethylbenz(a)anthracenetreated hamster cheek pouch were used as a living animal carcinogenesis model. And the autofluorescence signals of NADH, collagen and elastin were recorded by a time- and spectral- resolved detection system. The results show that there are obvious differences in the morphology of three-dimensional autofluorescence images between normal and precancerous epithelial tissues. The fluorescence lifetime of NADH and the SHG signal from collagen could provide additional approaches to identify cancer from normal tissue.

  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. 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.

    2015-11-01

    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.

  19. Super-resolution image transfer by a vortex-like metamaterial

    CERN Document Server

    Dong, Hui Yuan; Fung, Kin Hung; Cui, Tie Jun

    2013-01-01

    We propose a vortex-like metamaterial device that is capable of transferring image along a spiral route without losing subwavelength information of the image. The super-resolution image can be guided and magnified at the same time with one single design. Our design may provide insights in manipulating super-resolution image in a more flexible manner. Examples are given and illustrated with numerical simulations.

  20. Redundant Discrete Wavelet Transform Based Super-Resolution Using Sub-Pixel Image Registration

    Science.gov (United States)

    2003-03-01

    AFIT/GE/ENG/03-18 REDUNDANT DISCRETE WAVELET TRANSFORM BASED SUPER-RESOLUTION USING SUB-PIXEL IMAGE REGISTRATION THESIS Daniel L. Ward Second...position of the United States Air Force, Department of Defense, or the United States Government. AFIT/GE/ENG/03-18 REDUNDANT DISCRETE WAVELET TRANSFORM BASED...O3-18 REDUNDANT DISCRETE WAVELET TRANSFORM BASED SUPER-RESOLUTION USING SUB-PIXEL IMAGE REGISTRATION THESIS Daniel Lee Ward, B.S.E.E. Second

  1. Experimental Study of Super-Resolution Using a Compressive Sensing Architecture

    Science.gov (United States)

    2015-03-01

    Experimental study of super-resolution using a compressive sensing architecture J. Christopher Flakea,c, Gary Eulissa, John B. Greerb, Stephanie...laboratory imaging system was constructed following an architecture that has become familiar from the theory of compressive sensing . The system uses...choices in system design will become increasingly more important. We present a compressive sensing image system designed for super-resolution: the

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

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-22

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

  4. Quantitative high dynamic range beam profiling for fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, T. J., E-mail: t.j.mitchell@dur.ac.uk; Saunter, C. D.; O’Nions, W.; Girkin, J. M.; Love, G. D. [Centre for Advanced Instrumentation and Biophysical Sciences Institute, Department of Physics, Durham University, Durham DH1 3LE (United Kingdom)

    2014-10-15

    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.

  5. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    Science.gov (United States)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  6. Fluorescence microscopy test in porphyrias, photodermatoses and lead exposed persons.

    Science.gov (United States)

    Kansky, A

    1975-07-18

    Fluorescence microscopy tests were carried out in different groups of patients Peripheral blood diluted with saline was used and 200 high power fields were inspected in every case. The results were presented as the number of fluorescing erythrocytes (FE) per 100000 red blood cells (or 200 fields). In the controls, porphyria cutanea tarda patients and patients with photodermatoses other than erythopoietic protoporphyria and pellagra almost no FE were detected. In erythropoietic protoporphyria the mean value was 10600, in lead poisoning 1032, in patients exposed to lead 48.2, in sideropenic anaemia 123 and in patients with pellagra 8.1 FE/100000 red blood cells. The conclusion is made that one has to take care, when using this test for detection of latent carriers in genetic studies of the relatives of patients with erythropoietic protoporphyria. The test is useful for the confirmation of the diagnosis of erythropoietic protoporphyria.

  7. Three-dimensional super-resolution imaging of the midplane protein FtsZ in live Caulobacter crescentus cells using astigmatism.

    Science.gov (United States)

    Biteen, Julie S; Goley, Erin D; Shapiro, Lucy; Moerner, W E

    2012-03-01

    Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring. We quantify these dynamics and determine the FtsZ depolymerization time to be divisional stage.

  8. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    Science.gov (United States)

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

  9. Comparison between two super-resolution implementations in PET imaging.

    Science.gov (United States)

    Chang, Guoping; Pan, Tinsu; Qiao, Feng; Clark, John W; Mawlawi, Osama R

    2009-04-01

    Super-resolution (SR) techniques are used in PET imaging to generate a high-resolution image by combining multiple low-resolution images that have been acquired from different points of view (POV). In this article, the authors propose a novel implementation of the SR technique whereby the required multiple low-resolution images are generated by shifting the reconstruction pixel grid during the image reconstruction process rather than being acquired from different POVs. The objective of this article is to compare the performances of the two SR implementations using theoretical and experimental studies. A mathematical framework is first provided to support the hypothesis that the two SR implementations have similar performance in current PET/CT scanners that use block detectors. Based on this framework, a simulation study, a point source study, and a NEMA/IEC phantom study were conducted to compare the performance of these two SR implementations with respect to contrast, resolution, noise, and SNR. For reference purposes, a comparison with a native reconstruction (NR) image using a high-resolution pixel grid was also performed. The mathematical framework showed that the two SR implementations are expected to achieve similar contrast and resolution but different noise contents. These results were confirmed by the simulation and experimental studies. The simulation study showed that the two SR implementations have an average contrast difference of 2.3%, while the point source study showed that their average differences in contrast and resolution were 0.5% and 1.2%, respectively. Comparisons between the SR and NR images for the point source study showed that the NR image exhibited averages of 30% and 8% lower contrast and resolution, respectively. The NEMA/IEC phantom study showed that the three images (two SR and NR) exhibited different noise structures. The SNR of the new SR implementation was, on average, 21.5% lower than the original implementation largely due to an

  10. 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

    2015-01-22

    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.

  11. Super-resolution with Toraldo pupils: analysis with electromagnetic numerical simulations

    Science.gov (United States)

    Olmi, Luca; Bolli, Pietro; Cresci, Luca; Mugnai, Daniela; Natale, Enzo; Nesti, Renzo; Panella, Dario; Stefani, Lorenzo

    2016-07-01

    The concept of super-resolution refers to various methods for improving the angular resolution of an optical imaging system beyond the classical diffraction limit. In optical microscopy, several techniques have been developed with the aim of narrowing the central lobe of the illumination Point Spread Function (PSF). In Astronomy a few methods have been proposed to achieve reflector telescopes and antennas with resolution significantly better than the diffraction limit but, to our best knowledge, no working system is in operation. A possible practical approach consists of using the so-called "Toraldo Pupils" (TPs) or variable transmittance filters. These pupils were introduced by G. Toraldo di Francia in 1952,1 and consist of a series of discrete, concentric circular coronae providing specific optical transparency and dephasing in order to engineer the required PSF. The first successful laboratory test of TPs in the microwaves was achieved in 2003,2 and in the present work we build upon these initial measurements to perform electromagnetic (EM) numerical simulations of TPs, using a commercial full-wave software tool. These simulations were used to study various EM effects that can mask and/or affect the performance of the pupils and to analyze the near-field as well as the far-field response. Our EM analysis confirms that at 20 GHz the width of the central lobe in the far-field generated by a TP significantly decreases compared to a clear circular aperture with the same diameter.

  12. 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.

  13. A new probe for super-resolution imaging of membranes elucidates trafficking pathways.

    Science.gov (United States)

    Revelo, Natalia H; Kamin, Dirk; Truckenbrodt, Sven; Wong, Aaron B; Reuter-Jessen, Kirsten; Reisinger, Ellen; Moser, Tobias; Rizzoli, Silvio O

    2014-05-26

    The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.

  14. Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis

    Directory of Open Access Journals (Sweden)

    Nilima Prakash

    2011-01-01

    Full Text Available Context and Aims: The oral cavity is the most predominant location in the head and neck region for primary malignant epithelial tumors. Oral cancer is estimated to be the sixth most common malignancy. Early recognition is imperative for successful treatment and good prognosis. Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological specimen. Materials and Methods: Smears were collected from 20 individuals with oral lesions suspicious of malignancy, oral lesions not suggestive of malignancy and normal buccal mucosa. One smear was stained with Papanicolaou stain and another one with acridine orange stain. The differences in the study group and control group were compared by means of the χ2 (Chi-square test. The results were considered statistically significant whenever P was <0.05. Results: The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. However, the acridine orange fluorescence stain did not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content. Conclusion: The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases.

  15. Signal enhanced holographic fluorescence microscopy with guide-star reconstruction

    Science.gov (United States)

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

    2016-01-01

    We propose a signal enhanced guide-star reconstruction method for holographic fluorescence microscopy. In the late 00’s, incoherent digital holography started to be vigorously studied by several groups to overcome the limitations of conventional digital holography. The basic concept of incoherent digital holography is to acquire the complex hologram from incoherent light by utilizing temporal coherency of a spatially incoherent light source. The advent of incoherent digital holography opened new possibility of holographic fluorescence microscopy (HFM), which was difficult to achieve with conventional digital holography. However there has been an important issue of low and noisy signal in HFM which slows down the system speed and degrades the imaging quality. When guide-star reconstruction is adopted, the image reconstruction gives an improved result compared to the conventional propagation reconstruction method. The guide-star reconstruction method gives higher imaging signal-to-noise ratio since the acquired complex point spread function provides optimal system-adaptive information and can restore the signal buried in the noise more efficiently. We present theoretical explanation and simulation as well as experimental results. PMID:27446653

  16. Tools and techniques to measure mitophagy using fluorescence microscopy.

    Science.gov (United States)

    Dolman, Nick J; Chambers, Kevin M; Mandavilli, Bhaskar; Batchelor, Robert H; Janes, Michael S

    2013-11-01

    Mitophagy is a specialized form of autophagy that removes damaged mitochondria, thereby maintaining efficient cellular metabolism and reducing cellular stress caused by aberrant oxidative bursts. Deficits in mitophagy underlie several diseases, and a substantial body of research has elucidated key steps in the pathways that lead to and execute autophagic clearance of mitochondria. Many of these studies employ fluorescence microscopy to visualize mitochondrial morphology, mass, and functional state. Studies in this area also examine colocalization/recruitment of accessory factors, components of the autophagic machinery and signaling molecules to mitochondria. In this review, we provide a brief summary of the current understanding about the processes involved in mitophagy followed by a discussion of probes commonly employed and important considerations of the methodologies to study and analyze mitophagy using fluorescence microscopy. Representative data, where appropriate, are provided to highlight the use of key probes to monitor mitophagy. The review will conclude with a consideration of new possibilities for mitophagy research and a discussion of recently developed technologies for this emerging area of cell biology.

  17. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Beaudet A.

    1998-01-01

    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. Non-linear image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Gregor, Ingo; Ros, Robert; Enderlein, Jörg

    2017-02-01

    Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates. We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging. 1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101. 2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53-54.

  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

    2008-04-15

    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 oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) 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-gp91(phox). 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-gp91(phox) are approximately 1.38 and approximately 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.

  20. Saturated pattern-illuminated Fourier ptychography microscopy

    Science.gov (United States)

    Fang, Yue; Chen, Youhua; Kuang, Cuifang; Xiu, Peng; Liu, Qiulan; Ge, Baoliang; Liu, Xu

    2017-01-01

    We report a series of simulation studies which extends pattern-illuminated Fourier ptychography microscopy by integrating with the nonlinearity arising from saturation of the fluorophore excited state for super-resolution fluorescence imaging. This extended technique, termed Saturated pattern-illuminated Fourier ptychography (SpiFP) microscopy, could achieve a resolution four times that of wide field when the illuminating light intensity approaches the saturation threshold in simulations. Increasing light intensity leads to further resolution enhancement. In order to demonstrate the performance of SpiFP, we make a comparison between SpiFP and saturated structure illumination microscopy in simulations, and prove that the SpiFP exhibits superior robustness to noise, aberration correcting ability, and pattern’s flexibility. Introducing the saturation of the fluorescent emission brings in notable improvements in imaging performance, implying its potential in nanoscale-sized biological observations by wide-field microscopy.

  1. Infrared super-resolution imaging method based on retina micro-motion

    Science.gov (United States)

    Sui, Xiubao; Gao, Hang; Sun, Yicheng; Chen, Qian; Gu, Guohua

    2013-09-01

    With the wide application of infrared focal plane arrays (IRFPA), military, aerospace, public security and other applications have higher and higher requirements on the spatial resolution of infrared images. However, traditional super-resolution imaging methods have increasingly unable to meet this requirement in technology. In this paper, we adopt the achievement that the human retina micro-motion is the important reason why the human has the hyperacuity ability. Based on the achievement, we bring forward an infrared super-resolution imaging method based on retina micro-motion. In the method, we use the piezoelectric ceramic equipment to control the infrared detector moving variably within a plane parallel to the focal plane. The motion direction is toward each other into a direction of 90°. In the four directions of the movement, we get four sub-images and generate a high spatial resolution infrared image by image interpolation method. In the process of the shifting movement of the detector, we set the threshold of the detector response and record the response time difference when adjacent pixel responses are up to the threshold. By the method, we get the object's edges, enhance them in the high resolution infrared image and get the super-resolution infrared image. The experimental results show that our proposed super-resolution imaging methods can improve the spatial resolution of the infrared image effectively. The method will offer a new idea for the super-resolution reconstruction of infrared images.

  2. Pairwise Operator Learning for Patch Based Single-image Super-resolution.

    Science.gov (United States)

    Tang, Yi; Shao, Ling

    2016-12-14

    Motivated by the fact that image patches could be inherently represented by matrices, single-image super-resolution is treated as a problem of learning regression operators in a matrix space in this paper. The regression operators that map low-resolution image patches to high-resolution image patches are generally defined by left and right multiplication operators. The pairwise operators are respectively used to extract the raw and column information of low-resolution image patches for recovering high-resolution estimations. The patch based regression algorithm possesses three favorable properties. Firstly, the proposed super-resolution algorithm is efficient during both training and testing, because image patches are treated as matrices. Secondly, the data storage requirement of the optimal pairwise operator is far less than most popular single-image super-resolution algorithms because only two small sized matrices need to be stored. Lastly, the super-resolution performance is competitive with most popular single-image super-resolution algorithms because both raw and column information of image patches is considered. Experimental results show the efficiency and effectiveness of the proposed patch-based single-image superresolution algorithm.

  3. 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

  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. Directional bilateral filters for smoothing fluorescence microscopy images

    Directory of Open Access Journals (Sweden)

    Manasij Venkatesh

    2015-08-01

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

  6. Detecting inactivated endospores in fluorescence microscopy using propidium monoazide

    Science.gov (United States)

    Probst, Alexander; Mahnert, Alexander; Weber, Christina; Haberer, Klaus; Moissl-Eichinger, Christine

    2012-04-01

    The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

  7. 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.

  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. Near-field focusing of dielectric microspheres: Super-resolution and field-invariant parameter scaling

    CERN Document Server

    Wang, Zengbo

    2013-01-01

    Optical near-fields of small dielectric particles are of particular importance and interests for nanoscale optical engineering such as field localization, fabrication, characterization, sensing and imaging. This paper represents a systematic investigation on the focusing characteristics (focal length, field enhancement, spot size) for a given refractive-index microsphere (n=1.6) with a varying size parameter pisuper-resolution foci were analysised in details. Particularly strong super-resolution foci with spot size falling at least 50% below the diffraction limit were identified and possible new applications were suggested. To understand how the super-resolution conditions could be scaled to other refractive-index particles or background medium, principles of field-invariant parameters scaling (size, wavelength, and refractive index) were revealed and demonstrated with example cases. It offers the new freedom to choose particles and background medium to gai...

  10. Super-resolution Image Created from a Sequence of Images with Application of Character Recognition

    Directory of Open Access Journals (Sweden)

    Leandro Luiz de Almeida

    2013-12-01

    Full Text Available Super-resolution techniques allow combine multiple images of the same scene to obtain an image with increased geometric and radiometric resolution, called super-resolution image. In this image are enhanced features allowing to recover important details and information. The objective of this work is to develop efficient algorithm, robust and automated fusion image frames to obtain a super-resolution image. Image registration is a fundamental step in combining several images that make up the scene. Our research is based on the determination and extraction of characteristics defined by the SIFT and RANSAC algorithms for automatic image registration. We use images containing characters and perform recognition of these characters to validate and show the effectiveness of our proposed method. The distinction of this work is the way to get the matching and merging of images because it occurs dynamically between elements common images that are stored in a dynamic matrix.

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

    Science.gov (United States)

    Li, Jun-Bao; Liu, Jing; Pan, Jeng-Shyang; Yao, Hongxun

    2017-06-01

    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.

  12. APES-based procedure for super-resolution SAR imagery with GPU parallel computing

    Science.gov (United States)

    Jia, Weiwei; Xu, Xiaojian; Xu, Guangyao

    2015-10-01

    The amplitude and phase estimation (APES) algorithm is widely used in modern spectral analysis. Compared with conventional Fourier transform (FFT), APES results in lower sidelobes and narrower spectral peaks. However, in synthetic aperture radar (SAR) imaging with large scene, without parallel computation, it is difficult to apply APES directly to super-resolution radar image processing due to its great amount of calculation. In this paper, a procedure is proposed to achieve target extraction and parallel computing of APES for super-resolution SAR imaging. Numerical experimental are carried out on Tesla K40C with 745 MHz GPU clock rate and 2880 CUDA cores. Results of SAR image with GPU parallel computing show that the parallel APES is remarkably more efficient than that of CPU-based with the same super-resolution.

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

    Science.gov (United States)

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

    2006-02-01

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

  14. Efficient super-resolution image reconstruction applied to surveillance video captured by small unmanned aircraft systems

    Science.gov (United States)

    He, Qiang; Schultz, Richard R.; Chu, Chee-Hung Henry

    2008-04-01

    The concept surrounding super-resolution image reconstruction is to recover a highly-resolved image from a series of low-resolution images via between-frame subpixel image registration. In this paper, we propose a novel and efficient super-resolution algorithm, and then apply it to the reconstruction of real video data captured by a small Unmanned Aircraft System (UAS). Small UAS aircraft generally have a wingspan of less than four meters, so that these vehicles and their payloads can be buffeted by even light winds, resulting in potentially unstable video. This algorithm is based on a coarse-to-fine strategy, in which a coarsely super-resolved image sequence is first built from the original video data by image registration and bi-cubic interpolation between a fixed reference frame and every additional frame. It is well known that the median filter is robust to outliers. If we calculate pixel-wise medians in the coarsely super-resolved image sequence, we can restore a refined super-resolved image. The primary advantage is that this is a noniterative algorithm, unlike traditional approaches based on highly-computational iterative algorithms. Experimental results show that our coarse-to-fine super-resolution algorithm is not only robust, but also very efficient. In comparison with five well-known super-resolution algorithms, namely the robust super-resolution algorithm, bi-cubic interpolation, projection onto convex sets (POCS), the Papoulis-Gerchberg algorithm, and the iterated back projection algorithm, our proposed algorithm gives both strong efficiency and robustness, as well as good visual performance. This is particularly useful for the application of super-resolution to UAS surveillance video, where real-time processing is highly desired.

  15. Recent developments in fluorescence-based microscopy applied in biomedical sciences

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The present short review aims to give an overview of the most recent de velopments in fluorescence microscopy and its applications in biomedical science s. Apart from improvements in well-established methods based on conventional fl u orescence microscopy and confocal microscopy (fluorescence in situ hybridisa tion (FISH), tyramide signal amplification (TSA) in immunocytochemistry, new fluorop hores), more recently introduced techniques like fluorescence resonance energy t ransfer (FRET), fluorescence recovery after photobleaching (FRAP), multiphoton m icroscopy and fluorescence correlation spectroscopy (FCS) will be discussed.

  16. Single image super-resolution reconstruction method based on LC-KSVD algorithm

    Science.gov (United States)

    Zhang, Yaolan; Liu, Yijun

    2017-05-01

    A good dictionary has direct impact to the result of super-resolution image reconstruction. For solving the problem that dictionary learning only contains representation ability but no class information using K-SVD algorithm, this paper proposes single image super-resolution algorithm based on LC-KSVD (Label consist K-SVD). The algorithm adds classifier parameter constraints into the process of dictionary learning and classifier parameters in the process, making the dictionary possess good representation and discrimination ability. The experimental results show that the algorithm has high reconstruction results and good robustness.

  17. Time-reversed two-photon interferometry for phase super-resolution

    CERN Document Server

    Ogawa, Kazuhisa; Kobayashi, Hirokazu; Nakanishi, Toshihiro; Kitano, Masao

    2013-01-01

    We observed two-photon phase super-resolution in an unbalanced Michelson interferometer with classical Gaussian laser pulses. Our work is a time-reversed version of a two-photon interference experiment using an unbalanced Michelson interferometer. A measured interferogram exhibits two-photon phase super-resolution with a high visibility of 97.9% \\pm 0.4%. Its coherence length is about 22 times longer than that of the input laser pulses. It is a classical analogue to the large difference between the one- and two-photon coherence lengths of entangled photon pairs.

  18. High Resolution Pulse Compression Imaging Using Super Resolution FM-Chirp Correlation Method (SCM)

    Science.gov (United States)

    Fujiwara, M.; Okubo, K.; Tagawa, N.

    This study addresses the issue of the super-resolution pulse compression technique (PCT) for ultrasound imaging. Time resolution of multiple ultrasonic echoes using the FM-Chirp PCT is limited by the bandwidth of the sweep-frequency. That is, the resolution depends on the sharpness of auto-correlation function. We propose the Super resolution FM-Chirp correlation Method (SCM) and evaluate its performance. This method is based on the multiple signal classification (MUSIC) algorithm. Our simulations were made for the model assuming multiple signals reflected from some scatterers. We confirmed that SCM detects time delay of complicated reflected signals successfully with high resolution.

  19. Design of Super-resolution Filters with a Gaussian Beam in Optical Data Storage Systems

    Institute of Scientific and Technical Information of China (English)

    WANG Sha-Sha; ZHAO Xiao-Feng; LI Cheng-Fang; RUAN Hao

    2008-01-01

    @@ Super-resolution filters based on a Ganssian beam are proposed to reduce the focusing spot in optical data storage systems.Both of amplitude filters and pure-phase filters are designed respectively to gain the desired intensity distributions.Their performances are analysed and compared with those based on plane wave in detail.The energy utilizations are presented.The simulation results show that our designed super-resolution filters are favourable for use in optical data storage systems in terms of performance and energy utilization.

  20. Enhanced resolution in Argon and Neon spectra using a Super-Resolution algorithm

    CERN Document Server

    Hoyos-Campo, L M; Capella, A

    2016-01-01

    This paper presents the principles and application of a super-resolution (SR) technique aimed to obtain high resolution spectra obtained from the optogalvanic effect in Neon and Argon discharges over the 413-423 nm wavelength range. By applying the super-resolution algorithm to the experimental data, a surprising 70-fold reduction of the linewidth is achieved allowing to resolve prior indistinguishable peaks. In addition to this, the limits on the applicability of this powerful mathematical technique, mainly the signal to noise ratio of the original spectra, as well as the potential applications of the SR algorithm in other spectroscopic applications are discussed upon.

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

    Science.gov (United States)

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

    2012-02-28

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

  2. 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.

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

    OpenAIRE

    Chao Liu; Xinwei Wang; Yan Zhou; Yuliang Liu

    2013-01-01

    Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM) can provide high resolution and high imaging frame during mature FLIM methods. An abstract ti...

  4. Correlative photoactivated localization and scanning electron microscopy.

    Directory of Open Access Journals (Sweden)

    Benjamin G Kopek

    Full Text Available The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM and scanning electron microscope (SEM system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

  5. Correlative photoactivated localization and scanning electron microscopy.

    Science.gov (United States)

    Kopek, Benjamin G; Shtengel, Gleb; Grimm, Jonathan B; Clayton, David A; Hess, Harald F

    2013-01-01

    The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

  6. Multimode fibres: a pathway towards deep-tissue fluorescence microscopy

    Science.gov (United States)

    Plöschner, Martin; Tyc, TomáÅ.¡; Čižmár, TomáÅ.¡

    2015-12-01

    Fluorescence microscopy has emerged as a pivotal platform for imaging in the life sciences. In recent years, the overwhelming success of its different modalities has been accompanied by various efforts to carry out imaging deeper inside living tissues. A key challenge of these efforts is to overcome scattering and absorption of light in such environments. Multiple strategies (e.g. multi-photon, wavefront correction techniques) extended the penetration depth to the current state-of-the-art of about 1000μm at the resolution of approximately 1μm. The only viable strategy for imaging deeper than this is by employing a fibre bundle based endoscope. However, such devices lack resolution and have a significant footprint (1mm in diameter), which prohibits their use in studies involving tissues deep in live animals. We have recently demonstrated a radically new approach that delivers the light in/out of place of interest through an extremely thin (tens of microns in diameter) cylindrical glass tube called a multimode optical fibre (MMF). Not only is this type of delivery much less invasive compared to fibre bundle technology, it also enables higher resolution and has the ability to image at any plane behind the fibre without any auxiliary optics. The two most important limitations of this exciting technology are (i) the lack of bending flexibility and (ii) high demands on computational power, making the performance of such systems slow. We will discuss how to overcome these limitations.

  7. Structural Configuration of Myelin Figures Using Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Lobat Tayebi

    2012-01-01

    Full Text Available Using epifluorescence microscopy, the configuration of myelin figures that are formed upon hydration of lipid stack was studied qualitatively. Little knowledge is currently available for conditions that determine the diameter of myelin figures and their degree of multilamellarity. Examining more than 300 samples, we realized that there are distinct populations of myelin figures protruding from discrete regions of lipid stack. Each population contains myelin figures with similar diameters. This indicates a direct relationship between local characteristics of parent lipid stack and the diameter of myelin figures. Evidenced by fluorescent images, we classified all the observed myelin figures into three major groups of (1 solid tubes, (2 thin tethers, and (3 hollow tubes. Solid tubes are the most common structure of myelin figures which appeared as dense shiny cylinders. Thin tethers, with long hair-shaped structure, were observed protruding from part of lipid plaque which is likely to be under tension. Hollow tubes were protruded from the parts that are unpinned from the substrate and possibly under low or no tension. The abrupt change in the configuration of myelin figures from solid tubes to hollow ones was described in a reproducible experiment where the pinned region of the parent stack became unpinned. Our observations can indicate a relation between the membrane tension of the source material and the diameter of the myelin figures.

  8. 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

  9. Spatiotonal adaptivity in super-resolution of under-sampled image sequences

    NARCIS (Netherlands)

    Pham, T.Q.

    2006-01-01

    This thesis concerns the use of spatial and tonal adaptivity in improving the resolution of aliased image sequences under scene or camera motion. Each of the five content chapters focuses on a different subtopic of super-resolution: image registration (chapter 2), image fusion (chapter 3 and 4), sup

  10. An Effective Multi-Frame Super Resolution of Image from Blurry and Noisy Images Using PCA

    Directory of Open Access Journals (Sweden)

    Swati A. Patil

    2014-01-01

    Full Text Available Image super-resolution are techniques aiming restoration of a high-resolution image from one or several low-resolution observation images, which offer the advantages overcoming some of the inherent resolution limitations of low-cost imaging sensors (e.g., satellite image, cell phone, camera’s or surveillance camera’s, and allow better utilization of the growing capability and noise free image of HR displays. Conventional image super-resolution approaches normally require multiple LR inputs of the same scene with sub-pixel motions. This paper attempts to undertake the study of the super-resolution restoration problem and improved resolution image is restored from several geometrically warped, blurred, noisy images. The super-resolution restoration problem is modeled and analyzed from the filters such as Median Filter, Adaptive Wiener Filter, Gaussian Filter these different noise densities have been removed between 10% to 65%. The Principal Component analysis (PCA is the technique which is useful for improving the image sharpness after the process of de-blurring

  11. Performance Evaluation of Super-Resolution Reconstruction Methods on Real-World Data

    NARCIS (Netherlands)

    Eekeren, A.W.M. van; Schutte, K.; Oudegeest, O.R.; Vliet, L.J. van

    2007-01-01

    The performance of a super-resolution (SR) reconstruction method on real-world data is not easy to measure, especially as a ground-truth (GT) is often not available. In this paper, a quantitative performance measure is used, based on triangle orientation discrimination (TOD). The TOD measure, simula

  12. Sparse spikes super-resolution on thin grids II: the continuous basis pursuit

    Science.gov (United States)

    Duval, Vincent; Peyré, Gabriel

    2017-09-01

    This article analyzes the performance of the continuous basis pursuit (C-BP) method for sparse super-resolution. The C-BP has been recently proposed by Ekanadham, Tranchina and Simoncelli as a refined discretization scheme for the recovery of spikes in inverse problems regularization. One of the most well known discretization scheme, the basis pursuit (BP, also known as \

  13. Sparsity and super-resolution in sound source localization with sensor arrays

    DEFF Research Database (Denmark)

    Xenaki, Angeliki; Gerstoft, Peter; Mosegaard, Klaus

    2014-01-01

    Sound source localization with sensor arrays involves the estimation of the direction-of-arrival (DOA) from a limited number of observations. Compressive sensing (CS) is a method for solving such undetermined problems which achieves simultaneously sparsity, thus super-resolution, and computational...

  14. Sparsity and super-resolution in sound source localization with sensor arrays

    DEFF Research Database (Denmark)

    Xenaki, Angeliki; Gerstoft, Peter; Mosegaard, Klaus

    2014-01-01

    Sound source localization with sensor arrays involves the estimation of the direction-of-arrival (DOA) from a limited number of observations. Compressive sensing (CS) is a method for solving such undetermined problems which achieves simultaneously sparsity, thus super-resolution, and computational...

  15. Small-Animal Imaging Using Clinical Positron Emission Tomography/Computed Tomography and Super-Resolution

    Directory of Open Access Journals (Sweden)

    Frank P. DiFilippo

    2012-05-01

    Full Text Available Considering the high cost of dedicated small-animal positron emission tomography/computed tomography (PET/CT, an acceptable alternative in many situations might be clinical PET/CT. However, spatial resolution and image quality are of concern. The utility of clinical PET/CT for small-animal research and image quality improvements from super-resolution (spatial subsampling were investigated. National Electrical Manufacturers Association (NEMA NU 4 phantom and mouse data were acquired with a clinical PET/CT scanner, as both conventional static and stepped scans. Static scans were reconstructed with and without point spread function (PSF modeling. Stepped images were postprocessed with iterative deconvolution to produce super-resolution images. Image quality was markedly improved using the super-resolution technique, avoiding certain artifacts produced by PSF modeling. The 2 mm rod of the NU 4 phantom was visualized with high contrast, and the major structures of the mouse were well resolved. Although not a perfect substitute for a state-of-the-art small-animal PET/CT scanner, a clinical PET/CT scanner with super-resolution produces acceptable small-animal image quality for many preclinical research studies.

  16. Integrating super resolution mapping and SEBS modeling for evapotranspiration mapping at the field scale

    NARCIS (Netherlands)

    Mahour, M.; Stein, A.; Sharifi, M.A.; Tolpekin, V.A.

    2015-01-01

    This study addresses the use of super resolution mapping (SRM) for precision agriculture. SRM was applied to a high resolution GeoEye image of a vineyard in Iran with the aim to determine the actual evapotranspiration (AET) and potential evapotranspiration (PET). The Surface Energy Balance System

  17. Spatiotonal adaptivity in super-resolution of under-sampled image sequences

    NARCIS (Netherlands)

    Pham, T.Q.

    2006-01-01

    This thesis concerns the use of spatial and tonal adaptivity in improving the resolution of aliased image sequences under scene or camera motion. Each of the five content chapters focuses on a different subtopic of super-resolution: image registration (chapter 2), image fusion (chapter 3 and 4),

  18. Group-based single image super-resolution with online dictionary learning

    Science.gov (United States)

    Lu, Xuan; Wang, Dingwen; Shi, Wenxuan; Deng, Dexiang

    2016-12-01

    Recently, sparse representation has been successfully used in single image super-resolution reconstruction. Unlike the traditional single image super-resolution methods such as image interpolation, the super-resolution with sparse representation reconstructs image with one or several constant dictionaries learned from external databases. However, the contents can vary significantly across different patches in a single image, and the fixed dictionaries cannot suit for every patch. This paper presents a novel approach for single image super-resolution based on sparse representation, which uses group as the basic unit, and trains dictionary with external database and the input low-resolution image itself for each group to ensure that the dictionary is suitable for the patches in the group. Simultaneous sparse coding algorithm is used to accelerate the processing and improve the result. Extensive experiments on natural images show that our method achieves better results than some state-of-the-art algorithms in terms of both objective and human visual evaluations.

  19. A fluorescence microscopy study of quantum dots as fluorescent probes for brain tumor diagnosis

    Science.gov (United States)

    Wang, Jingjing; Vernier, P. Thomas; Sun, Yinghua; Gundersen, Martin A.; Marcu, Laura

    2005-03-01

    In vivo fluorescent spectroscopy and imaging using endogenous and exogenous sources of contrast can provide new approaches for enhanced demarcation of brain tumor margins and infiltration. Quantum dots (QDs), nanometer-size fluorescent probes, represent excellent contrast agents for biomedical imaging due to their broader excitation spectrum, narrower emission spectra, and higher sensitivity and stability. The epidermal growth factor receptor (EGFR) is implicated in the development and progression of a number of human solid tumors including brain tumors and thus a potential target for brain tumor diagnosis. In this study, we investigate the up-take of ODs by brain tumor cells and the potential use of EGFR-targeted QDs for enhanced optical imaging of brain tumors. We conducted fluorescence microscopy studies of the up-take mechanism of the anti-EGFR-ODs complexes by Human U87, and SKMG-3 glioblastoma cells. Our preliminary results show that QDs can enter into glioma cells through anti-EGFR mediated endocytosis, suggesting that these nano-size particles can tag brain tumor cells.

  20. Fluorescence and fluorescence-lifetime imaging microscopy (FLIM) to characterize yeast strains by autofluorescence

    Science.gov (United States)

    Bhatta, H.; Goldys, E. M.; Ma, J.

    2006-02-01

    We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.