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Sample records for super-resolution optical microscopy

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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...

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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 ...

  14. 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.

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

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

    Science.gov (United States)

    McEvoy, Andrea Lynn

    All cells spatially organize their interiors, and this arrangement is necessary for cell viability. Until recently, it was believed that only eukaryotic cells spatially segregate their components. However, it is becoming increasingly clear that bacteria also assemble their proteins into complex patterns. In eukaryotic cells, spatial organization arises from membrane bound organelles as well as motor transport proteins which can move cargos within the cell. To date, there are no known motor transport proteins in bacteria and most microbes lack membrane bound organelles, so it remains a mystery how bacterial spatial organization emerges. In hind-sight it is not surprising that bacteria also exhibit complex spatial organization considering much of what we have learned about the basic processes that take place in all cells, such as transcription and translation was first discovered in prokaryotic cells. Perhaps the fundamental principles that govern spatial organization in prokaryotic cells may be applicable in eukaryotic cells as well. In addition, bacteria are attractive model organism for spatial organization studies because they are genetically tractable, grow quickly and much biochemical and structural data is known about them. A powerful tool for observing spatial organization in cells is the fluorescence microscope. By specifically tagging a protein of interest with a fluorescent probe, it is possible to examine how proteins organize and dynamically assemble inside cells. A significant disadvantage of this technology is its spatial resolution (approximately 250 nm laterally and 500 nm axially). This limitation on resolution causes closely spaced proteins to look blurred making it difficult to observe the fine structure within the complexes. This resolution limit is especially problematic within small cells such as bacteria. With the recent invention of new optical microscopies, we now can surpass the existing limits of fluorescence imaging. In some cases, we can

  7. 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.

  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. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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...

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Zhaokun Zhou

    2015-06-01

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

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

    Science.gov (United States)

    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.

  17. 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 ...

  18. 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.

  19. 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.

  20. 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.

  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. 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.

  3. 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.

  4. 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).

  5. Aptamer Stainings for Super-resolution Microscopy.

    Science.gov (United States)

    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.

  6. 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...

  7. 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.

  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. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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...

  15. 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....

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  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. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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).

  13. 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.

  14. 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.

  15. 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

  16. 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.

  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. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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)

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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. 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.

  19. 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...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  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. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  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. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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.

  17. 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

  18. 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...

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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.

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

    Science.gov (United States)

    Liu, Cheng-Yang; Lo, Wei-Chieh

    2017-09-01

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

  19. 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.

  20. 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.

  1. 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.

  2. 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).

  3. 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.

  4. Design and preparation of film for microsphere based optical super-resolution imaging

    Science.gov (United States)

    Pang, Hui; Du, Chunlei; Qiu, Qi; Yin, Shaoyun; Zhang, Man; Deng, Qiling

    2014-08-01

    In this paper, a novel thin film was proposed for optical super-resolution imaging, which contains a layer of closely-arranged barium titanate glass microsphere with diameter about 30-100μm embedded in a transparent polydimethylsiloxane soft mold. Then the imaging mechanism was analyzed by the finite-difference time-domain (FDTD) simulation and spectrum analysis method. Finally, the thin film was prepared and used to image the sample with sub-wavelength feature to confirm the capability of super-resolution imaging. The experimental result shows that an irresolvable Blu-ray DVD disk with feature size of 300nm can be resolved by placing a thin-film on its surface and then look through it with a conventional microscope. The thin film presented here is flexible, lightweight, easy to carry and can be used in the nanophotonics, nanoplasmonics, and biomedical imaging areas.

  5. 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...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  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. 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.

  15. 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.

  16. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Alexandrov, A., E-mail: andrey.alexandrov@na.infn.it [INFN - Napoli, I-80125 Napoli (Italy); LPI - Lebedev Physical Institute of the Russian Academy of Sciences, RUS-119991 Moscow (Russian Federation); Asada, T. [Nagoya University, J-464-8602 Nagoya (Japan); Consiglio, L.; D' Ambrosio, N. [INFN - Laboratori Nazionali del Gran Sasso, I-67010 Assergi (AQ) (Italy); De Lellis, G. [INFN - Napoli, I-80125 Napoli (Italy); University of Naples, I-80125 Napoli (Italy); Di Crescenzo, A. [INFN - Napoli, I-80125 Napoli (Italy); Di Marco, N. [INFN - Laboratori Nazionali del Gran Sasso, I-67010 Assergi (AQ) (Italy); Furuya, S.; Hakamata, K.; Ishikawa, M.; Katsuragawa, T.; Kuwabara, K.; Machii, S.; Naka, T. [Nagoya University, J-464-8602 Nagoya (Japan); Pupilli, F. [INFN - Laboratori Nazionali di Frascati, I-00044 Frascati (RM) (Italy); Sirignano, C. [University of Padova and INFN, Padova (PD), 35131 Italy (Italy); Tawara, Y. [Nagoya University, J-464-8602 Nagoya (Japan); Tioukov, V. [INFN - Napoli, I-80125 Napoli (Italy); Umemoto, A.; Yoshimoto, M. [Nagoya University, J-464-8602 Nagoya (Japan)

    2016-07-11

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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Meiyu Liang

    2013-01-01

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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Influence of standing wave phase error on super-resolution optical inspection for periodic microstructures

    Science.gov (United States)

    Kudo, R.; Usuki, S.; Takahashi, S.; Takamasu, K.

    2012-05-01

    The miniaturization of microfabricated structures such as patterned semiconductor wafers continues to advance, thereby increasing the demand for a high-speed, nondestructive and high-resolution measurement technique. We propose a novel optical inspecting method for a microfabricated structure using the standing wave illumination (SWI) shift as such a measurement technique. This method is based on a super-resolution algorithm in which the inspection system's resolution exceeds the diffraction limit by shifting the SWI. Resolution beyond the diffraction limit has previously been studied theoretically and realized experimentally. The influence of various experimental error factors needs to be investigated and calibration needs to be performed accordingly when actual applications that utilize the proposed method are constructed. These error factors include errors related to the phase, pitch and shift step size of the standing wave. Identifying the phase accurately is extremely difficult and greatly influences the resolution result. Hence, the SWI phase was focused upon as an experimental error factor. The effect of the phase difference between the actual experimental standing wave and the computationally set standing wave was investigated using a computer simulation. The periodic structure characteristic of a microfabricated structure was analyzed. The following findings were obtained as a result. The influence of an error is divided into three modes depending on the pitch of the periodic structure: (1) if the pitch is comparatively small, the influence of the error is cancelled, allowing the structure of a sample to be resolved correctly; (2) if the pitch of the structure is from 150 to 350 nm, the reconstructed solution shifts in a transverse direction corresponding to a phase gap of SWI; and (3) if it is a comparatively large pitch, then it is difficult to reconstruct the right pitch. Verification was experimentally attempted for mode (2), and the same result as

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Dylan Myers Owen

    2013-12-01

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

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. Diffractive super-resolution elements applied to near-field optical data storage with solid immersion lens

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yaoju [Department of Physics, Wenzhou Normal College, Wenzhou 325027 (China); Xiao Huaceng [Department of Biology, Wenzhou Normal College, Wenzhou 325027 (China); Zheng Chongwei [Department of Physics, Wenzhou Normal College, Wenzhou 325027 (China)

    2004-07-01

    The intensity distribution in near-field optical data storage with a solid immersion lens (SIL) and a binary phase-only diffractive super-resolution element (DSE) is expressed in a single definite integral by using angular spectrum theory. The super-resolution of binary two-zone phase DSEs for SIL systems is numerically studied for low and high numerical aperture (NA) systems. The results for the low-NA systems show that optimizing the zone boundary and phase of binary two-zone phase DSEs can decrease the spot size. The Strehl ratio, sidelobe intensity and axial characteristic length are also discussed. In addition, a binary two-zone phase filter can change the position of focus that shifts from the SIL-air interface to air, but the spot size increases. For the high-NA systems, the y- and z-polarized components of the transmitted field increase as the boundary and depth of phase of the DSE increase. When the phase boundary is smaller and the depth of phase depth is close to {pi}, super-resolving effect of DSE is more obvious but the intensity of sidelobes is larger for the high-NA system. In this way, it may be possible to improve both the resolution and focal depth of the SIL with high-NA systems.

  18. Super-resolution nanofabrication with metal-ion doped hybrid material through an optical dual-beam approach

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Yaoyu; Li, Xiangping; Gu, Min, E-mail: mgu@swin.edu.au [Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, P.O. Box 218, Hawthorn, Victoria 3122 (Australia)

    2014-12-29

    We apply an optical dual-beam approach to a metal-ion doped hybrid material to achieve nanofeatures beyond the optical diffraction limit. By spatially inhibiting the photoreduction and the photopolymerization, we realize a nano-line, consisting of polymer matrix and in-situ generated gold nanoparticles, with a lateral size of sub 100 nm, corresponding to a factor of 7 improvement compared to the diffraction limit. With the existence of gold nanoparticles, a plasmon enhanced super-resolution fabrication mechanism in the hybrid material is observed, which benefits in a further reduction in size of the fabricated feature. The demonstrated nanofeature in hybrid materials paves the way for realizing functional nanostructures.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  6. 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...

  7. Ptychographic reconstruction algorithm for frequency resolved optical gating: super-resolution and supreme robustness

    CERN Document Server

    Sidorenko, Pavel; Avnat, Zohar; Cohen, Oren

    2016-01-01

    Frequency-resolved optical gating (FROG) is probably the most popular technique for complete characterization of ultrashort laser pulses. In FROG, a reconstruction algorithm retrieves the pulse from a measured spectrogram, yet current FROG reconstruction algorithms require and exhibit several restricting features that weaken FROG performances. For example, the delay step must correspond to the spectral bandwidth measured with large enough SNR a condition that limits the temporal resolution of the reconstructed pulse, obscures measurements of weak broadband pulses, and makes measurement of broadband mid-IR pulses hard and slow because the spectrograms become huge. We develop a new approach for FROG reconstruction, based on ptychography (a scanning coherent diffraction imaging technique), that removes many of the algorithmic restrictions. The ptychographic reconstruction algorithm is significantly faster and more robust to noise than current FROG algorithms, which are based on generalized projections (GP). We d...

  8. Investigating sub-spine actin dynamics in rat hippocampal neurons with super-resolution optical imaging.

    Directory of Open Access Journals (Sweden)

    Vedakumar Tatavarty

    Full Text Available Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM-based single-molecule tracking technique to analyze F-actin movements with approximately 30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow and kinetic (F-actin turn-over dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of approximately 138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

  9. Spectral estimation optical coherence tomography for axial super-resolution (Conference Presentation)

    Science.gov (United States)

    Liu, Xinyu; Yu, Xiaojun; Wang, Nanshuo; Bo, En; Luo, Yuemei; Chen, Si; Cui, Dongyao; Liu, Linbo

    2016-03-01

    The sample depth reflectivity profile of Fourier domain optical coherence tomography (FD-OCT) is estimated from the inverse Fourier transform of the spectral interference signals (interferograms). As a result, the axial resolution is fundamentally limited by the coherence length of the light source. We demonstrate an axial resolution improvement method by using the autoregressive spectral estimation technique to instead of the inverse Fourier transform to analyze the spectral interferograms, which is named as spectral estimation OCT (SE-OCT). SE-OCT improves the axial resolution by a factor of up to 4.7 compared with the corresponding FD-OCT. Furthermore, SE-OCT provides a complete sidelobe suppression in the point-spread function. Using phantoms such as an air wedge and micro particles, we prove the ability of resolution improvement. To test SE-OCT for real biological tissue, we image the rat cornea and demonstrate that SE-OCT enables clear identification of corneal endothelium anatomical details ex vivo. We also find that the performance of SE-OCT is depended on SNR of the feature object. To evaluate the potential usage and define the application scope of SE-OCT, we further investigate the property of SNR dependence and the artifacts that may be caused. We find SE-OCT may be uniquely suited for viewing high SNR layer structures, such as the epithelium and endothelium in cornea, retina and aorta. Given that SE-OCT can be implemented in the FD-OCT devices easily, the new capabilities provided by SE-OCT are likely to offer immediate improvements to the diagnosis and management of diseases based on OCT imaging.

  10. 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...

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. Tip-enhanced near-field optical microscope with side-on and ATR-mode sample excitation for super-resolution Raman imaging of surfaces

    Science.gov (United States)

    Heilman, A. L.; Gordon, M. J.

    2016-06-01

    A tip-enhanced near-field optical microscope with side-on and attenuated total reflectance (ATR) excitation and collection is described and used to demonstrate sub-diffraction-limited (super-resolution) optical and chemical characterization of surfaces. ATR illumination is combined with an Au optical antenna tip to show that (i) the tip can quantitatively transduce the optical near-field (evanescent waves) above the surface by scattering photons into the far-field, (ii) the ATR geometry enables excitation and characterization of surface plasmon polaritons (SPPs), whose associated optical fields are shown to enhance Raman scattering from a thin layer of copper phthalocyanine (CuPc), and (iii) SPPs can be used to plasmonically excite the tip for super-resolution chemical imaging of patterned CuPc via tip-enhanced Raman spectroscopy (TERS). ATR-illumination TERS is also quantitatively compared with the more conventional side-on illumination scheme. In both cases, spatial resolution was better than 40 nm and tip on/tip off Raman enhancement factors were >6500. Furthermore, ATR illumination was shown to provide similar Raman signal levels at lower "effective" pump powers due to additional optical energy delivered by SPPs to the active region in the tip-surface gap.

  16. 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.

  17. 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

  18. Optical Photon Reassignment Microscopy (OPRA)

    CERN Document Server

    Roth, Stephan; Wicker, Kai; Heintzmann, Rainer

    2013-01-01

    To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used [C. J. R. Sheppard, Super-resolution in confocal imaging, Optik 80, 5354 (1988)]. This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and resolution is obtained. Here we present optical photon reassignment microscopy (OPRA) which realises this concept in an all-optical way obviating the need for image-processing. With the help of an additional intermediate optical beam expansion between descanning and a further rescanning of the detected light, an image with the advantages of photon reassignment can be acquired. Due to its simplicity and flexibility this m...

  19. 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...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  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. 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...

  16. 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.

  17. Numerical study of super-resolved optical microscopy with partly staggered beams

    Science.gov (United States)

    He, Jinping; Wang, Nan; Kobayashi, Takayoshi

    2016-12-01

    The resolving power of optical microscopy involving two or even more beams, such as pump-probe microscopy and nonlinear optical microscopy, can be enhanced both laterally and longitudinally with partly staggered beams. A numerical study of the new super-resolution imaging technology is performed with vector diffraction theory. The influence of polarization is discussed. A resolving power of sub-100 nm and sub-300 nm in the lateral and longitudinal directions, respectively, is achievable.

  18. 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。

  19. Holography based super resolution

    Science.gov (United States)

    Hussain, Anwar; Mudassar, Asloob A.

    2012-05-01

    This paper describes the simulation of a simple technique of superresolution based on holographic imaging in spectral domain. The input beam assembly containing 25 optical fibers with different orientations and positions is placed to illuminate the object in the 4f optical system. The position and orientation of each fiber is calculated with respect to the central fiber in the array. The positions and orientations of the fibers are related to the shift of object spectrum at aperture plane. During the imaging process each fiber is operated once in the whole procedure to illuminate the input object transparency which gives shift to the object spectrum in the spectral domain. This shift of the spectrum is equal to the integral multiple of the pass band aperture width. During the operation of single fiber (ON-state) all other fibers are in OFF-state at that time. The hologram recorded by each fiber at the CCD plane is stored in computer memory. At the end of illumination process total 25 holograms are recorded by the whole fiber array and by applying some post processing and specific algorithm single super resolved image is obtained. The superresolved image is five times better than the band-limited image. The work is demonstrated using computer simulation only.

  20. Study of the shape of an optical window in a super-resolution state by electromagnetic-thermal coupled simulation: Effects of melting of an active layer in an optical disc

    Science.gov (United States)

    Sano, Haruyuki; Shima, Takayuki; Kuwahara, Masashi; Fujita, Yoshiya; Uchiyama, Munehisa; Aono, Yoshiyuki

    2014-04-01

    We performed a multi-physics simulation for the propagation of electromagnetic waves and heat conduction in a super-resolution optical disc that includes an active layer of InSb. Because the change in the optical constant of InSb due to the phase transition is taken into account, the melting of the active layer can be realistically simulated in our calculation. It was found that in the case of an incident light power (P) of 2 mW, a profile of the electric field intensity transmitted through the InSb layer has an asymmetric shape with a narrow peak. This beam-narrowing was suggested to be an essential mechanism of the super-resolution, because a narrower light beam allows the detection of a smaller pit structure than the optical diffraction limit. This beam-narrowing was found to be originating from a small molten region produced in the InSb layer, which works as a mask for light exposure.

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods.

    Science.gov (United States)

    Tam, Johnny; Merino, David

    2015-11-01

    Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy are two super-resolution optical microscopy approaches that have rapidly gained popularity in recent years. Both modalities offer super-resolution imaging capabilities with the potential for imaging in multiple colors, three-dimensions, and the possibility to image in live cells. In this review, we focus on the specific advantages and disadvantages of each technique in the context of each other. STORM has been reported to achieve higher spatial resolution when compared to STED, but a lengthy acquisition may be required. STED utilizes relatively higher laser intensities, but is able to generate a super-resolution image immediately after acquisition without the need for any additional data processing. Ultimately, the choice between STORM and STED will depend not only on the specific application, but also on the users' ability to understand and optimize the various parameters ranging from sample preparation to image acquisition, which determine the quality of the final image. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) are two super-resolution microscopy approaches that have rapidly gained popularity in recent years. STORM is based on the precise localization of a large number of individual molecules that together form a super-resolved image (bottom), whereas STED is based on the scanning of two super-imposed light sources which together allow for a super-resolved spot on the sample to be imaged (top). We discuss the specific advantages and disadvantages of each technique and explain the various parameters that affect image quality, which should be taken into consideration when planning experiments.

  14. 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

  15. 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.

  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. 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.

  19. 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].

  20. Optical imaging. Expansion microscopy.

    Science.gov (United States)

    Chen, Fei; Tillberg, Paul W; Boyden, Edward S

    2015-01-30

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~10(7) cubic micrometers of the mouse hippocampus with a conventional confocal microscope.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  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. 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

  9. 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...

  10. 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.

  11. Perfect optical vortex enhanced surface plasmon excitation for plasmonic structured illumination microscopy imaging

    Science.gov (United States)

    Zhang, Chonglei; Min, Changjun; Du, Luping; Yuan, X.-C.

    2016-05-01

    We propose an all-optical technique for plasmonic structured illumination microscopy (PSIM) with perfect optical vortex (POV). POV can improve the efficiency of the excitation of surface plasma and reduce the background noise of the excited fluorescence. The plasmonic standing wave patterns are excited by POV with fractional topological charges for accurate phase shift of {-2π/3, 0, and 2π/3}. The imaging resolution of less than 200 nm was produced. This PSIM technique is expected to be used as a wide field, super resolution imaging technique in dynamic biological imaging.

  12. 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

  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. 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.

  16. 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...

  17. 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.

  18. Improved 3D Superresolution Localization Microscopy Using Adaptive Optics

    CERN Document Server

    Piro, Nicolas; Olivier, Nicolas; Manley, Suliana

    2014-01-01

    We demonstrate a new versatile method for 3D super-resolution microscopy by using a deformable mirror to shape the point spread function of our microscope in a continuous and controllable way. We apply this for 3D STORM imaging of microtubules.

  19. 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.

  20. 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.

  1. Quantum Optics in Diamond Nanophotonic Chips

    Science.gov (United States)

    2014-07-01

    techniques [12]. Using a CCD camera, this “deterministic emitter switch microscopy ” (DESM) technique enables super - resolution imaging with localization down...selective optical transitions allow individual NV electron spins to be easily observed using standard confocal microscopy . The NV has two unpaired...record-precision magnetometry with diamond nanocrystals [11]. 1.3 Wide-Field Multispectral Super - Resolution Imaging Using Spin- Dependent Fluorescence in

  2. 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.

  3. 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.

  4. 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.

  5. 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...

  6. 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.

  7. 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

  8. 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.

  9. In掩膜层超分辨光盘的读出研究%Study on Super-Resolution Readout with Indium Thin Film as Mask Layer

    Institute of Scientific and Technical Information of China (English)

    张奎; 耿永友; 施宏仁

    2012-01-01

    在超分辨光存储技术中,掩膜层材料是决定其性能优劣的关键.In薄膜可以作为掩膜层用来实现超分辨信息点的动态读出.采用直流磁控溅射法制备不同厚度的In薄膜,用台阶仪测量薄膜厚度随时间的变化关系,用原子力显微镜观察不同厚度薄膜样品的表面形貌.在预刻有尺寸为390 nm信息点的光盘盘基上制备In薄膜,从而形成In掩膜超分辨光盘.利用光盘动态测试仪进行动态读出,最高读出载噪比(CNR)达到26 dB.为了进一步分析超分辨动态读出的物理机理,采用变温椭圆偏振光谱仪测量In薄膜在不同温度下的光学常数,得到In薄膜在不同温度下的反射率和吸收系数.分析表明In掩膜超分辨光盘的读出机理符合孔径型超分辨读出模型.%The key performance of super-resolution optical disk storage technique is mainly determined by the mask material. In this work, the super resolution pits on the optical disk are dynamically read out with indium thin film as a functional mask layer. Thin films with different thicknesses are deposited by direct current magnetron sputtering method, the thickness changes with time of the In thin films are measured by profilometer and the different surface morphologies are observed by atomic force microscopy. In thin film in the pre-engraved super-resolution optical disk with pit size of 390 nm is created to form the In mask super-resolution optical disk. The readout is tested by the optical disk dynamic tester, and the largest carrier to noise ratio (CNR) of 26 dB is obtained. To explore the internal mechanism of this super-resolution readout ability, the optical constant under different temperatures is studied by variable temperature ellipsometer. The reflectivity and absorption coefficiency are also obtained. The experimental results indicate that the mechanism of the super-resolution readout with In thin film as mask can be explained by the model of aperture-type super-resolution.

  10. 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.

  11. Near-field Optical Microscopy

    NARCIS (Netherlands)

    Ruiter, A.G.T.

    1997-01-01

    Near-field scanning optical microscopy (NSOM) is one of the most recent scanning probe techniques. In this technique, an optical probe is brought in the vicinity of the sample surface, in the near-field zone. The microscope can either work in illumination mode, in which the probe consists of a

  12. Near-field Optical Microscopy

    NARCIS (Netherlands)

    Ruiter, Anthonius Gerardus Theodorus

    1997-01-01

    Near-field scanning optical microscopy (NSOM) is one of the most recent scanning probe techniques. In this technique, an optical probe is brought in the vicinity of the sample surface, in the near-field zone. The microscope can either work in illumination mode, in which the probe consists of a sub-w

  13. 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.

  14. 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.

  15. 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

  16. Super-Resolution Optical Subtraction Microscopy Using Optical Scattering Imaging%基于散射信号的超分辨光学相减显微镜

    Institute of Scientific and Technical Information of China (English)

    周前; 于建强; 赵立波; 李德胜; 吴魁; 朱建华; 袁景和; 方晓红

    2016-01-01

    现有的光学超分辨显微成像技术主要依赖于特殊的荧光标记物,其对于大多数非荧光样品的超分辨成像就变得无能为力.因此我们提出将光学相减显微技术应用到非荧光样品的成像当中,利用普通共聚焦光斑和面包圈型光斑分别激发样品的散射光成像,从而得到样品同一区域的两幅图像,再通过图像相减的方法提高了图像空间分辨率.不同于一般的超分辨成像方法,这种光学相减显微镜不需要特殊的样品预处理过程,同时两次成像的激发光强度可以保持在一个较低水平,避免了样品损伤的影响.随后金纳米小球和有机聚合物微丝的散射成像实验证明了光学相减显微镜可以将空间分辨率提高到215 nm (0.33λ,1λ=650 nm),并且通过探测散射信号得到更多的样品细节信息.

  17. All-optical photoacoustic microscopy

    Directory of Open Access Journals (Sweden)

    Sung-Liang Chen

    2015-12-01

    Full Text Available Three-dimensional photoacoustic microscopy (PAM has gained considerable attention within the biomedical imaging community during the past decade. Detecting laser-induced photoacoustic waves by optical sensing techniques facilitates the idea of all-optical PAM (AOPAM, which is of particular interest as it provides unique advantages for achieving high spatial resolution using miniaturized embodiments of the imaging system. The review presents the technology aspects of optical-sensing techniques for ultrasound detection, such as those based on optical resonators, as well as system developments of all-optical photoacoustic systems including PAM, photoacoustic endoscopy, and multi-modality microscopy. The progress of different AOPAM systems and their representative applications are summarized.

  18. 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.

  19. 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-...

  20. 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...

  1. 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.

  2. 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

  3. Re-scan confocal microscopy: scanning twice for better resolution

    NARCIS (Netherlands)

    De Luca, G.M.R.; Breedijk, R.M.P.; Brandt, R.A.J.; Zeelenberg, C.H.C.; De Jong, B.E.; Timmermans, W.; Nahidi Azar, L.; Hoebe, R.A.; Stallinga, S.; Manders, E.M.M.

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity wh

  4. Re-scan confocal microscopy : scanning twice for better resolution

    NARCIS (Netherlands)

    De Luca, G.M.R.; Breedijk, R.M.P.; Brandt, R.A.J.; Zeelenberg, C.H.C.; de Jong, B.E.; Timmermans, W.; Azar, L.N.; Hoebe, R.A.; Stallinga, S.; Manders, E.M.M.

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity wh

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

    OpenAIRE

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

    2016-01-01

    Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the- art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate --- hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image fr...

  6. 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.

  7. 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...

  8. 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.

  9. 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.

  10. 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.

  11. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations.

    Science.gov (United States)

    Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M; Pereira, Pedro Matos; Henriques, Ricardo

    2016-08-12

    Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours.

  12. Quantitative Characterization of Super-Resolution Infrared Imaging Based on Time-Varying Focal Plane Coding

    Science.gov (United States)

    Wang, X.; Yuan, Y.; Zhang, J.; Chen, Y.; Cheng, Y.

    2014-10-01

    High resolution infrared image has been the goal of an infrared imaging system. In this paper, a super-resolution infrared imaging method using time-varying coded mask is proposed based on focal plane coding and compressed sensing theory. The basic idea of this method is to set a coded mask on the focal plane of the optical system, and the same scene could be sampled many times repeatedly by using time-varying control coding strategy, the super-resolution image is further reconstructed by sparse optimization algorithm. The results of simulation are quantitatively evaluated by introducing the Peak Signal-to-Noise Ratio (PSNR) and Modulation Transfer Function (MTF), which illustrate that the effect of compressed measurement coefficient r and coded mask resolution m on the reconstructed image quality. Research results show that the proposed method will promote infrared imaging quality effectively, which will be helpful for the practical design of new type of high resolution ! infrared imaging systems.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 超分辨光学显微镜的生物学应用%Application of super-resolution optical microscopy in biology

    Institute of Scientific and Technical Information of China (English)

    毛秀海; 杜建聪; 黄庆; 樊春海; 邓素辉

    2013-01-01

    生物学家一直渴望使用无损、可实时成像的远场光学显微镜对细胞内动力学行为或者纳米尺度的蛋白质-蛋白质相互作用进行研究.由于光学衍射极限的存在,传统的远场光学显微镜无法对200纳米尺度内的这些生命活动进行观察.近年来,克服光学衍射极限的超分辨成像技术快速发展,将分辨率提高至数十纳米级别.基于超分辨技术的荧光显微镜包括受激发射损耗显微镜(STED)、光敏定位显微镜(PALM)以及随机光重建显微镜(STORM).这些技术日趋成熟并成功应用于细胞生物学、微生物学、神经生物学等研究,为整个生命科学的发展带来全新的认识.

  3. 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.

  4. 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.

  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. 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.

  7. Visual-servoing optical microscopy

    Science.gov (United States)

    Callahan, Daniel E.; Parvin, Bahram

    2009-06-09

    The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time: quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

  8. 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.

  9. Structured illumination microscopy and its new developments

    Directory of Open Access Journals (Sweden)

    Jianling Chen

    2016-05-01

    Full Text Available Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM, a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

  10. 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.

  11. 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...

  12. Supporting lander and rover operation: a novel super-resolution restoration technique

    Science.gov (United States)

    Tao, Yu; Muller, Jan-Peter

    2015-04-01

    Higher resolution imaging data is always desirable to critical rover engineering operations, such as landing site selection, path planning, and optical localisation. For current Mars missions, 25cm HiRISE images have been widely used by the MER & MSL engineering team for rover path planning and location registration/adjustment. However, 25cm is not high enough resolution to be able to view individual rocks (≤2m in size) or visualise the types of sedimentary features that rover onboard cameras might observe. Nevertheless, due to various physical constraints (e.g. telescope size and mass) from the imaging instruments themselves, one needs to be able to tradeoff spatial resolution and bandwidth. This means that future imaging systems are likely to be limited to resolve features larger than 25cm. We have developed a novel super-resolution algorithm/pipeline to be able to restore higher resolution image from the non-redundant sub-pixel information contained in multiple lower resolution raw images [Tao & Muller 2015]. We will demonstrate with experiments performed using 5-10 overlapped 25cm HiRISE images for MER-A, MER-B & MSL to resolve 5-10cm super resolution images that can be directly compared to rover imagery at a range of 5 metres from the rover cameras but in our case can be used to visualise features many kilometres away from the actual rover traverse. We will demonstrate how these super-resolution images together with image understanding software can be used to quantify rock size-frequency distributions as well as measure sedimentary rock layers for several critical sites for comparison with rover orthorectified image mosaic to demonstrate optimality of using our super-resolution resolved image to better support future lander and rover operation in future. We present the potential of super-resolution for virtual exploration to the ˜400 HiRISE areas which have been viewed 5 or more times and the potential application of this technique to all of the ESA Exo

  13. Spectral super-resolution in metamaterial composites

    CERN Document Server

    Helsing, Johan; Milton, Graeme W

    2011-01-01

    We investigate the optical properties of periodic composites containing metamaterial inclusions in a normal material matrix. We consider the case where these inclusions have sharp corners, and following Hetherington and Thorpe, use analytic results to argue that it is then possible to deduce the shape of the corner (its included angle) by measurements of the absorptance of such composites when the scale size of the inclusions and period cell is much finer than the wavelength. These analytic arguments are supported by highly accurate numerical results for the effective permittivity function of such composites as a function of the permittivity ratio of inclusions to matrix. The results show that this function has a continuous spectral component with limits independent of the area fraction of inclusions, and with the same limits for both square and staggered square arrays.

  14. 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

  15. Scattering-type scanning near-field optical microscopy with reconstruction of vertical interaction

    Science.gov (United States)

    Wang, Le; Xu, Xiaoji G.

    2015-11-01

    Scattering-type scanning near-field optical microscopy provides access to super-resolution spectroscopic imaging of the surfaces of a variety of materials and nanostructures. In addition to chemical identification, it enables observations of nano-optical phenomena, such as mid-infrared plasmons in graphene and phonon polaritons in boron nitride. Despite the high lateral spatial resolution, scattering-type near-field optical microscopy is not able to provide characteristics of near-field responses in the vertical dimension, normal to the sample surface. Here, we present an accurate and fast reconstruction method to obtain vertical characteristics of near-field interactions. For its first application, we investigated the bound electromagnetic field component of surface phonon polaritons on the surface of boron nitride nanotubes and found that it decays within 20 nm with a considerable phase change in the near-field signal. The method is expected to provide characterization of the vertical field distribution of a wide range of nano-optical materials and structures.

  16. Super-Resolution Imaging by Arrays of High-Index Spheres Embedded in Transparent Matrices

    CERN Document Server

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

    2014-01-01

    We fabricated thin-films made from polydimethylsiloxane (PDMS) with embedded high-index (n~1.9-2.2) microspheres for super-resolution imaging applications. To control the position of microspheres, such films can be translated along the surface of the nanoplasmonic structure to be imaged. Microsphere-assisted imaging, through these matrices, provided lateral resolution of ~{\\lambda}/7 in nanoplasmonic dimer arrays with an illuminating wavelength {\\lambda}=405 nm. Such thin films can be used as contact optical components to boost the resolution capability of conventional microscopes.

  17. Correct self-assembling of spatial frequencies in super-resolution synthetic aperture digital holography.

    Science.gov (United States)

    Paturzo, Melania; Ferraro, Pietro

    2009-12-01

    Synthetic aperture enlargement is obtained, in lensless digital holography, by introducing a diffraction grating between the object and the CCD camera with the aim of getting super-resolution. We demonstrate here that the spatial frequencies are naturally self-assembled in the reconstructed image plane when the NA is increased synthetically at its maximum extent of three times. By this approach it possible to avoid the use of the grating transmission formula in the numerical reconstruction process, thus reducing significantly the noise in the final super-resolved image. Demonstrations are reported in 1D and 2D with an optical target and a biological sample, respectively.

  18. Design of the annular binary filters with super-resolution based on the genetic algorithm

    Institute of Scientific and Technical Information of China (English)

    YU Qi-lei; LE Zi-chun; ZHU Hong-ying

    2006-01-01

    To improve the density of information storage,this paper introduces a kind of annular binary filters with super-resolution,Several of these filters have been designed based on the genetic algorithm,the simulations demonstrate that the transverse gain of the filters can reach the value of 1.37.Thus they can remarkably decrease the recording spot size,which is helpful to improve the density of information storage and to make the depth of focus longer,and therefore they can avoid the mistake caused by the small undulation of the optical disk in the process of recording/reading the information.

  19. Advanced Microscopy of Microbial Cells

    DEFF Research Database (Denmark)

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

    2011-01-01

    microscopy, super-resolution optical microscopy (STED, SIM, PALM) as well as atomic force microscopy and Raman spectroscopy. Using examples of bistability in microbial populations as well as biofilm development and differentiation in bacterial and yeast consortia, we demonstrate the importance of microscopy......Growing awareness of heterogeneity in cells of microbial populations has emphasized the importance of advanced microscopy for visualization and understanding of the molecular mechanisms underlying cell-to-cell variation. In this review, we highlight some of the recent advances in confocal...

  20. 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...

  1. 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...

  2. Disposable optics for microscopy diagnostics.

    Science.gov (United States)

    Vilmi, Pauliina; Varjo, Sami; Sliz, Rafal; Hannuksela, Jari; Fabritius, Tapio

    2015-11-20

    The point-of-care testing (POCT) is having increasing role on modern health care systems due to a possibility to perform tests for patients conveniently and immediately. POCT includes lot of disposable devices because of the environment they are often used. For a disposable system to be reasonably utilized, it needs to be high in quality but low in price. Optics based POCT systems are interesting approach to be developed, and here we describe a low-cost fabrication process for microlens arrays for microscopy. Lens arrays having average lens diameter of 222 μm with 300 μm lens pitch were fabricated. The lenses were characterized to have standard deviation of 0.06 μm in height and 4.61 μm in diameter. The resolution limit of 3.9μm is demonstrated with real images, and the images were compared with ones made with glass and polycarbonate lens arrays. The image quality is at the same level than with the glass lenses and the manufacturing costs are very low, thus making them suitable for POCT applications.

  3. Disposable optics for microscopy diagnostics

    Science.gov (United States)

    Vilmi, Pauliina; Varjo, Sami; Sliz, Rafal; Hannuksela, Jari; Fabritius, Tapio

    2015-11-01

    The point-of-care testing (POCT) is having increasing role on modern health care systems due to a possibility to perform tests for patients conveniently and immediately. POCT includes lot of disposable devices because of the environment they are often used. For a disposable system to be reasonably utilized, it needs to be high in quality but low in price. Optics based POCT systems are interesting approach to be developed, and here we describe a low-cost fabrication process for microlens arrays for microscopy. Lens arrays having average lens diameter of 222 μm with 300 μm lens pitch were fabricated. The lenses were characterized to have standard deviation of 0.06 μm in height and 4.61 μm in diameter. The resolution limit of 3.9μm is demonstrated with real images, and the images were compared with ones made with glass and polycarbonate lens arrays. The image quality is at the same level than with the glass lenses and the manufacturing costs are very low, thus making them suitable for POCT applications.

  4. 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.

  5. 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

  6. Two-photon luminescence contrast by tip-sample coupling in femtosecond near-field optical microscopy

    Science.gov (United States)

    Horneber, Anke; Wackenhut, Frank; Braun, Kai; Wang, Xiao; Wang, Jiyong; Zhang, Dai; Meixner, Alfred J.

    2017-01-01

    We investigate the role of tip-sample interaction in nonlinear optical scanning near-field microscopy. The experiment was performed by tightly focusing femtosecond laser pulses onto a sharp gold tip that was positioned in close proximity to the surface of a sample with gold nanostructures on a Si-substrate by shear force feedback. The nonlinear optical signal consists of two-photon photoluminescence and second harmonic signal from the gold tip and the gold nanostructures. These signals can be used to characterize different coupling parameters such as geometry, material and width of the tip-sample gap and enable to reveal the mechanism responsible for the image contrast. Under the excitation with 776-nm and 110-fs laser pulses nonlinear imaging is almost background free and yields super resolution showing features with dimensions significantly below the diffraction limit with a signal intensity following quadratic excitation power law.

  7. Super-Resolution Imaging by using a Metallic Rod Array in the Near Infrared Region

    Institute of Scientific and Technical Information of China (English)

    YAO Jie; YE Yong-Hong

    2012-01-01

    An array of metallic rods can transport details below the diffraction limit of an object from the front face to the back face. This super-resolution imaging system has been studied in the microwave, mid-infrared and optical range. We investigate its performance in the near infrared (1550 nm) region. Numerical simulations show that the near-field components of dipole sources are transferred by the excitation and propagation of the surface plasmon mode of the rods. The appropriate length of rods is determined by the excited surface plasmon mode. The spatial resolution is greatly affected by the loss of metal.%An array of metallic rods can transport details below the diffraction limit of an object from the front face to the back face.This super-resolution imaging system has been studied in the microwave,mid-infrared and optical range.We investigate its performance in the near infrared (1550nm) region.Numerical simulations show that the near-field components of dipole sources are transferred by the excitation and propagation of the surface plasmon mode of the rods.The appropriate length of rods is determined by the excited surface plasmon mode.The spatial resolution is greatly affected by the loss of metal.

  8. 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.

  9. 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.

  10. 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.

  11. Fibre-optic nonlinear optical microscopy and endoscopy.

    Science.gov (United States)

    Fu, L; Gu, M

    2007-06-01

    Nonlinear optical microscopy has been an indispensable laboratory tool of high-resolution imaging in thick tissue and live animals. Rapid developments of fibre-optic components in terms of growing functionality and decreasing size provide enormous opportunities for innovations in nonlinear optical microscopy. Fibre-based nonlinear optical endoscopy is the sole instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible to a conventional optical microscope. This article reviews the current development of fibre-optic nonlinear optical microscopy and endoscopy, which includes crucial technologies for miniaturized nonlinear optical microscopy and their embodiments of endoscopic systems. A particular attention is given to several classes of photonic crystal fibres that have been applied to nonlinear optical microscopy due to their unique properties for ultrashort pulse delivery and signal collection. Furthermore, fibre-optic nonlinear optical imaging systems can be classified into portable microscopes suitable for imaging behaving animals, rigid endoscopes that allow for deep tissue imaging with minimally invasive manners, and flexible endoscopes enabling imaging of internal organs. Fibre-optic nonlinear optical endoscopy is coming of age and a paradigm shift leading to optical microscope tools for early cancer detection and minimally invasive surgery.

  12. 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.

  13. Rotary-scanning optical resolution photoacoustic microscopy

    Science.gov (United States)

    Qi, Weizhi; Xi, Lei

    2016-10-01

    Optical resolution photoacoustic microscopy (ORPAM) is currently one of the fastest evolving photoacoustic imaging modalities. It has a comparable spatial resolution to pure optical microscopic techniques such as epifluorescence microscopy, confocal microscopy, and two-photon microscopy, but also owns a deeper penetration depth. In this paper, we report a rotary-scanning (RS)-ORPAM that utilizes a galvanometer scanner integrated with objective to achieve rotary laser scanning. A 15 MHz cylindrically focused ultrasonic transducer is mounted onto a motorized rotation stage to follow optical scanning traces synchronously. To minimize the loss of signal to noise ratio, the acoustic focus is precisely adjusted to reach confocal with optical focus. Black tapes and carbon fibers are firstly imaged to evaluate the performance of the system, and then in vivo imaging of vasculature networks inside the ears and brains of mice is demonstrated using this system.

  14. 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...

  15. 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.

  16. 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.

  17. 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.

  18. Single-Image Super Resolution for Multispectral Remote Sensing Data Using Convolutional Neural Networks

    Science.gov (United States)

    Liebel, L.; Körner, M.

    2016-06-01

    In optical remote sensing, spatial resolution of images is crucial for numerous applications. Space-borne systems are most likely to be affected by a lack of spatial resolution, due to their natural disadvantage of a large distance between the sensor and the sensed object. Thus, methods for single-image super resolution are desirable to exceed the limits of the sensor. Apart from assisting visual inspection of datasets, post-processing operations—e.g., segmentation or feature extraction—can benefit from detailed and distinguishable structures. In this paper, we show that recently introduced state-of-the-art approaches for single-image super resolution of conventional photographs, making use of deep learning techniques, such as convolutional neural networks (CNN), can successfully be applied to remote sensing data. With a huge amount of training data available, end-to-end learning is reasonably easy to apply and can achieve results unattainable using conventional handcrafted algorithms. We trained our CNN on a specifically designed, domain-specific dataset, in order to take into account the special characteristics of multispectral remote sensing data. This dataset consists of publicly available SENTINEL-2 images featuring 13 spectral bands, a ground resolution of up to 10m, and a high radiometric resolution and thus satisfying our requirements in terms of quality and quantity. In experiments, we obtained results superior compared to competing approaches trained on generic image sets, which failed to reasonably scale satellite images with a high radiometric resolution, as well as conventional interpolation methods.

  19. Hyperspectral imagery super-resolution by compressive sensing inspired dictionary learning and spatial-spectral regularization.

    Science.gov (United States)

    Huang, Wei; Xiao, Liang; Liu, Hongyi; Wei, Zhihui

    2015-01-19

    Due to the instrumental and imaging optics limitations, it is difficult to acquire high spatial resolution hyperspectral imagery (HSI). Super-resolution (SR) imagery aims at inferring high quality images of a given scene from degraded versions of the same scene. This paper proposes a novel hyperspectral imagery super-resolution (HSI-SR) method via dictionary learning and spatial-spectral regularization. The main contributions of this paper are twofold. First, inspired by the compressive sensing (CS) framework, for learning the high resolution dictionary, we encourage stronger sparsity on image patches and promote smaller coherence between the learned dictionary and sensing matrix. Thus, a sparsity and incoherence restricted dictionary learning method is proposed to achieve higher efficiency sparse representation. Second, a variational regularization model combing a spatial sparsity regularization term and a new local spectral similarity preserving term is proposed to integrate the spectral and spatial-contextual information of the HSI. Experimental results show that the proposed method can effectively recover spatial information and better preserve spectral information. The high spatial resolution HSI reconstructed by the proposed method outperforms reconstructed results by other well-known methods in terms of both objective measurements and visual evaluation.

  20. Compressed sensing for super-resolution spatial and temporal laser detection and ranging

    Science.gov (United States)

    Laurenzis, Martin; Schertzer, Stephane; Christnacher, Frank

    2016-10-01

    In the past decades, laser aided electro-optical sensing has reached high maturity and several commercial systems are available at the market for various but specific applications. These systems can be used for detection i.e. imaging as well as ranging. They cover laser scanning devices like LiDAR and staring full frame imaging systems like laser gated viewing or LADAR. The sensing capabilities of these systems is limited by physical parameter (like FPA array size, temporal band width, scanning rate, sampling rate) and is adapted to specific applications. Change of system parameter like an increase of spatial resolution implies the setup of a new sensing device with high development cost or the purchase and installation of a complete new sensor unit. Computational imaging approaches can help to setup sensor devices with flexible or adaptable sensing capabilities. Especially, compressed sensing is an emerging computational method which is a promising candidate to realize super-resolution sensing with the possibility to adapt its performance to various sensing tasks. It is possible to increase sensing capabilities with compressed sensing to gain either higher spatial and/or temporal resolution. Then, the sensing capabilities depend no longer only on the physical performance of the device but also on the computational effort and can be adapted to the application. In this paper, we demonstrate and discuss laser aided imaging using CS for super-resolution tempo-spatial imaging and ranging.

  1. Characterization of Polymer Blends: Optical Microscopy (*Polarized, Interference and Phase Contrast Microscopy*) and Confocal Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ramanathan, Nathan Muruganathan [ORNL; Darling, Seth B. [Argonne National Laboratory (ANL)

    2015-01-01

    Chapter 15 surveys the characterization of macro, micro and meso morphologies of polymer blends by optical microscopy. Confocal Microscopy offers the ability to view the three dimensional morphology of polymer blends, popular in characterization of biological systems. Confocal microscopy uses point illumination and a spatial pinhole to eliminate out-of focus light in samples that are thicker than the focal plane.

  2. Optical tweezers for confocal microscopy

    Science.gov (United States)

    Hoffmann, A.; Meyer zu Hörste, G.; Pilarczyk, G.; Monajembashi, S.; Uhl, V.; Greulich, K. O.

    2000-11-01

    In confocal laser scanning microscopes (CLSMs), lasers can be used for image formation as well as tools for the manipulation of microscopic objects. In the latter case, in addition to the imaging lasers, the light of an extra laser has to be focused into the object plane of the CLSM, for example as optical tweezers. Imaging as well as trapping by optical tweezers can be done using the same objective lens. In this case, z-sectioning for 3D imaging shifts the optical tweezers with the focal plane of the objective along the optical axis, so that a trapped object remains positioned in the focal plane. Consequently, 3D imaging of trapped objects is impossible without further measures. We present an experimental set-up keeping the axial trapping position of the optical tweezers at its intended position whilst the focal plane can be axially shifted over a distance of about 15 μm. It is based on fast-moving correctional optics synchronized with the objective movement. First examples of application are the 3D imaging of chloroplasts of Elodea densa (Canadian waterweed) in a vigorous cytoplasmic streaming and the displacement of zymogen granules in pancreatic cancer cells (AR42 J).

  3. Super resolution nano-information recording in a new hydrazone metal complex material

    Science.gov (United States)

    Zhang, Kui; Wei, Jingsong; Chen, Zhimin; Wei, Tao; Geng, Yongyou; Wang, Yang; Wu, Yiqun

    2016-10-01

    Laser thermal lithography has been proposed for a few years, which has the advantages of breaking through the optical diffraction limit, operation in far-field and in air, and low production cost. In this paper, a new hydrazone metal complex is used as the laser thermal lithography material due to its feature of the one-step fabrication of micro/nano structure without mask and wet-etching process. Based on the laser thermal lithography method, super resolution nano-information pits are directly written on the surface of hydrazone metal complex thin films. Pits with a minimum feature size of about 79 nm are successfully obtained, which is only about 1/7 of the writing spot size. Moreover, the reactive ion etching method can be applied to transfer the pits onto a silica substrate. These results suggest the potential applications of the new material in high density optical data storage and semiconductor industries.

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

    Science.gov (United States)

    Tang, Heng-He; Liu, Pu-Kun

    2015-09-07

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

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Re-scan confocal microscopy: scanning twice for better resolution.

    Science.gov (United States)

    De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

  15. A novel super resolution scheme to acquire and process satellite images

    Science.gov (United States)

    Yin, Dong-yu; Su, Xiao-feng; Lin, Jian-chun; Wang, Gan-quan; Kuang, Ding-bo

    2013-09-01

    Geosynchronous satellite has obvious limitations for the weight and the scale of payloads, and large aperture optical system is not permitted. The optical diffraction limit of small aperture optical system has an adverse impact on the resolution of the acquired images. Therefore, how to get high resolution images using super-resolution technique with the acquired low resolution images becomes a popular problem investigated by researchers. Here, we present a novel scheme to acquire low resolution images and process them to achieve a high resolution image. Firstly, to acquire low resolution images, we adopt a special arrangement pattern of four CCD staggered arrays on the focal plane in the remote sensing satellite framework .These four CCD linear arrays are parallelized with a 0.25√2 pixel shift along the CCD direction and a 1.25 pixel shift along the scanning direction. The rotation angle between the two directions is 45 degree. The tilting sampling mode and the special arrangement pattern allow the sensor to acquire images with a smaller sampling interval which can give the resolution a greater enhancement. Secondly, to reconstruct a high resolution image of pretty good quality with a magnification factor 4, we propose a novel algorithm based on the iterative-interpolation super resolution algorithm (IISR) and the new edge-directed interpolation algorithm (NEDI). The new algorithm makes a critical improvement to NEDI and introduces it into the multi-frame interpolation in IISR. The algorithm can preserve the edges well and requires a relatively small number of low-resolution images to achieve better reconstruction accuracy .In the last part of the paper, we carry out a simulation experiment, and use MSE as the quality measure. The results demonstrate that our new scheme substantially improves the image resolution with both better quantitative quality and visual quality compared with some previous normal methods.

  16. 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.

  17. A novel method for surface exploration: Super-resolution restoration of Mars repeat-pass orbital imagery

    Science.gov (United States)

    Tao, Y.; Muller, J.-P.

    2016-02-01

    Higher resolution imaging data of planetary surfaces is considered desirable by the international community of planetary scientists interested in improving understanding of surface formation processes. However, given various physical constraints from the imaging instruments through to limited bandwidth of transmission one needs to trade-off spatial resolution against bandwidth. Even given optical communications, future imaging systems are unlikely to be able to resolve features smaller than 25 cm on most planetary bodies, such as Mars. In this paper, we propose a novel super-resolution restoration technique, called Gotcha-PDE-TV (GPT), taking advantage of the non-redundant sub-pixel information contained in multiple raw orbital images in order to restore higher resolution imagery. We demonstrate optimality of this technique in planetary image super-resolution restoration with example processing of 8 repeat-pass 25 cm HiRISE images covering the MER-A Spirit rover traverse in Gusev crater to resolve a 5 cm resolution of the area. We assess the "true" resolution of the 5 cm super-resolution restored images using contemporaneous rover Navcam imagery on the surface and an inter-comparison of landmarks in the two sets of imagery.

  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. Label-Free Nanoscopy with Contact Microlenses: Super-Resolution Mechanisms and Limitations

    CERN Document Server

    Astratov, Vasily N; Brettin, Aaron; Allen, Kenneth W; Maslov, Alexey V; Limberopoulos, Nicholaos I; Walker, Dennis E; Urbas, Augustine M

    2016-01-01

    Despite all the success with developing super-resolution imaging techniques, the Abbe limit poses a severe fundamental restriction on the resolution of far-field imaging systems based on diffraction of light. Imaging with contact microlenses, such as microspheres or microfibers, can increase the resolution by a factor of two beyond the Abbe limit. The theoretical mechanisms of these methods are debated in the literature. In this work, we focus on the recently expressed idea that optical coupling between closely spaced nanoscale objects can lead to the formation of the modes that drastically impact the imaging properties. These coupling effects emerge in nanoplasmonic or nanocavity clusters, photonic molecules, or various arrays under resonant excitation conditions. The coherent nature of imaging processes is key to understanding their physical mechanisms. We used a cluster of point dipoles, as a simple model system, to study and compare the consequences of coherent and incoherent imaging. Using finite differe...

  2. Super-resolution reconstruction algorithm based on adaptive convolution kernel size selection

    Science.gov (United States)

    Gao, Hang; Chen, Qian; Sui, Xiubao; Zeng, Junjie; Zhao, Yao

    2016-09-01

    Restricted by the detector technology and optical diffraction limit, the spatial resolution of infrared imaging system is difficult to achieve significant improvement. Super-Resolution (SR) reconstruction algorithm is an effective way to solve this problem. Among them, the SR algorithm based on multichannel blind deconvolution (MBD) estimates the convolution kernel only by low resolution observation images, according to the appropriate regularization constraints introduced by a priori assumption, to realize the high resolution image restoration. The algorithm has been shown effective when each channel is prime. In this paper, we use the significant edges to estimate the convolution kernel and introduce an adaptive convolution kernel size selection mechanism, according to the uncertainty of the convolution kernel size in MBD processing. To reduce the interference of noise, we amend the convolution kernel in an iterative process, and finally restore a clear image. Experimental results show that the algorithm can meet the convergence requirement of the convolution kernel estimation.

  3. Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

    Science.gov (United States)

    Tamada, Yosuke; Murata, Takashi; Hattori, Masayuki; Oya, Shin; Hayano, Yutaka; Kamei, Yasuhiro; Hasebe, Mitsuyasu

    2014-04-01

    In astronomy, adaptive optics (AO) can be used to cancel aberrations caused by atmospheric turbulence and to perform diffraction-limited observation of astronomical objects from the ground. AO can also be applied to microscopy, to cancel aberrations caused by cellular structures and to perform high-resolution live imaging. As a step toward the application of AO to microscopy, here we analyzed the optical properties of plant cells. We used leaves of the moss Physcomitrella patens, which have a single layer of cells and are thus suitable for optical analysis. Observation of the cells with bright field and phase contrast microscopy, and image degradation analysis using fluorescent beads demonstrated that chloroplasts provide the main source of optical degradations. Unexpectedly, the cell wall, which was thought to be a major obstacle, has only a minor effect. Such information provides the basis for the application of AO to microscopy for the observation of plant cells.

  4. 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.

  5. 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.

  6. Single spin stochastic optical reconstruction microscopy

    CERN Document Server

    Pfender, Matthias; Waldherr, Gerald; Wrachtrup, Jörg

    2014-01-01

    We experimentally demonstrate precision addressing of single quantum emitters by combined optical microscopy and spin resonance techniques. To this end we utilize nitrogen-vacancy (NV) color centers in diamond confined within a few ten nanometers as individually resolvable quantum systems. By developing a stochastic optical reconstruction microscopy (STORM) technique for NV centers we are able to simultaneously perform sub diffraction-limit imaging and optically detected spin resonance (ODMR) measurements on NV spins. This allows the assignment of spin resonance spectra to individual NV center locations with nanometer scale resolution and thus further improves spatial discrimination. For example, we resolved formerly indistinguishable emitters by their spectra. Furthermore, ODMR spectra contain metrology information allowing for sub diffraction-limit sensing of, for instance, magnetic or electric fields with inherently parallel data acquisition. As an example, we have detected nuclear spins with nanometer sca...

  7. 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.

  8. 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.

  9. 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.

  10. A Super-resolution Reconstruction Algorithm for Surveillance Video

    Directory of Open Access Journals (Sweden)

    Jian Shao

    2017-01-01

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

  11. Formation of super-resolution spot through nonlinear Fabry-Perot cavity structures: theory and simulation.

    Science.gov (United States)

    Wei, Jingsong; Wang, Rui; Yan, Hui; Fan, Yongtao

    2014-04-07

    This study explores how interference manipulation breaks through the diffraction limit and induces super-resolution nano-optical hot spots through the nonlinear Fabry-Perot cavity structure. The theoretical analytical model is established, and the numerical simulation results show that when the thickness of the nonlinear thin film inside the nonlinear Fabry-Perot cavity structure is adjusted to centain value, the constructive interference effect can be formed in the central point of the spot, which causes the nanoscale optical hot spot in the central region to be produced. The simulation results also tell us that the hot spot size is sensitive to nonlinear thin film thickness, and the accuracy is required to be up to nanometer or even subnanometer scale, which is very large challenging for thin film deposition technique, however, slightly changing the incident laser power can compensate for drawbacks of low thickness accuracy of nonlinear thin films. Taking As(2)S(3) as the nonlinear thin film, the central hot spot with a size of 40nm is obtained at suitable nonlinear thin film thickness and incident laser power. The central hot spot size is only about λ/16, which is very useful in super-high density optical recording, nanolithography, and high-resolving optical surface imaging.

  12. Virtual k -Space Modulation Optical Microscopy

    Science.gov (United States)

    Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Zheng, Guoan; Fang, Yue; Xu, Yingke; Liu, Xu; So, Peter T. C.

    2016-07-01

    We report a novel superresolution microscopy approach for imaging fluorescence samples. The reported approach, termed virtual k -space modulation optical microscopy (VIKMOM), is able to improve the lateral resolution by a factor of 2, reduce the background level, improve the optical sectioning effect and correct for unknown optical aberrations. In the acquisition process of VIKMOM, we used a scanning confocal microscope setup with a 2D detector array to capture sample information at each scanned x -y position. In the recovery process of VIKMOM, we first modulated the captured data by virtual k -space coding and then employed a ptychography-inspired procedure to recover the sample information and correct for unknown optical aberrations. We demonstrated the performance of the reported approach by imaging fluorescent beads, fixed bovine pulmonary artery endothelial (BPAE) cells, and living human astrocytes (HA). As the VIKMOM approach is fully compatible with conventional confocal microscope setups, it may provide a turn-key solution for imaging biological samples with ˜100 nm lateral resolution, in two or three dimensions, with improved optical sectioning capabilities and aberration correcting.

  13. A near-field optical microscopy nanoarray

    Energy Technology Data Exchange (ETDEWEB)

    Semin, D.J.; Ambrose, W.P.; Goodwin, P.M.; Kwller, A. [Los Alamos National Lab., NM (United States); Wendt, J.R. [Sandia National Labs., Albuquerque, NM (United States)

    1996-12-31

    Multiplexing near-field scanning optical microscopy (NSOM) by the use of a nanoarray with parallel imaging is studied. The fabrication, characterization, and utilization of nanoarrays with {approximately} 100 nm diameter apertures spaced 500 nm center-to- center is presented. Extremely uniform nanoarrays with {approximately} 10{sup 8} apertures were fabricated by electron beam lithography and reactive ion etching. The nanoarrays were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). In this paper we utilize these nanoarrays in a laser-illuminated microscope with parallel detection on a charge- coupled device (CCD). Detection of B-phycoerythrin (B-PE) molecules using near-field illumination is presented. In principle, our system can be used to obtain high lateral resolution NSOM images over a wide-field of view (e.g. 50-100 {mu}m) within seconds.

  14. Introduction to Modern Methods in Light Microscopy.

    Science.gov (United States)

    Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

    2017-01-01

    For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  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. Multiparallel Three-Dimensional Optical Microscopy

    Science.gov (United States)

    Nguyen, Lam K.; Price, Jeffrey H.; Kellner, Albert L.; Bravo-Zanoquera, Miguel

    2010-01-01

    Multiparallel three-dimensional optical microscopy is a method of forming an approximate three-dimensional image of a microscope sample as a collection of images from different depths through the sample. The imaging apparatus includes a single microscope plus an assembly of beam splitters and mirrors that divide the output of the microscope into multiple channels. An imaging array of photodetectors in each channel is located at a different distance along the optical path from the microscope, corresponding to a focal plane at a different depth within the sample. The optical path leading to each photodetector array also includes lenses to compensate for the variation of magnification with distance so that the images ultimately formed on all the photodetector arrays are of the same magnification. The use of optical components common to multiple channels in a simple geometry makes it possible to obtain high light-transmission efficiency with an optically and mechanically simple assembly. In addition, because images can be read out simultaneously from all the photodetector arrays, the apparatus can support three-dimensional imaging at a high scanning rate.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    2017-03-01

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

  9. Performance Evaluation of Super-Resolution Reconstruction Methods on Real-World Data

    Directory of Open Access Journals (Sweden)

    L. J. van Vliet

    2007-01-01

    Full Text Available 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, simulating a real-observer task, is capable of determining the performance of a specific SR reconstruction method under varying conditions of the input data. It is shown that the performance of an SR reconstruction method on real-world data can be predicted accurately by measuring its performance on simulated data. This prediction of the performance on real-world data enables the optimization of the complete chain of a vision system; from camera setup and SR reconstruction up to image detection/recognition/identification. Furthermore, different SR reconstruction methods are compared to show that the TOD method is a useful tool to select a specific SR reconstruction method according to the imaging conditions (camera's fill-factor, optical point-spread-function (PSF, signal-to-noise ratio (SNR.

  10. Experimental evidence of super-resolution better than λ/105 with positive refraction

    Science.gov (United States)

    Miñano, Juan C.; Sánchez-Dehesa, José; González, Juan C.; Benítez, P.; Grabovičkić, D.; Carbonell, Jorge; Ahmadpanahi, H.

    2014-03-01

    Super-resolution (SR) systems surpassing the Abbe diffraction limit have been theoretically and experimentally demonstrated using a number of different approaches and technologies: using materials with a negative refractive index, utilizing optical super-oscillation, using a resonant metalens, etc. However, recently it has been proved theoretically that in the Maxwell fish-eye lens (MFE), a device made of positive refractive index materials, the same phenomenon takes place. Moreover, using a simpler device equivalent to the MFE called the spherical geodesic waveguide (SGW), an SR of up to λ/3000 was simulated in COMSOL. Until now, only one piece of experimental evidence of SR with positive refraction has been reported (up to λ/5) for an MFE prototype working at microwave frequencies. Here, experimental results are presented for an SGW prototype showing an SR of up to λ/105. The SGW prototype consists of two concentric metallic spheres with an air space in between and two coaxial ports acting as an emitter and a receiver. The prototype has been analyzed in the range 1 GHz to 1.3 GHz.

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

    Directory of Open Access Journals (Sweden)

    Xiwei Huang

    2016-11-01

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

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

    Science.gov (United States)

    Huang, Xiwei; Jiang, Yu; Liu, Xu; Xu, Hang; Han, Zhi; Rong, Hailong; Yang, Haiping; Yan, Mei; Yu, Hao

    2016-11-02

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

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

    Science.gov (United States)

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

    2011-02-01

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

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

    CERN Document Server

    Chan, Antony C S; Bogaraju, Sharat C V; So, Hayden K H; Lam, Edmund Y; Tsia, Kevin K

    2016-01-01

    Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the- art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate --- hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image frames inherently introduced by asynchronous digital sampling of the continuous time-stretch imaging process. Precise pixel registration is thus accomplished without any active opto-mechanical subpixel-shift control or other additional hardware. Here, we present the experimental pixel-SR image reconstruction pipeline that restores high-resolution time-stretch images of microparticles and biological cells (phytoplankton) at a relaxed sampling rate (approx. 2--5 GSa/s) --- more than four times lower than the originally requir...

  15. Portable fiber-optic taper coupled optical microscopy platform

    Science.gov (United States)

    Wang, Weiming; Yu, Yan; Huang, Hui; Ou, Jinping

    2017-04-01

    The optical fiber taper coupled with CMOS has advantages of high sensitivity, compact structure and low distortion in the imaging platform. So it is widely used in low light, high speed and X-ray imaging systems. In the meanwhile, the peculiarity of the coupled structure can meet the needs of the demand in microscopy imaging. Toward this end, we developed a microscopic imaging platform based on the coupling of cellphone camera module and fiber optic taper for the measurement of the human blood samples and ascaris lumbricoides. The platform, weighing 70 grams, is based on the existing camera module of the smartphone and a fiber-optic array which providing a magnification factor of 6x.The top facet of the taper, on which samples are placed, serves as an irregular sampling grid for contact imaging. The magnified images of the sample, located on the bottom facet of the fiber, are then projected onto the CMOS sensor. This paper introduces the portable medical imaging system based on the optical fiber coupling with CMOS, and theoretically analyzes the feasibility of the system. The image data and process results either can be stored on the memory or transmitted to the remote medical institutions for the telemedicine. We validate the performance of this cell-phone based microscopy platform using human blood samples and test target, achieving comparable results to a standard bench-top microscope.

  16. 超分辨近场结构的研究进展及其应用%Development and applications of super-resolution near-field structures

    Institute of Scientific and Technical Information of China (English)

    刘前; 曹四海; 郭传飞

    2009-01-01

    Super-resolution near-field structures have promising applications in nanostorage, nanofabrica-tion and biosensors, due to their very high resolution beyond the optical diffraction limit. A brief review will be presented of their history, operation mechanism, recent new developments, as well as their applications in super-high density optical storage and super-resolution near-field lithography.%超分辨近场结构(super-resolution near-field structure,super-RENS)由于突破了传统远场光学衍射极限的限制,在纳米光储存、微纳米加工、基于局域表面等离子体增强的生物传感器方面展现出良好的应用前景,因此吸引了众多研究者的目光.文章简要介绍了超分辨近场结构的发展历程、相应的工作机理、最新研究动态及其在超高密度光存储、近场光刻中的应用状况,并对未来的工作重点做出展望.

  17. 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.

  18. 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.

  19. Volumetric optical coherence microscopy enabled by aberrated optics (Conference Presentation)

    Science.gov (United States)

    Mulligan, Jeffrey A.; Liu, Siyang; Adie, Steven G.

    2017-02-01

    Optical coherence microscopy (OCM) is an interferometric imaging technique that enables high resolution, non-invasive imaging of 3D cell cultures and biological tissues. Volumetric imaging with OCM suffers a trade-off between high transverse resolution and poor depth-of-field resulting from defocus, optical aberrations, and reduced signal collection away from the focal plane. While defocus and aberrations can be compensated with computational methods such as interferometric synthetic aperture microscopy (ISAM) or computational adaptive optics (CAO), reduced signal collection must be physically addressed through optical hardware. Axial scanning of the focus is one approach, but comes at the cost of longer acquisition times, larger datasets, and greater image reconstruction times. Given the capabilities of CAO to compensate for general phase aberrations, we present an alternative method to address the signal collection problem without axial scanning by using intentionally aberrated optical hardware. We demonstrate the use of an astigmatic spectral domain (SD-)OCM imaging system to enable single-acquisition volumetric OCM in 3D cell culture over an extended depth range, compared to a non-aberrated SD-OCM system. The transverse resolution of the non-aberrated and astigmatic imaging systems after application of CAO were 2 um and 2.2 um, respectively. The depth-range of effective signal collection about the nominal focal plane was increased from 100 um in the non-aberrated system to over 300 um in the astigmatic system, extending the range over which useful data may be acquired in a single OCM dataset. We anticipate that this method will enable high-throughput cellular-resolution imaging of dynamic biological systems over extended volumes.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. A numerical study of super-resolution through fast 3D wideband algorithm for scattering in highly-heterogeneous media

    KAUST Repository

    Létourneau, Pierre-David

    2016-09-19

    We present a wideband fast algorithm capable of accurately computing the full numerical solution of the problem of acoustic scattering of waves by multiple finite-sized bodies such as spherical scatterers in three dimensions. By full solution, we mean that no assumption (e.g. Rayleigh scattering, geometrical optics, weak scattering, Born single scattering, etc.) is necessary regarding the properties of the scatterers, their distribution or the background medium. The algorithm is also fast in the sense that it scales linearly with the number of unknowns. We use this algorithm to study the phenomenon of super-resolution in time-reversal refocusing in highly-scattering media recently observed experimentally (Lemoult et al., 2011), and provide numerical arguments towards the fact that such a phenomenon can be explained through a homogenization theory.

  11. Phase measurements exhibiting super sensitivity and super resolution features

    DEFF Research Database (Denmark)

    Schäfermeier, Clemens; Jezek, Miroslav; Gehring, Tobias

    2016-01-01

    By using an optical squeezed state and a post-processed homodyne detection scheme we show that phase measurements can overcome Rayleigh's resolution criterion and beat the quantum shot noise limit simultaneously......By using an optical squeezed state and a post-processed homodyne detection scheme we show that phase measurements can overcome Rayleigh's resolution criterion and beat the quantum shot noise limit simultaneously...

  12. 3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

    KAUST Repository

    Zhang, Yibo

    2017-08-12

    High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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 \

  18. 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...

  19. 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...

  20. 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.

  1. 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

  2. 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),

  3. 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.

  4. 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...

  5. Projection based image restoration, super-resolution and error correction codes

    Science.gov (United States)

    Bauer, Karl Gregory

    Super-resolution is the ability of a restoration algorithm to restore meaningful spatial frequency content beyond the diffraction limit of the imaging system. The Gerchberg-Papoulis (GP) algorithm is one of the most celebrated algorithms for super-resolution. The GP algorithm is conceptually simple and demonstrates the importance of using a priori information in the formation of the object estimate. In the first part of this dissertation the continuous GP algorithm is discussed in detail and shown to be a projection on convex sets algorithm. The discrete GP algorithm is shown to converge in the exactly-, over- and under-determined cases. A direct formula for the computation of the estimate at the kth iteration and at convergence is given. This analysis of the discrete GP algorithm sets the stage to connect super-resolution to error-correction codes. Reed-Solomon codes are used for error-correction in magnetic recording devices, compact disk players and by NASA for space communications. Reed-Solomon codes have a very simple description when analyzed with the Fourier transform. This signal processing approach to error- correction codes allows the error-correction problem to be compared with the super-resolution problem. The GP algorithm for super-resolution is shown to be equivalent to the correction of errors with a Reed-Solomon code over an erasure channel. The Restoration from Magnitude (RFM) problem seeks to recover a signal from the magnitude of the spectrum. This problem has applications to imaging through a turbulent atmosphere. The turbulent atmosphere causes localized changes in the index of refraction and introduces different phase delays in the data collected. Synthetic aperture radar (SAR) and hyperspectral imaging systems are capable of simultaneously recording multiple images of different polarizations or wavelengths. Each of these images will experience the same turbulent atmosphere and have a common phase distortion. A projection based restoration

  6. Computationally efficient image restoration and super-resolution algorithns for real-time implementation

    Science.gov (United States)

    Sundareshan, Malur K.

    2002-07-01

    Computational complexity is a major impediment to the real- time implementation of image restoration and super- resolution algorithms. Although powerful restoration algorithms have been developed within the last few years utilizing sophisticated mathematical machinery (based on statistical optimization and convex set theory), these algorithms are typically iterative in nature and require enough number of iterations to be executed to achieve desired resolution gains in order to meaningfully perform detection and recognition tasks in practice. Additionally, recent technological breakthroughs have facilitated novel sensor designs (focal plane arrays, for instance) that make it possible to capture mega-pixel imagery data at video frame rates. A major challenge in the processing of these large format images is to complete the execution of the image processing steps within the frame capture times and to keep up with the output rate of the sensor so that all data captured by the sensor can be efficiently utilized. Consequently, development of novel methods that facilitate real-time implementation of image restoration and super- resolution algorithms is of significant practical interest and will be the primary focus of this paper. The key to designing computationally efficient processing schemes lies in strategically introducing appropriate pre-processing and post-processing steps together with the super-resolution iterations in order to tailor optimized overall processing sequences for imagery data of specific formats. Three distinct methods for tailoring a pre-processing filter and integrating it with the super-resolution processing steps will be outlined in this paper. These methods consist of a Region-of-Interest (ROI) extraction scheme, a background- detail separation procedure, and a scene-derived information extraction step for implementing a set-theoretic restoration of the image that is less demanding in computation compared to the super-resolution iterations. A

  7. Super-resolution imaging based on virtual Airy spot

    Science.gov (United States)

    Liu, Zhengjun; Guo, Cheng; Cui, Junning; Wu, Qun

    2015-02-01

    Based on the theoretical model of Airy spot, a method is proposed for improving the imaging speed from confocal microscopy. The virtual Airy spot is designed for obtaining the pattern on CCD at detecting plane. Here the size of the spot is determined by the parameters of imaging system and intensity data from point detector, which can receive data quicker than CCD. The treatment can improve the speed of imaging comparing with CCD at receiving end. The virtual structured detection is also utilized for generating high-resolution image. Some numerical simulation results are provided for demonstrating the validity of the proposed method.

  8. Super-resolution photoacoustic imaging through a scattering wall

    Science.gov (United States)

    Conkey, Donald B.; Caravaca-Aguirre, Antonio M.; Dove, Jake D.; Ju, Hengyi; Murray, Todd W.; Piestun, Rafael

    2015-08-01

    The use of wavefront shaping to compensate for scattering has brought a renewed interest as a potential solution to imaging through scattering walls. A key to the practicality of any imaging through scattering technique is the capability to focus light without direct access behind the scattering wall. Here we address this problem using photoacoustic feedback for wavefront optimization. By combining the spatially non-uniform sensitivity of the ultrasound transducer to the generated photoacoustic waves with an evolutionary competition among optical modes, the speckle field develops a single, high intensity focus significantly smaller than the acoustic focus used for feedback. Notably, this method is not limited by the size of the absorber to form a sub-acoustic optical focus. We demonstrate imaging behind a scattering medium using two different imaging modalities with up to ten times improvement in signal-to-noise ratio and five to six times sub-acoustic resolution.

  9. Super-resolution photoacoustic imaging through a scattering wall

    CERN Document Server

    Conkey, Donald B; Dove, Jacob D; Ju, Hengyi; Murray, Todd W; Piestun, Rafael

    2013-01-01

    Imaging through opaque, highly scattering walls is a long sought after capability with potential applications in a variety of fields. The use of wavefront shaping to compensate for scattering has brought a renewed interest as a potential solution to this problem. A key to the practicality of any imaging technique is the capability to focus light without direct access behind the scattering wall. Here, we address this problem using photoacoustic feedback for wavefront optimization. By combining the spatially non-uniform sensitivity of the ultrasound transducer to the generated photoacoustic waves with an evolutionary competition among optical modes, the speckle field develops a single, high intensity focus significantly smaller than the acoustic focus used for feedback. Notably, this method is not limited by the size of the absorber to form a sub-acoustic optical focus. We demonstrate imaging behind a scattering medium with up to ten times improvement in signal-to-noise ratio (SNR) and five to six times sub-aco...

  10. Optical imaging of individual biomolecules in densely packed clusters

    Science.gov (United States)

    Dai, Mingjie; Jungmann, Ralf; Yin, Peng

    2016-09-01

    Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to 10-20 nm in size to be imaged. However, the direct optical observation of individual molecular targets (∼5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography)—a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridization—on synthetic DNA nanostructures. We examined the effects of a high photon count, high blinking statistics and an appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves optical nanodisplay with 5 × 5 nm pixel size and three distinct colours with <1 nm cross-channel registration accuracy.

  11. Effect of optical surface flatness performance on spatial-light-modulator-based imaging system

    Science.gov (United States)

    Zhou, Hongqiang; Wan, Yuhong; Man, Tianlong; Han, Ying

    2016-10-01

    Spatial light modulator (SLM) has various of applications in the field of imaging, beam shaping, adaptive optics and so on. While SLM is used as an aberration correction element in super-resolution microscopy, the surface flatness of SLM could affect the imaging performance of the system due to the higher sensitivity to aberrations of these kind microscopic techniques. In this paper, the optical surface flatness of SLM is measured experimentally by employing the image plane digital holography. The topography of SLM is retrieved from the captured hologram. Aiming to the application of SLM as an adaptive correction element in super resolution microscopy, the aberrations introduced by the surface flatness of SLM are further evaluated and corrected in the same optical system.

  12. 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

  13. Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime

    OpenAIRE

    Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro; Lihong V. Wang

    2012-01-01

    Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method...

  14. Super-Resolution Reconstruction of High-Resolution Satellite ZY-3 TLC Images.

    Science.gov (United States)

    Li, Lin; Wang, Wei; Luo, Heng; Ying, Shen

    2017-05-07

    Super-resolution (SR) image reconstruction is a technique used to recover a high-resolution image using the cumulative information provided by several low-resolution images. With the help of SR techniques, satellite remotely sensed images can be combined to achieve a higher-resolution image, which is especially useful for a two- or three-line camera satellite, e.g., the ZY-3 high-resolution Three Line Camera (TLC) satellite. In this paper, we introduce the application of the SR reconstruction method, including motion estimation and the robust super-resolution technique, to ZY-3 TLC images. The results show that SR reconstruction can significantly improve both the resolution and image quality of ZY-3 TLC images.

  15. Robust super-resolution by minimizing a Gaussian-weighted L{sub 2} error norm

    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, P. O. 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 via regularization is often required to obtain a stable solution. While adding a regularization term to 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 Gaussian-weighted L{sub 2}-norm in the data misfit term that does not response to intensity outliers. With the outliers suppressed, our solution behaves similarly to a maximum-likelihood solution in 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.

  16. Super-resolution phase reconstruction technique in electron holography with a stage-scanning system

    Science.gov (United States)

    Lei, Dan; Mitsuishi, Kazutaka; Harada, Ken; Shimojo, Masayuki; Ju, Dongying; Takeguchi, Masaki

    2014-02-01

    Super-resolution image reconstruction is a digital signal processing technique that allows creating a high-resolution image from multiple low-resolution images taken at slightly different positions. We introduce the super-resolution image reconstruction technique into electron holography for reconstructing phase images as follows: the studied specimen is shifted step-wise with a high-precision piezo holder, and a series of holograms is recorded. When the step size is not a multiple of the CCD pixel size, processing of the acquired series results in a higher pixel density and spatial resolution as compared to the phase image obtained with conventional holography. The final resolution exceeds the limit of the CCD pixel size divided by the magnification.

  17. Radial Basis Function Neural Network Based Super-Resolution Restoration for an Underspled Image

    Institute of Scientific and Technical Information of China (English)

    苏秉华; 金伟其; 牛丽红

    2004-01-01

    To achieve restoration of high frequency information for an underspled and degraded low-resolution image, a nonlinear and real-time processing method-the radial basis function (RBF) neural network based super-resolution method of restoration is proposed. The RBF network configuration and processing method is suitable for a high resolution restoration from an underspled low-resolution image. The soft-competition learning scheme based on the k-means algorithm is used, and can achieve higher mapping approximation accuracy without increase in the network size. Experiments showed that the proposed algorithm can achieve a super-resolution restored image from an underspled and degraded low-resolution image, and requires a shorter training time when compared with the multiplayer perception (MLP) network.

  18. Super resolution reconstruction of moving objects from low resolution surveillance video

    Institute of Scientific and Technical Information of China (English)

    Wang Suyu; Shen Lansun; David Daganfeng; Li Xiaoguang

    2008-01-01

    Construction of high resolution images from low resolution sequences having rigid or semi-rigid objects with unified motions is often important in surveillance and other applications. In this paper a novel object-based super resolution reconstruction scheme was proposed, in which a six-parameter affine model-based object tracking and registration method was first used to segment and match objects among a sequence of low resolution frames. The motion model was then further extended to the traditional maximum a posterior (MAP) super resolution algorithm. The proposed object tracking and registration method was evaluated by both simulated and real acquired sequences. The results have demonstrated the high accuracy of the proposed object based method and the enhanced reconstruction performance of the extended approach.

  19. Super-Resolution Reconstruction of Image Sequence Using Multiple Motion Estimation Fusion

    Institute of Scientific and Technical Information of China (English)

    Cheng Wang; Run-Sheng Wang

    2004-01-01

    Super-resolution reconstruction algorithm produces a high-resolution image from a low-resolution image sequence. The accuracy and the stability of the motion estimation (ME) are essential for the whole restoration. In this paper, a new super-resolution reconstruction algorithm is developed using a robust ME method, which fuses multiple estimated motion vectors within the sequence. The new algorithm has two major improvements compared with the previous research. First, instead of only two frames, the whole sequence is used to obtain a more accurate and stable estimation of the motion vector of each frame; second, the reliability of the ME is quantitatively measured and introduced into the cost function of the reconstruction algorithm. The algorithm is applied to both synthetic and real sequences, and the results are presented in the paper.

  20. Wavelet Transform of Super-Resolutions Based on Radar and Infrared Sensor Fusion

    Science.gov (United States)

    1998-05-01

    0I Q’UAL1 INwPO¶= I VI STATEMB r AApproved for public release; Distribution Unlimited NAVY CASE 77545 WAVELET TRANSFORM OF SUPER-RESOLUTIONS BASED ON...INVENTION It is, therefore, an object of the present invention to provide a structure and method for applying the forward and reverse Wavelet Transform (WT...invention, the noisy super- 10 resolution of infrared imaging is combined with the Wavelet transform for radar corner back-scattering size information

  1. Super-resolution with complex masks using a phase-only LCD.

    Science.gov (United States)

    Alcalá Ochoa, Noé; Pérez-Santos, Carlos

    2013-12-15

    Two methods to achieve super-resolution with complex masks displayed in one-phase-only liquid crystal display (LCD) are described. The first method decomposes the complex mask into two phase-only elements with a posterior recombination using an interferometer, and the second method simultaneously encodes the amplitude and the phase by modulating the amplitude of the carrier phase. Experimental results are presented using a liquid crystal on silicon spatial light modulator under reflection.

  2. One-dimensional Fibonacci grating for far-field super-resolution imaging.

    Science.gov (United States)

    Wu, Kedi; Wang, Guo Ping

    2013-06-15

    One-dimensional Fibonacci gratings are used to transform evanescent waves into propagating waves for far-field super-resolution imaging. By detecting far-field intensity distributions of light through objects in front of the Fibonacci grating in free space, we can observe the objects with nearly λ/9 spatial resolution. Analytical results are verified by numerical simulations. We also discuss the effect of sampling error on imaging resolution of the system.

  3. A Bayesian Super-Resolution Approach to Demosaicing of Blurred Images

    OpenAIRE

    Molina Rafael; Katsaggelos Aggelos K; Vega Miguel

    2006-01-01

    Most of the available digital color cameras use a single image sensor with a color filter array (CFA) in acquiring an image. In order to produce a visible color image, a demosaicing process must be applied, which produces undesirable artifacts. An additional problem appears when the observed color image is also blurred. This paper addresses the problem of deconvolving color images observed with a single coupled charged device (CCD) from the super-resolution point of view. Utilizing the Bayes...

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

    OpenAIRE

    Chong Fan; Xushuai Chen; Lei Zhong; Min Zhou; Yun Shi; Yulin Duan

    2017-01-01

    A sub-block algorithm is usually applied in the super-resolution (SR) reconstruction of images because of limitations in computer memory. However, the sub-block SR images can hardly achieve a seamless image mosaicking because of the uneven distribution of brightness and contrast among these sub-blocks. An effectively improved weighted Wallis dodging algorithm is proposed, aiming at the characteristic that SR reconstructed images are gray images with the same size and overlapping region. This ...

  5. Internal Defect Measurement of Scattering Media by Optical Coherence Microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHU Yong-kai; ZHAO Hong; WANG Zhao; WANG Jun-li

    2005-01-01

    Optical coherence microscopy is applied to measure scattering media's internal defect, which based on low coherence interferometry and confocal microscopy. Optical coherence microscopy is more effective in the rejection of out of focus and multiple scattered photons originating further away of the focal plane. With the three-dimension scanning, the internal defect is detected by measuring the thickness of different points on the sample. The axial resolution is 6 μm and lateral resolution is 1.2 μm. This method is possessed of the advantages over the other measurement method of scattering media, such as non-destruction and highresolution.

  6. Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy

    NARCIS (Netherlands)

    Antonello, J.; Werkhoven, T. van; Verhaegen, M.; Truong, H.H.; Keller, C.U.; Gerritsen, H.C.

    2014-01-01

    Optical aberrations have detrimental effects in multiphoton microscopy. These effects can be curtailed by implementing model-based wavefront sensorless adaptive optics, which only requires the addition of a wavefront shaping device, such as a deformable mirror (DM) to an existing microscope. The abe

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

    Directory of Open Access Journals (Sweden)

    Tariq Jamil Saifullah Khanzada

    2011-10-01

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

  8. Bayesian Deconvolution for Angular Super-Resolution in Forward-Looking Scanning Radar

    Directory of Open Access Journals (Sweden)

    Yuebo Zha

    2015-03-01

    Full Text Available Scanning radar is of notable importance for ground surveillance, terrain mapping and disaster rescue. However, the angular resolution of a scanning radar image is poor compared to the achievable range resolution. This paper presents a deconvolution algorithm for angular super-resolution in scanning radar based on Bayesian theory, which states that the angular super-resolution can be realized by solving the corresponding deconvolution problem with the maximum a posteriori (MAP criterion. The algorithm considers that the noise is composed of two mutually independent parts, i.e., a Gaussian signal-independent component and a Poisson signal-dependent component. In addition, the Laplace distribution is used to represent the prior information about the targets under the assumption that the radar image of interest can be represented by the dominant scatters in the scene. Experimental results demonstrate that the proposed deconvolution algorithm has higher precision for angular super-resolution compared with the conventional algorithms, such as the Tikhonov regularization algorithm, the Wiener filter and the Richardson–Lucy algorithm.

  9. A novel super-resolution image fusion algorithm based on improved PCNN and wavelet transform

    Science.gov (United States)

    Liu, Na; Gao, Kun; Song, Yajun; Ni, Guoqiang

    2009-10-01

    Super-resolution reconstruction technology is to explore new information between the under-sampling image series obtained from the same scene and to achieve the high-resolution picture through image fusion in sub-pixel level. The traditional super-resolution fusion methods for sub-sampling images need motion estimation and motion interpolation and construct multi-resolution pyramid to obtain high-resolution, yet the function of the human beings' visual features are ignored. In this paper, a novel resolution reconstruction for under-sampling images of static scene based on the human vision model is considered by introducing PCNN (Pulse Coupled Neural Network) model, which simplifies and improves the input model, internal behavior and control parameters selection. The proposed super-resolution image fusion algorithm based on PCNN-wavelet is aimed at the down-sampling image series in a static scene. And on the basis of keeping the original features, we introduce Relief Filter(RF) to the control and judge segment to overcome the effect of random factors(such as noise, etc) effectively to achieve the aim that highlighting interested object though the fusion. Numerical simulations show that the new algorithm has the better performance in retaining more details and keeping high resolution.

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

    Science.gov (United States)

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

    2011-12-01

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

  11. Infrared chemical imaging: Spatial resolution evaluation and super-resolution concept

    Energy Technology Data Exchange (ETDEWEB)

    Offroy, Marc [Laboratoire de Spectrochimie Infrarouge et Raman, LASIR, CNRS UMR 8516, Bat. C5, Universite des Sciences et Technologies de Lille, 59655 Villeneuve d' Ascq Cedex (France); Roggo, Yves [F. Hoffmann-La Roche A.G., Basel (Switzerland); Milanfar, Peyman [Multi-Dimensional Signal Processing Laboratory, Electrical Engineering Department, Baskin School of Engineering, University of California, 1156 High Street, Mailcode SOE2, Santa Cruz, CA 95064 (United States); Duponchel, Ludovic, E-mail: ludovic.duponchel@univ-lille1.fr [Laboratoire de Spectrochimie Infrarouge et Raman, LASIR, CNRS UMR 8516, Bat. C5, Universite des Sciences et Technologies de Lille, 59655 Villeneuve d' Ascq Cedex (France)

    2010-08-03

    Chemical imaging systems help to solve many challenges in various scientific fields. Able to deliver rapid spatial and chemical information, modern infrared spectrometers using Focal Plane Array detectors (FPA) are of great interest. Considering conventional infrared spectrometers with a single element detector, we can consider that the diffraction-limited spatial resolution is more or less equal to the wavelength of the light (i.e. 2.5-25 {mu}m). Unfortunately, the spatial resolution of FPA spectroscopic setup is even lower due to the detector pixel size. This becomes a real constraint when micron-sized samples are analysed. New chemometrics methods are thus of great interest to overcome such resolution drawback, while keeping our far-field infrared imaging spectrometers. The aim of the present work is to evaluate the super-resolution concept in order to increase the spatial resolution of infrared imaging spectrometers using FPA detectors. The main idea of super-resolution is the fusion of several low-resolution images of the same sample to obtain a higher-resolution image. Applying the super-resolution concept on a relatively low number of FPA acquisitions, it was possible to observe a 30% decrease in spatial resolution.

  12. Dictionary learning based noisy image super-resolution via distance penalty weight model.

    Science.gov (United States)

    Han, Yulan; Zhao, Yongping; Wang, Qisong

    2017-01-01

    In this study, we address the problem of noisy image super-resolution. Noisy low resolution (LR) image is always obtained in applications, while most of the existing algorithms assume that the LR image is noise-free. As to this situation, we present an algorithm for noisy image super-resolution which can achieve simultaneously image super-resolution and denoising. And in the training stage of our method, LR example images are noise-free. For different input LR images, even if the noise variance varies, the dictionary pair does not need to be retrained. For the input LR image patch, the corresponding high resolution (HR) image patch is reconstructed through weighted average of similar HR example patches. To reduce computational cost, we use the atoms of learned sparse dictionary as the examples instead of original example patches. We proposed a distance penalty model for calculating the weight, which can complete a second selection on similar atoms at the same time. Moreover, LR example patches removed mean pixel value are also used to learn dictionary rather than just their gradient features. Based on this, we can reconstruct initial estimated HR image and denoised LR image. Combined with iterative back projection, the two reconstructed images are applied to obtain final estimated HR image. We validate our algorithm on natural images and compared with the previously reported algorithms. Experimental results show that our proposed method performs better noise robustness.

  13. Image super resolution using deep convolutional network based on topology aggregation structure

    Science.gov (United States)

    Yang, Fan; Xu, Wei; Tian, Yapeng

    2017-08-01

    In this paper, we propose a new architecture of the deep convolutional network for single-image super-resolution (SR). Our convolutional network is inspired by GoogLeNet and Res Ne Xt, improved on VDSR which is a representative state-of-the-art method for deep learning-based SR approach. In the field of image super-resolution, we pioneer using the topology aggregation method to improve the network structure. Our network is constructed by repeating the same blocks and each block has the same uniform topology aggregation structure. This design results in reducing the amount of network parameters, so as to increase the depth of the network, thereby enhancing the image super-resolution performance. The design of the network is to take into account both computing performance and practicality. In addition to the performance, the size of model is also important. Experiments show that if the depth of our network is 20 layers, as same as VDSR, our model size is smaller than VDSR 1/3 and the performance is as good as VDSR. Moreover, if we set our model size is as same as VDSR's model size, the depth of our network can be increased to 32 layers, and the performance is better than VDSR.

  14. Bayesian deconvolution for angular super-resolution in forward-looking scanning radar.

    Science.gov (United States)

    Zha, Yuebo; Huang, Yulin; Sun, Zhichao; Wang, Yue; Yang, Jianyu

    2015-03-23

    Scanning radar is of notable importance for ground surveillance, terrain mapping and disaster rescue. However, the angular resolution of a scanning radar image is poor compared to the achievable range resolution. This paper presents a deconvolution algorithm for angular super-resolution in scanning radar based on Bayesian theory, which states that the angular super-resolution can be realized by solving the corresponding deconvolution problem with the maximum a posteriori (MAP) criterion. The algorithm considers that the noise is composed of two mutually independent parts, i.e., a Gaussian signal-independent component and a Poisson signal-dependent component. In addition, the Laplace distribution is used to represent the prior information about the targets under the assumption that the radar image of interest can be represented by the dominant scatters in the scene. Experimental results demonstrate that the proposed deconvolution algorithm has higher precision for angular super-resolution compared with the conventional algorithms, such as the Tikhonov regularization algorithm, the Wiener filter and the Richardson-Lucy algorithm.

  15. Towards the Use of Super-Resolution in Biomedical Systems-on-Chip

    Directory of Open Access Journals (Sweden)

    Gustavo M. Callico

    2013-08-01

    Full Text Available Super-resolution is a smart process capable of generating images with a higher resolution than the resolution of the sensor used to acquire the images. Due to this reason, it has acquired a significant relevance within the medical community during the last years, especially for those specialties closely related with the medical imaging field. However, the super-resolution algorithms used in this field are normally extremely complex and thus, they tend to be slow and difficult to be implemented in hardware. This paper proposes a new super-resolution algorithm for video sequences that, while maintaining excellent levels in the objective and subjective visual quality of the processed images, presents a reduced computational cost due to its non-iterative nature and the use of fast motion estimation techniques. Additionally, the algorithm has been successfully implemented in a low-cost hardware platform, which guarantees the viability of the proposed solution for real-time biomedical systems-on-chip.

  16. Single face image reconstruction for super resolution using support vector regression

    Science.gov (United States)

    Lin, Haijie; Yuan, Qiping; Chen, Zhihong; Yang, Xiaoping

    2016-10-01

    In recent years, we have witnessed the prosperity of the face image super-resolution (SR) reconstruction, especially the learning-based technology. In this paper, a novel super-resolution face reconstruction framework based on support vector regression (SVR) about a single image is presented. Given some input data, SVR can precisely predict output class labels. We regard the SR problem as the estimation of pixel labels in its high resolution version. It's effective to put local binary pattern (LBP) codes and partial pixels into input vectors during training models in our work, and models are learnt from a set of high and low resolution face image. By optimizing vector pairs which are used for learning model, the final reconstructed results were advanced. Especially to deserve to be mentioned, we can get more high frequency information by exploiting the cyclical scan actions in the process of both training and prediction. A large number of experimental data and visual observation have shown that our method outperforms bicubic interpolation and some stateof- the-art super-resolution algorithms.

  17. Time multiplexing super resolution using a 2D Barker-based array

    Science.gov (United States)

    Ilovitsh, Asaf; Ilovitsh, Tali; Preter, Eyal; Levanon, Nadav; Zalevsky, Zeev

    2016-03-01

    We propose the use of a two dimensional Barker-based array in order to improve the performance of the standard time multiplexing super resolution system. The Barker-based array is a 2D generalization of the standard 1D Barker code. It enables achieving a two dimensional super resolution image using only one dimensional scan, by exploiting its unique auto correlation property. A sequence of low resolution images are captured at different lateral positions of the array, and are decoded properly using the same array. In addition, we present the use of a mismatched array for the decoding process. The cross correlation between the Barker-based array and the mismatched array has a perfect peak to sidelobes ratio, making it ideal for the super resolution process. Also, we propose the projection of this array onto the object using a phase-only spatial light modulator. Projecting the array eliminates the need for printing it, mechanically shifting it, and having a direct contact with the object, which is not feasible in many imaging applications. The proposed method is presented analytically, demonstrated via numerical simulation, and validated by laboratory experiments.

  18. Super-resolution infrared time measurement method based on target dynamic characteristics

    Science.gov (United States)

    Li, Bin; Hu, Qiuping; Tang, Zili; Zhang, Sanxi; Zhang, Hua; Yue, Peng; Liu, Biao

    2016-10-01

    High precision time control in the use of weapons and equipment is an important part of product design and development. In order to satisfy the data acquisition requirement of high accuracy and reliability in the rapid flight process, the super-resolution time measurement method based on target dynamic characteristics was put forward and proved by the cabin opening time measurement experiment. First, the changes of explosion pressure wave and image in the cabin opening process were analyzed in detail. The change regulation of explosion flame shape was analyzed by the characteristics of typical pressure wave, and then the high frequency images of the explosion process were shot by high speed camera. The change regulation of the infrared image was obtained through the comparison of visible and infrared image mechanism. Then, combined with the target motion features, and the observed station parameters, the observation model of movement process was built. On the basis of the above research, the infrared characteristic and the movement characteristic were transformed, and the super resolution model was established. For test method, combined with the actual class time measuring process in experimental design, to obtain the special radar for measuring high precision open class time as the true value of the precision appraisal. Experimental results show that the infrared feature and motion feature can realize open class time super resolution measurement, can effectively improve the accuracy and reliability of the data, to achieve specific action of high accuracy measurement that plays an important role by making use of the target dynamic characteristics.

  19. 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

  20. Nonlinear optical microscopy for imaging thin films and surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Smilowitz, L.B.; McBranch, D.W.; Robinson, J.M.

    1995-03-01

    We have used the inherent surface sensitivity of second harmonic generation to develop an instrument for nonlinear optical microscopy of surfaces and interfaces. We have demonstrated the use of several nonlinear optical responses for imaging thin films. The second harmonic response of a thin film of C{sub 60} has been used to image patterned films. Two photon absorption light induced fluorescence has been used to image patterned thin films of Rhodamine 6G. Applications of nonlinear optical microscopy include the imaging of charge injection and photoinduced charge transfer between layers in semiconductor heterojunction devices as well as across membranes in biological systems.