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检索条件"任意字段=Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX"

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Tomographic STED microscopy to study bone resorption 22

Tomographic STED microscopy to study bone resorption

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Deguchi, Takahiro Koho, Sami V. Nareoja, Tuomas Peltonen, Juha Hanninen, Pekka Univ Turku Inst Biomed Dept Cell Biol & Anat Lab Biophys SF-20500 Turku Finland Univ Turku Medic Res Labs SF-20500 Turku Finland Karolinska Inst Dept Neurosci Stockholm Sweden Univ Turku Inst Biomed Dept Cell Biol & Anat SF-20500 Turku Finland

ISBN: (纸本)9781628414202

We present a tomographic Stimulated Emission Depletion (STED) microscopy method with three-dimensional super-resolution, and its application to osteoclast bone resorption study. In order to improve axial resolution in standard STED system by tomography, two axial projections were obtained by imaging a sample at two different angles;one conventionally from below and another from the side. The second observation was acquired via a metal-coated silicon mirror, positioned above the region of interest by a custom-built micro-positioner. The acquired two sets of 3D stacks were computationally registered and fused, with our own in-house-developed software, to produce a 3D tomogram with three-dimensional super-resolution. With the presented tomographic super-resolution method we optically investigated actin cytoskeleton through thin and smooth bone layer, particularly at ruffled boarders (RB), which are directly associated with active bone resorption in osteoclasts. Tomographic STED microscopy at RB of osteoclast, cultured on thin bone layer, demonstrated axial resolution of approx. 210 nm, revealing fine axial structures of actin cytoskeleton at RB. Further investigation of the cytoskeleton at RB in relation with associated proteins would provide understanding in the protein roles during the bone resorption.

关键词： fluorescence STED super-resolution tomography osteoclast bone

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Computational modeling of STED microscopy through multiple biological cells under one- and two-photon excitation 22

Computational modeling of STED microscopy through multiple b...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Mark, Andrew E. Davis, Mitchell A. Starosta, Matthew S. Dunn, Andrew K. Univ Texas Austin Dept Elect & Comp Engn Austin TX 78712 USA Sandia Natl Labs Albuquerque NM 87185 USA Univ Texas Austin Dept Biomed Engn Austin TX 78712 USA

ISBN: (纸本)9781628414202

While superresolution optical microscopy techniques afford enhanced resolution for biological applications, they have largely been used to study structures in isolated cells. We use the FDTD method to simulate the propagation of focused beams for STED microscopy through multiple biological cells. We model depletion beams that provide 2D and 3D confinement of the fluorescence spot and assess the effective PSF of the system as a function of focal depth. We compare the relative size of the STED effective PSF under one- and two-photon excitation. PSF calculations suggest that imaging is possible up to the maximum simulation depth if the fluorescence emission remains detectable.

关键词： STED microscopy superresolution computational modeling FDTD method light scattering

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Resolving power in direct oblique plane imaging 22

Resolving power in direct oblique plane imaging

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Kim, Jeongmin Li, Tongcang Wang, Yuan Zhang, Xiang NSF Nanoscale Sci & Engn Ctr Berkeley CA 94720 USA Purdue Univ Dept Phys & Astron W Lafayette IN 47907 USA Univ Calif Berkeley Lawrence Berkeley Natl Lab Div Mat Sci Berkeley CA 94720 USA

ISBN: (纸本)9781628414202

Direct oblique plane imaging is a high-speed microscopy technique that observes a sample's plane that is inclined to the focal plane of the microscope objective lens. This wide-field microscopy is suitable for a study of fast dynamics of living samples where the principle plane of interest is tilted to the focal plane. A way to implement this imaging technique is to use remote focusing together with a tilted mirror, which involves asymmetrical pupil function of the imaging system. We rigorously study the anisotropic resolving power of the oblique plane imaging using a vectorial diffraction theory. From the derived effective pupil function, we calculate vectorial point spread function (PSF) and optical transfer function (OTF). We show that the two-dimensional (2D) PSF of the direct oblique plane imaging is not merely an oblique cross-section of the 3D PSF of circular aperture system. Similarly, 2D OTF of the oblique plane imaging is different from 2D oblique projection of conventional 3D OTF in circular aperture system.

关键词： Oblique plane imaging oblique plane microscopy vectorial diffraction theory asymmetric pupil function point spread function optical transfer function optical resolution remote focusing

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3D widefield light microscope image reconstruction without dyes 22

3D widefield light microscope image reconstruction without d...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Larkin, S. Larson, J. Holmes, C. Vaicik, M. Turturro, M. Jurkevich, A. Sinha, S. Ezashi, T. Papavasiliou, G. Brey, E. Holmes, T. Lickenbrock Technol LLC St Louis MO USA IIT Chicago IL 60616 USA Univ Missouri Columbia MO 65211 USA

ISBN: (纸本)9781628414202

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample's 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

关键词： three dimensional phase contrast live imaging image reconstruction light microscopy quantitative phase imaging phase imaging

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A block-based forward imaging model for improved sample volume representation in computational optical sectioning microscopy 22

A block-based forward imaging model for improved sample volu...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Ghosh, Sreya Preza, Chrysanthe Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA

ISBN: (纸本)9781628414202

In typical fluorescence imaging systems the refractive index (RI) variability between the immersion medium of the objective lens, the coverslip, and the specimen, changes the spherical wave-front of the emitted light and introduces spherical aberrations (SA) in the acquired 3D image. In existing computational optical sectioning algorithms (COSM) to simplify the complexity of the problem, the specimen is either assumed to be thin or in the case of depth-variant algorithms to have a constant RI which is an invalid assumption for biological samples. Accurate modeling of biological samples requires a space variant (SV) imaging system i.e. a different point spread functions (PSF) for each pixel. To reduce the computational load an approximate block-based forward model is introduced in this study. The entire object space is divided into a collection of small 3D blocks where the PSFs at the faces of the blocks are known. An optimized combination of overlap-save and overlap-add methods of interpolation are used to obtain the final SV (axially and laterally variant) image. Simulated SV images using the new imaging model, of a numerical object comprising of similar structures dispersed in a medium with spatially variant RI are discussed. images of fluorescent microspheres (6-mu m in diameter) dispersed in a controlled sample with two distinct RIs are compared to simulated images of a numerical object subjected to the same imaging condition, to evaluate the new model. The accuracy of the block-based forward model to model the effect of space variance within a specimen was assessed using intensity profiles through the microspheres. The qualitative similarities in the appearance of the experimental and simulated image indicate the validity of the block-based forward model to appropriately model samples with lateral variability in RI.

关键词： 3D modeling Point spread functions 3D image processing microscopy Statistical modeling Spherical lenses Confocal microscopy Imaging systems Computer simulations Data modeling

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High Brightness LED in Confocal microscopy 22

High Brightness LED in Confocal Microscopy

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Vakili, Ali Xiong, Daxi Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Boston MA 02115 USA Chinese Acad Sci Suzhou Inst Biomed Engn & Technol Jiangsu Key Lab Med Opt Suzhou Peoples R China Mem Sloan Kettering Canc Ctr New York NY USA

ISBN: (纸本)9781628414202

We have introduced a novel illumination system for line scanning confocal microscopy. Confocal microscopy is a popular imaging tool in many applications specifically in medical imaging. Line scanning confocal microscopes have been proven to provide images with resolution comparable to point scanning microscopes. In the point scanning microscopes, the light is focused onto a diffraction limited spot. A pinhole is placed conjugate to the diffraction limited spot, in front of the detector to reject the light coming from out-of-focus planes. Therefore, confocal microscopy can provide optical sectioning. The size of the pinhole determines the amount of light that reaches the detector. A large pinhole results in a blurry image since more of the out-of-focus light contribute to the image. On the other hand, a smaller pinhole rejects more of the light, leading to a lower signal-to-noise ratio. Ideally it is desired to deliver a larger amount of optical power to the diffraction limited spot to increase the signal-to-noise ratio and have a smaller pinhole to reject more of the out-of-focus light. This is the property of the illumination system. In order to get a good signal-to noise ratio in the image, the light source has to provide sufficient radiance. We have introduced a new illumination system utilizing a high brightness LED in the line scanning confocal microscope. High brightness LEDs provide more optical power compared to ordinary LEDs from a smaller area;they have higher radiance. Preliminary results from our line scanning confocal microscope show that the high brightness LED is able to provide enough radiance to obtain an image with resolution comparable with the same microscope utilizing the laser diode. However, in high frame-rate application higher radiance or lower-noise detection system is required.

关键词： High Brightness LED Confocal Line-scan Radiance

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Standing-wave excitation of fluorescence in a laser-scanning microscope allows precise contour mapping of the red blood cell membrane 22

Standing-wave excitation of fluorescence in a laser-scanning...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Amor, Rumelo Mahajan, Sumeet Amos, William Bradshaw McConnell, Gail Univ Strathclyde Strathclyde Inst Pharm & Biomed Sci Ctr Biophoton Glasgow G4 0RE Lanark Scotland Univ Southampton Inst Life Sci Southampton SO17 1BJ Hants England Univ Southampton Dept Chem Southampton SO17 1BJ Hants England MRC Mol Biol Lab Cambridge CB2 2QH England

ISBN: (纸本)9781628414202

We demonstrate fluorescence excitation at multiple planes in a laser-scanning microscope by using the standing wave from a mirror placed close to the specimen. We have observed precise modulation of the standing waves close to a mirror, with a frequency proportional to the Stokes shift, corresponding to a moire pattern between the excitation and emission standing-wave fields. We use standing-wave excitation to plot the exact contour maps of the red blood cell membrane, with an axial resolution of approximate to 90 nm. The method may prove useful in the study of diseases which involve the surface membrane of red blood cells.

关键词： Fluorescence microscopy standing-wave excitation laser-scanning cell membrane

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Multi-channel beam-scanning imaging at kHz frame rates by Lissajous trajectory microscopy 22

Multi-channel beam-scanning imaging at kHz frame rates by Li...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Newman, Justin A. Sullivan, Shane Z. Muir, Ryan D. Sreehari, Suhas Bouman, Charles A. Simpson, Garth J. Purdue Univ Dept Chem W Lafayette IN 47907 USA Purdue Univ Elect & Comp Engn W Lafayette IN 47907 USA

ISBN: (纸本)9781628414202

A beam-scanning microscope based on Lissajous trajectory imaging is described for achieving streaming 2D imaging with continuous frame rates up to 1.4 kHz. The microscope utilizes two fast-scan resonant mirrors to direct the optical beam on a circuitous trajectory through the field of view. By separating the full Lissajous trajectory time-domain data into sub-trajectories (partial, undersampled trajectories) effective frame-rates much higher than the repeat time of the Lissajous trajectory are achieved with many unsampled pixels present. A model-based image reconstruction (MBIR) 3D in-painting algorithm is then used to interpolate the missing data for the unsampled pixels to recover full images. The MBIR algorithm uses a maximum a posteriori estimation with a generalized Gaussian Markov random field prior model for image interpolation. Because images are acquired using photomultiplier tubes or photodiodes, parallelization for multi-channel imaging is straightforward. Preliminary results show that when combined with the MBIR in-painting algorithm, this technique has the ability to generate kHz frame rate images across 6 total dimensions of space, time, and polarization for SHG, TPEF, and confocal reflective birefringence data on a multimodal imaging platform for biomedical imaging. The use of a multi-channel data acquisition card allows for multimodal imaging with perfect image overlay. image blur due to sample motion was also reduced by using higher frame rates.

关键词： Lissajous multi-channel beam-scanning nonlinear optics second harmonic generation two-photon excited fluorescence image interpolation

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A software tool for STED-AFM correlative super-resolution microscopy 22

A software tool for STED-AFM correlative super-resolution mi...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXII

作者： Koho, Sami Deguehi, Takahiro Lohmus, Madis Nareoja, Tuomas Hanninen, Pekka E. Univ Turku Biophys Lab FI-20520 Turku Finland Karolinska Inst Dept Neurosci SE-17177 Stockholm Sweden

ISBN: (纸本)9781628414202

Multi-modal correlative microscopy allows combining the strengths of several imaging techniques to provide unique contrast. However it is not always straightforward to setup instruments for such customized experiments, as most microscope manufacturers use their own proprietary software, with limited or no capability to interface with other instruments - this makes correlation of the multi-modal data extremely challenging. We introduce a new software tool for simultaneous use of a STimulated Emission Depletion (STED) microscope with an Atomic Force Microscope (AFM). In our experiments, a Leica TCS STED commercial super-resolution microscope, together with an Agilent 5500ilm AFM microscope was used. With our software, it is possible to synchronize the data acquisition between the STED and AFM instruments, as well as to perform automatic registration of the AFM images with the super-resolution STED images. The software was realized in LabVIEW;the registration part was also implemented as an imageJ script. The synchronization was realized by controlling simple trigger signals, also available in the commercial STED microscope, with a low-cost National Instruments USB-6501 digital I/O card. The registration was based on detecting the positions of the AFM tip inside the STED field-of-view, which were then used as registration landmarks. The registration should work on any STED and tip-scanning AFM microscope combination, at nanometer-scale precision. Our STED-AFM correlation method has been tested with a variety of nanoparticle and fixed cell samples. The software will be released under BSD open-source license.

关键词： AFM STED super-resolution correlative imaging microscopy open-source software

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Quantitative imaging of intact cardiac tissue using remote focusing microscopy 22

Quantitative imaging of intact cardiac tissue using remote f...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXII

作者： Corbett, A.D. Burton, R.A.B. Bub, G. Wilson, T. Department of Engineering Science University of Oxford Parks Road OxfordOX1 3PJ United Kingdom Department of Physiology Anatomy and Genetics University of Oxford South Parks Road OxfordOX1 3QX United Kingdom

ISBN: (纸本)9781628414202

Remote focussing microscopy offers many advantages when acquiring volumetric data from living tissue. The all-optical means of refocussing does not agitate the specimen by moving either the stage or imaging objective. Aberration compensated imaging extends over volumes as large as 450 μm × 450 μm × 200 μm (X, Y and Z) allowing data to be collected from hundreds of cells. The speed with which refocussing can be achieved is limited only by the mechanical movement of a small (2 mm diameter) mirror. Using a pair of oblique imaging planes to rapidly acquire ( © 2015 SPIE.

关键词： Calibration

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