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Interferometric computed-microtomography of 3D phase objects

Interferometric computed-microtomography of 3D phase objects

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Vishnyakov, GN Levin, GG Zakarian, CS Russian Research Institute for Optophysical Measurements (Russia) Moscow State Technical Univ. (Russia)

ISBN: (纸本)0819423955

Measurements of refractive index spatial distribution within optically transparent phase 3-D samples with computerized interferomertric microscopes is proposed. For the reconstruction of the phase information the phase shifting interferometry is used, Interferometric computed-tomography microscope are fully automated integrated systems that incorporate CCD camera, frame grabber, computed operated PZT mirror as well as intelligible software for interferogram processing, tomographic reconstruction and refractive index distribution are displayed in 2-D and 3-D, sections, isolines, etc. Experimental results for interferometric microscopy and tomography of blood cells are presented.

关键词： computed tomography 3D microscopy interferometry phase object phase-shifting method refractive index erythrocyte

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Quantitative DIC microscopy using a geometric phase shifter

Quantitative DIC microscopy using a geometric phase shifter

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Cogswell, CJ Smith, NI Larkin, KG Hariharan, P Univ. of Sydney (United States) Univ. of Sydney (Japan) Univ. of Sydney (Australia)

ISBN: (纸本)0819423955

In this paper we investigate the use of a geometric phase-shifting (GPS) technique which allows us to convert conventional transmission or reflection differential interference contrast (DIC) microscopy into a quantitative mode. A phase-shifting algorithm is employed to extract the specimen phase gradient from the mixture of phase and amplitude information which is common in DIC. Fourier techniques are then used to recover the exact phase (i.e. optical path length variations) throughout the biological specimen viewed. In addition to this quantitative ''phase map,'' we demonstrate that the GPS process simultaneously yields an ''amplitude-only'' representation in which-various absorption and transmission properties of the specimen are displayed as intensity variations in the image, similar to brightfield microscopy. These two resulting images can then be analysed or further processed in a number of ways that are not possible with conventional DIC and which improve the microscopist's ability to correctly identify, interpret and measure features in the specimen.

关键词： 3D microscopy interference DIC phase-shifting image processing biomedical microscopy

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Comparison of 3D microscopy methods by imaging a well characterized test object

Comparison of 3D microscopy methods by imaging a well charac...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： McNally, JG Cogswell, CJ Fekete, PW Conchello, JA Washington Univ. (United States) Univ. of Sydney (United States) Univ. of Sydney (Australia)

ISBN: (纸本)0819423955

We used a 10-mu m-diameter fluorescent bead as a test object for 3D microscopy, and independently determined its structure by examining 1-mu m-thick physical sections of the bead. images of the full bead on different 3D microscopes revealed a number of aberrations and distortions. images also showed evidence for absorption and/or scattering on all. confocal and wide-field microscopes tested, but not on a two-photon microscope. The best 3D images of the bead came from deconvolution of either wide-field or certain confocal images. For deconvolution of partially confocal data, a region extending 7 mu m beyond the top and bottom of the bead contained out-of-focus-light information essential for correct restoration. Fully confocal images required 10-fold less computer time for deconvolution, but 1000-fold more excitation light than a wide-field image, even though the resultant restorations were reasonably comparable. Laser imaging of the bead appeared to produce an artifactual image for which we currently have no explanation.

关键词： 3D microscopy comparison confocal wide field two photon

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Tradeoffs in regularized maximum-likelihood image restoration

Tradeoffs in regularized maximum-likelihood image restoratio...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Markham, J Conchello, JA Washington Univ. (United States)

ISBN: (纸本)0819423955

All algorithms for three-dimensional deconvolution of fluorescence microscopical images have as a common goal the estimation of a specimen function (SF) that is consistent with the recorded image and the process for image formation and recording. To check for consistency, the image of the estimated SF predicted by the imaging operator is compared to the recorded image, and the similarity between them is used as a figure of merit (FOM) in the algorithm to improve the specimen function estimate. Commonly used FOM's include squared differences, maximum entropy, and. maximum likelihood (Mi,). The imaging operator is usually characterized by the point-spread function (PSF), the image of a point source of light, or its Fourier transform, the optical transfer function (OTF). Because the OTF is non-zero only over a small region of the spatial-frequency domain, the inversion of the image formation operator is non-unique and the estimated SF is potentially artifactual. Adding a term to the FOM that penalizes some unwanted behavior of the estimated SF effectively ameliorates potential artifacts, but at the same time biases the estimation process. For example, an intensity penalty avoids overly large pixel values but biases the SF to small pixel values. A roughness penalty avoids rapid pixel to pixel variations bur biases the SF to be smooth. In this article we assess the effects of the roughness and intensity penalties on maximum likelihood image estimation.

关键词： 3D microscopy maximum penalized likelihood deconvolution evaluation

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Restoration of three-dimensional quasi-binary images from confocal microscopy and its application to dendritic trees

Restoration of three-dimensional quasi-binary images from co...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Herzog, A Krell, G Michaelis, B Wang, JZ Zuschratter, W Braun, K Otto-von-Guericke Univ. Magdeburg (FRG) and Federal Institute of Neurobiology (Germany)

ISBN: (纸本)0819423955

For the analysis of learning processes and the underlying changes of the shape of excitatory synapses (spines), 3-D volume samples of selected dendritic segments are scanned by a confocal laser scanning microscope. For a :more detailed analysis, such as the classification of spine types, binary images of higher resolution are required. Simple threshold methods have disadvantages for small structures because the microscope point spread function (PSF) causes a darkening and a spread. The direction-dependent PSF leads to shape errors. To reconstruct structures and edge positions with a resolution smaller that one voxel a parametric model for the dendrite and the spines is created. In our application we use the known tree-like structure of the nerve cell as a-priori information. To create the model, simple geometrical elements (cylinders with hemispheres at the ends) are connected. The model can be adapted for size and position in sub-pixel domain. To estimate the quadratic error between the microscope image and the model, the model is sampled with the same resolution as the microscope image and convolved by the microscope PSF. During an iterative process the parameters of the model are optimised. In contrast to other pixel-based methods the number of variable parameters is much slower. The influence of small deviations in the microscope image (caused by the inhomogeneous biological material) is reduced.

关键词： deconvolution binarisation restoration confocal microscopy

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Subpixel resolution in maximum likelihood image restoration

Subpixel resolution in maximum likelihood image restoration

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Conchello, JA McNally, JG Washington Univ. (United States)

ISBN: (纸本)0819423955

A number of algorithms have been developed for three-dimensional (3D) deconvolution of fluorescence microscopical images. These algorithms use a mathematical-physics model for the process of image formation and try estimate the specimen function, i.e. the distribution of fluorescent dye in the specimen. To keep the algorithms tractable and computational load practical, the algorithms rely on simplifying assumptions, and the extent to which these assumptions approximate the actual process of image formation and recording has a strong effect on the capabilities of the algorithms. The process of image formation is a continuous-space process, but the algorithms must be implemented using a discrete-space approximation to this process and render a sampled specimen function. A commonly-used assumption is that there is one pixel in the specimen for each pixel in the recorded image and that the pixel size in the recorded image is small compared to the size of the diffraction limited spot or Airy disk, a condition necessary to satisfy Nyquist sampling criterion. Modern CCD cameras, however, have large wells that integrate into a single pixel an area of the image that is significantly larger that the Airy disk. We derived a maximum-likelihood-based algorithm to accommodate for these large CCD pixel sizes. In this algorithm we assume that each pixel in the recorded image integrates several pixels that satisfy Nyquist criterion. The algorithm then attempts to estimate the specimen function at a resolution better than that allowed by the CCD camera. Preliminary results of this sub-pixel resolution algorithm are encouraging.

关键词： sub-pixel resolution missing information maximum-likelihood expectation-maximization super-resolution

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Forward scattering particle image velocimetry (FSPIV): Application of Mie and imaging theory to measure 3D velocities in microscopy flows using partially coherent illumination and high aperture optics

Forward scattering particle image velocimetry (FSPIV): Appli...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Ovryn, B Khaydarov, JD NASA Lewis Research Ctr. (United States) Ohio Aerospace Institute (United States)

ISBN: (纸本)0819423955

We have combined Mie scattering theory and image theory to predict the forward scattering of light from spherical particles in a seeded fluid using high numerical aperture collection optics. Using this method, it is possible to determine all three components of a fluid's velocity by measuring the scattering from homogeneous spherical particles without moving the optics. The transverse velocity component is determined by following the centroid of the scattering pattern (with respect to time), while the component along the optical axis is determined by comparing the experimental data with numerical computations. We have verified our theoretical model and computer code by measuring the scattering from polystyrene particles illuminated with partially coherent, Koehler illumination in a transmitted light microscope. The three-dimensional scattering data is in quite good agreement with our model. To further verify our approach, we have measured the three-dimensional (parabolic) profile of a parallel flow of a low viscosity, seeded fluid in a straight channel (6 mm x 48 mm x 0.314 mm). The channel was placed on the stage of a conventional microscope equipped with a long working distance microscope objective, with the narrow dimension along the optical axis (OA). Instead of directly imaging the seed particles, the forward scattered light is recorded from the spherical, polystyrene seed particles (7 mu m diameter).

关键词： Particles Scattering Light scattering Interfaces Microscopes Mie scattering Spherical lenses Data modeling Velocity measurements Objectives

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Visualization environment for electron microscopy

Visualization environment for electron microscopy

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Proceedings of the 1997 5th Pacific conference on Computer Graphics and Applications

作者： Boier Martin, Ioana M. Marinescu, Dan C. Indiana Univ South Bend United States

In this paper we describe an environment for visualization and processing of low-dose, low-contrast electron micrographs of biological specimens. We focus on image selection, the first step in the process of reconstruction of the 3D structure of a specimen from its projections. Noise from a variety of sources makes automatic detection of particle positions a difficult task. New image acquisition devices and modern electron microscopy methods require processing and rendering of very large images (50-100 million pixels). We describe techniques for processing large images, algorithms for detecting particle positions on compressed images using the Crosspoint method, and methods for position refinement. EMMA, an interactive visualization environment for experimenting with particle identification methods is presented.

关键词： Three dimensional computer graphics

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Three-dimensional imaging through millimeter thick tissue specimens

Three-dimensional imaging through millimeter thick tissue sp...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Jones, MG Halliwell, M Bull, DR Davies, D Chinyama, C Wells, PNT United Bristol & NHS Trust & Bristol General Hospital (United Kingdom) Univ. of Bristol (United Kingdom) Univ. of Bristol and Southmead Hospital (United Kingdom) Univ. of Bristol and NHS Trust (United Kingdom)

ISBN: (纸本)0819423955

A 780 nm stage-scanned confocal transmission microscope using compact disk (CD) player optics has been constructed and used to study test objects (including scattering effects) and breast tissue specimens (25-1500 mu m thick). Confocal alignment in transmission is achieved using the CD optical mechanism moving coils to scan the objective lens until intensity peaks;the specimen is present but motionless, Intensity variation as a 3D function of mis-alignment of the two foci may allow estimation of optical deterioration caused by the specimen;appropriate confocal apertures and sample spacings can then be selected. Experimental measurements have been supplemented by 2D Monte-Carlo modeling of photon transport for a scattering-limited confocal transmission system. Modeling has characterized: transmission as a function of focal depth in a slab;contrast produced by a range of embedded spherical target radii;and optical mis-alignment. Poor dye perfusion within thick stained specimens results in little visible internal structure, This may be improved using partially phase dependent imaging (congruent to split-detector), sensitive to specimen refractive index variations.

关键词： confocal transmission imaging thick-tissue Monte-Carlo scattering phase-sensitive imaging

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Optimization of the design of a multiple-photon excitation laser scanning fluorescence imaging system.

Optimization of the design of a multiple-photon excitation l...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Wokosin, DL White, JG Univ. of Wisconsin/Madison (United States)

ISBN: (纸本)0819423955

Multi-photon (two or more photon) excitation imaging offers three significant advantages compared to laser-scanning confocal fluorescence microscopy for 3-D and 4-D fluorescence microscopy: considerable reduction in total sample excitation, increased depth penetration, and increased detection sensitivity. All-solid-state ultra-fast lasers offer tremendous potential for affordable, reliable, ''turn-key'' multi-photon excitation sources. We have been developing a multi-photon system that utilizes an all-solid-state Nd:YLF excitation source. We have been evaluating the potential of this source for biological microscopy and have been optimizing system parameters for this application area. We have found that the 1047 nm radiation from these lasers can excite by two-photon fluorescence many commonly used fluorophores that are normally excited from blue to yellow light. In addition, we have found that this wavelength readily excites several normally UV excited fluorophores by the mechanism of three-photon excitation. The Nd:YLF laser has proven reliable in operation with nearly 6000 hours logged without significant loss of power. However, the original system produced rather long pulses for multi-photon excitation (300 fs) and a beam shape that was not ideal. We have recently commissioned the development of an improved pulse compressor from the manufacturers that gives narrower pulses (120 fs), improved beam shape, and a smaller insertion loss. This optimized excitation system has 6 times more potential two-photon excited fluorescence and 22 times more potential three-photon excited fluorescence than the prototype system. In addition, by optimizing coatings in the excitation and signal paths, we have improved the descanned detection sensitivity by 20% for two-photon excited fluorescence and 315% for three-photon excited fluorescence. The excitation optical transfer efficiency (1047 nm) of our imaging system is currently 60% to the back aperture of the objective. The emissio

关键词： 3-D 4-D laser scanning fluorescence microscopy confocal three-photon excitation two-photon excitation non-descanned emission collection

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