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

作者： Yoo, Hongki Song, Incheon Kim, Taehoon Gweon, Daegab Korea Adv Inst Sci & Technol Dept Mech Engn Lab Nano Optomechatron Taejon 305701 South Korea Hanyang Univ Dept Biomed Engn Hanyang Peoples R China

ISBN: (纸本)0819461326

Recently, stimulated emission depletion microscopy has achieved high resolution in fluorescent imaging. In this paper, we present effects of a pupil filter on the performances of stimulated emission depletion microscopy. In stimulated emission depletion microscopy, a saturated zero-centered spot is usually used to achieve a high lateral resolution. Using a half-coated phase plate, a zero-centered spot was made with a narrow and steep gap at the center. Numerical and experimental results show that by simply inserting a central obstacle as a pupil filter, it is possible to reduce the central gap of the zero-centered spot. However in order to compensate inevitable loss of light, which is blocked by the obstacle, increased laser power is required.

关键词： STED microscopy confocal microscopy high resolution pupil filter phase plate annular aperture

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Quantitative phase-contrast microscopy by angular spectrum digital holography

Quantitative phase-contrast microscopy by angular spectrum d...

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

作者： Mann, C. J. Kim, M. K. Univ S Florida Dept Phys Tampa FL 33620 USA

ISBN: (纸本)0819461326

Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating the holographic optical field is seen to have several advantages over the more commonly used Fresnel transformation or Huygens convolution method. Spurious noise and interference components can be tightly controlled through the analysis and filtering of the angular spectrum. The reconstruction distance does not have a lower limit and the off-axis angle between the object and reference can be lower than the Fresnel requirement and still be able to cleanly separate out the zero-order background. Holographic phase images are largely immune from the coherent noise common in amplitude images. Together with the use of a miniature pulsed laser, the resulting images have 0.5 mu m diffraction-limited lateral resolution and the phase profile is accurate to about 30 nm of optical path length. SKOV-3 (ovarian cancer cells) and HUVEC (human umbilical vein endothelial cells) are imaged that display intra-cellular and intra-nuclear organelles with clarity and quantitative accuracy. The technique clearly exceeds currently available methods in phase-contrast optical microscopy in the level of resolution and detail, and provides a new modality for imaging morphology of cellular and intracellular structures that is not currently available.

关键词： digital holography angular spectrum phase microscopy

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Measuring the surface topography at focal adhesions using laser feedback interferometry

Measuring the surface topography at focal adhesions using la...

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

作者： Bambino, Michael Ovryn, Ben Copper Union Adv Sci & Art Dept Engn Mech Cooper Sq New York NY 10003 USA Albert Einstein Coll Med Dept Anat & Struct Biol Bronx NY 10467 USA

ISBN: (纸本)0819461326

Reflection interference contrast microscopy of living cells has not yet fully matured. One goal would be to temporally resolve the distance between the cell and the substratum at each point over the cell surface. We have combined phase shifting laser feedback interferometry with a high numerical aperture inverted microscope in order to determine the topography of the ventral surface of a cell. We have obtained a map of both the topography of a cell as well as its reflectivity. Our data demonstrate that interference microscopy can be adapted to yield a measure of the distance between the cell and the substratum. We have quantified the ventral surface topology at focal adhesions and we have shown that these changes are correlated with markers for a focal adhesion adaptor protein. The laser feedback interferometer was used to determine the ventral surface topography of fixed metastatic mammary adenocarcinoma cells. The ventral surface of the cell was scanned by moving the sample with a piezoelectric stage. The height of the ventral surface, as well as the reflectivity, were determined using phase shifting interferometry. An overlay of a fluorescence image with the interference data shows that the prominent dark regions of the interference image correlate with the location of the paxillin.

关键词： Reflectivity Microscopes Visibility Geometrical optics Luminescence microscopy Interfaces Interferometers Interferometry Laser interferometry

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Algorithms for extracting true phase from rotationally-diverse and phase-shifted DIC images

Algorithms for extracting true phase from rotationally-diver...

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

作者： Preza, Chrysanthe King, Sharon V. Cogswell, Carol J. Washington Univ Dept Elect & Syst Engn 1 Brookings DrBox 1127 St Louis MO 63130 USA Univ Colorado Dept Elect & Comp Engn Boulder CO 80309 USA

ISBN: (纸本)0819461326

In this paper, we report on the status of our current algorithms and extensions for improved algorithms for extracting phase from images acquired with differential-interference-contrast (DIC) microscopy. Our algorithms are based on two different approaches for the computation of a specimen's phase function or optical path length (OPL) distribution from DIC images. The first approach uses an iterative phase-estimation method that minimizes the I-divergence discrepancy measure using the conjugate-gradient technique to estimate the OPL from multiple DIC images acquired at different specimen rotations. The method is based on the assumption that the specimen does not absorb light. The second approach is a non-iterative method that is based on a geometric-optics model and the phase-shifting technique that allows separation of the amplitude and phase gradient information from DIC images thereby allowing computation of the desired phase from its gradient. We show results from both methods and discuss the tradeoff between complexity (with respect to data-acquisitiona and computation) and accuracy. Our long term goal is to develop a new and improved method based on a combination of our two approaches.

关键词： DIC (Nomarski) microscopy phase estimation phase shifting

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En-face optical coherence imaging for three-dimensional microscopy

En-face optical coherence imaging for three-dimensional micr...

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Conference on 3-dimensional and multidimensional microscopy: image acquisition and processing IX

作者： Akiba, M Chan, KP Tanno, N Japan Sci & Technol Corp Reg Joint Res Project Yamagata 9902473 Japan

ISBN: (纸本)0819443603

En-face optical coherence tomography (OCT) employing parallel heterodyne detection technique is demonstrated for three-dimensional microscopy. To enable the use of commercially available CCD cameras as two-dimensional heterodyne detector arrays in OCT imaging, a frequency synchronous detection method is Employed. Depth-resolved, full-field images are acquired at 30 Hz video-rate without lateral scanning.

关键词： three-dimensional microscopy optical coherence tomography frequency synchronous detection

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Data compression in microscopy: a comparative study

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 158-168页

作者： Kriete, Andres Klein, Norbert Berger, Lars C. Inst of Anatomy and Cell Biology Giessen Germany

ISBN: (纸本)0819430757;9780819430755

image compression in microscopy is a valuable technique, in particular if applied to multidimensional data. Information-preserving and competitive information-losing compression is applied to microscopical data and the resulting image quality is evaluated on a quantitative basis both in the spatial and frequency domain. Included are image data featuring different signal-to-noise ratios, but also voxel data for volume representations as well as graphical data for surface representations. The effect of compression on 3-D visualization and image management with data bases is included.

关键词： image compression

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High-resolution three-dimensional imaging using multiple nanometric probes

High-resolution three-dimensional imaging using multiple nan...

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing X

作者： Dubois, A Moneron, G Lecaque, R Lequeux, F Boccara, AC Ecole Super Phys & Chim Ind Ville Paris CNRS UPR A0005 Lab Opt Phys F-75231 Paris 5 France

We present a new imaging modality using nanometric beads as multiple local probes. The positions of the beads are determined in three dimensions using white-light interference microscopy, by over-sampling and fitting ... 详细信息

ISBN: (纸本)0819447641

关键词： interference microscopy three-dimensional microscopy scanning probe microscopy

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Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D

Simultaneous imaging of several focal planes in fluorescence...

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

作者： Prabhat, Prashant Ram, Sripad Ward, E. Sally Ober, Raimund J. Univ Texas SW Med Ctr Ctr Immunol NB9-1066000 Harry Hines Blvd Dallas TX 75235 USA Univ Texas Dallas Dept Elect Engn Richardson TX 75083 USA Univ Texas Dallas Joint Biomed Engn Grad Program Richardson TX 75083 USA

ISBN: (纸本)0819461326

Fluorescence microscopy of live cells is an important tool to investigate cellular trafficking pathways. The existing microscope design is very well suited to image fast moving vesicles, tubules and organelles in one focal plane. More problematic is the imaging of cellular components that move between different focal planes. This is due to the fact that tracking of such cellular components requires that the focal plane of the microscope be changed. This has to be done with a focusing device, which is relatively slow. More importantly, only one focal plane can be imaged at a time. Therefore, while the cell is imaged at one focal plane, important events could be missed at other focal planes. To overcome these shortcomings, we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. In this design, the emission light collected by a single stationary objective lens is split into multiple channels. Light in each channel is focused on a CGD camera by a tube lens. By ensuring that the camera position with respect to the tube lens focal plane position is not the same in any two channels, distinct planes within the specimen can be simultaneously imaged. Here we discuss the implementation of a configuration with which four focal planes can be imaged simultaneously.

关键词： fluorescence microscopy three-dimensional (3-D) tracking multi-focal plane imaging

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Closing the spectral gap - the transition from fixed-parameter fluorescence to tunable devices in confocal microscopy.

Closing the spectral gap - the transition from fixed-paramet...

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

作者： Borlinghaus, Rolf Gugel, Hilmar Albertano, Patrizia Seyfried, Volker Leica Microsyst CMS Mannheim Germany Univ Rome Dept Biol Rome Italy

ISBN: (纸本)0819461326

Modem microscopy in life sciences is ruled by development and exploration of new dyes and stains (probes for histochemical staining, quantum dots, fluorescent proteins etc.) on one side, and technological improvements and innovations for fluorescence microscopy - especially high resolution and optical sectioning microscopy - on the other side. Concerning the technical innovations, several ingenious inventions have been made available for confocal microscopy. First, the acousto optical tunable filter, which allows switching and dimming of laser lines. Second the spectral detector, employing mirror sliders in front of the detectors which allow continuous tuning of the spectral emission band detected by the sensor. Third, the most challenging task: a substitute to the classical beam splitter - the device which is restricting fluorescence microscopy most. This was solved by introduction of the acousto optical beam splitter. The very last device which is still lacking flexibility is the laser source, operating only at non-equidistant frequencies and requiring a set of quite different laser sources as gas lasers, solid state lasers or diode lasers. A new approach by supercontinuum light sources is presented and discussed, which significantly enhances flexibility and coverage of the excitation spectra of typical, rare and natural fluorochromes.

关键词： confocal spectral white laser supercontinuum AOBS cyanobacteria excitation-emission correlation microscopy sensitivity multi-parameter fluorescence

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Biological cell (pollen grain) refractive index tomography with digital holographic microscopy

Biological cell (pollen grain) refractive index tomography w...

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

作者： Charriere, Florian Cuche, Etienne Marquet, Pierre Depeursinge, Christian Ecole Polytech Fed Lausanne Imaging & Appl Opt Inst CH-1015 Lausanne Switzerland Lyncee Tec SA PSE A CH-1015 Lausanne Switzerland CHU Vaudois Ctr Neurosci Psychiat Dept Psychiat CH-1008 Lausanne Switzerland

ISBN: (纸本)0819461326

This paper present a novel approach to perform the tomography of biological specimen based on Digital Holographic microscopy (DHM). A hologram results from the interference between a reference wave and an object wave reflected from or transmitted through a sample. In the hologram, both amplitude and phase of the field transmitted through the object are registered. In DHM, the object field is recovered when the hologram is processed by a digitally computed replica of the reference wave, allowing quantitative measurement of both phase and amplitude. Phase measurements provide high accuracy optical path length measurements across the specimen along the optical axis. To proceed to a tomographic reconstruction of the refractive index of the sample based on this quantitative phase measurement, such 2-dimentionnal data must be recorded for different sample orientations covering an angle of 180 degrees to cover all the object spatial frequencies in the reciprocal space. The representation of the data in function of the angle is known as a sinogram. The 3-dimentionnal refractive index can then be reconstructed from the sinograms by a filtered backprojection algorithm. In our system, the specimen is inserted in a glass micropipette to permit its rotation. To our knowledge, a quantitative tomography of the refractive index of a pollen cell with a resolution in the micron range is presented for the first time.

关键词： holography microscopy phase-contrast tomography refractive index cell imaging

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