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检索条件"任意字段=Conference on 3-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX"

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Optimal Pupil Design for Confocal microscopy

Optimal Pupil Design for Confocal Microscopy

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XVII

作者： Patel, Yogesh G. Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA Memorial Sloan Kettering Cancer Ctr Dept Dermatol New York NY 10022 USA

ISBN: (纸本)9780819479662

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided pupil may offer better sectioning and contrast. We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fill-factor, h, the ratio of the 1/e(2) diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02 (full) and 0.52 (half). Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in comparing the experimental performance of the different configurations.

关键词： confocal microscopy Fourier optics point-scanning line-scanning full-pupil divided-pupil

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Comparison of Optical Quadrature microscopy and Shack-Hartmann Wavefront Sensor

Comparison of Optical Quadrature Microscopy and Shack-Hartma...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Patel, Yogesh G. Warger, William C., II Ballesta, Jerome DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Imagine Optic SA F-914000 Orsay France

ISBN: (纸本)9780819474308

Several quantitative phase imaging techniques, such as digital holography, Hilbert-phase microscopy, and phase-shifting interferometry have applications in biological and medical imaging. Quantitative phase imaging measures the changes in the wavefront of the incident light due to refractive index variations throughout a 3-D specimen. We have developed a multimodal microscope which combines optical quadrature microscopy (OQM) and a Shack-Hartmann wavefront sensor for applications in biological imaging. OQM is an interferometric imaging modality that noninvasively measures the amplitude and phase of a signal beam that travels through a transparent specimen. The phase is obtained from interferograms with four different delayed reference wavefronts. The phase is then transformed into a quantitative image of optical path length difference. The Shack-Hartmann wavefront sensor measures the gradient of the wavefront at various points across a beam. A microlens array focuses the local wavefront onto a specific region of the CCD camera. The intensity is given by the maximum amplitude in the region and the phase is determined based on the exact pixel position within the region. We compare the amplitude and quantitative phase information of poly-methyl-meth-acrylate (PMMA) beads in oil and one-cell and two-cell mouse embryos with micrometer resolution using OQM and the Shack-Hartmann. Each pixel in OQM provides a phase measurement, whereas multiple pixels are used in Shack-Hartmann to determine the tilt. Therefore, the simple Shack-Hartmann system is limited by its resolution and field-of-view. Real-time imaging in Shack-Hartmann requires spatial averaging which smoothes the edges of the PMMA beads. The OQM has a greater field-of-view with good resolution;however, it is a complex system requiring multiple optical components and four cameras which may introduce additional artifacts in processing quantitative images. The OQM and Shack-Hartmann has certain advantages depending on

关键词： optical quadrature microscopy quantitative phase imaging shack-hartmann wavefront sensor

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Dual objective fluorescence microscopy for single molecule imaging applications

Dual objective fluorescence microscopy for single molecule i...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Ram, Sripad Prabhat, Prashant Ward, E. Sally Ober, Raimund J. Univ Texas SW Med Ctr Dallas Dept Immunol Dallas TX 75390 USA

ISBN: (纸本)9780819474308

Fluorescence microscopy is an invaluable tool for studying biological processes in cells. In the recent past there has been significant interest in imaging cellular processes at the single molecule level. Single molecule experiments remove ensemble averaging effects and provide information that is typically not accessible through bulk experiments. One of the major requirements in single molecule imaging applications is that a sufficient number of photons be detected from the single molecule. This is not only important for the visual identification of single molecules, but also plays a crucial role in the quantitative analysis of the acquired data. Here, we demonstrate the use of a dual objective imaging configuration for single molecule studies. The configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. The use of opposing objective lenses has been previously demonstrated in 4pi confocal microscopy and I5M to achieve high axial resolution when compared to confocal/widefield microscopes. Here we demonstrate that the dual objective imaging configuration provides higher photon collection efficiency when compared to a regular microscope for a given illumination condition. As a result, single molecules can be localized with better accuracy when imaged through opposing objective lenses than when imaged through a regular optical microscope. Analytical tools are introduced to estimate the 2D location of single molecules and to characterize the accuracy with which they can be determined.

关键词： Multifocal plane microscopy single molecule tracking localization accuracy Fisher information matrix

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A novel phase shifting structured illumination microscopy 13

A novel phase shifting structured illumination microscopy

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXIII

作者： Singh, Veena Dubey, Vishesh Ahmad, Azeem Singh, Gyanendra Mehta, D. S. Indian Inst Technol Delhi Dept Phys New Delhi 110016 India Indian Inst Technol Delhi Instrument Design & Dev Ctr New Delhi 110016 India

ISBN: (纸本)9781628419474

This paper describes a new and novel phase shifting technique for qualitative as well as quantitative measurement in microscopy. We have developed a phase shifting device which is robust, inexpensive and involves no mechanical movement. In this method, phase shifting is implemented using LED array, beam splitters and defocused projection of Ronchi grating. The light from the LEDs are made incident on the beam splitters at spatially different locations. Due to variation in the geometrical distances of LEDs from the Ronchi grating and by sequentially illuminating the grating by switching on one LED at a time the phase shifted grating patterns are generated. The phase shifted structured patterns are projected onto the sample using microscopic objective lens. The phase shifted deformed patterns are recorded by a CCD camera. The initial alignment of the setup involves a simple procedure for the calibration for equal fringe width and intensity such that the phase shifted fringes are at equal phase difference. Three frame phase shifting algorithm is employed for the reconstruction of the phase map. The method described here is fully automated so that the phase shifted images are recorded just by switching of LEDs and has been used for the shape measurement of microscopic industrial objects. The analysis of the phase shifted images provides qualitative as well as quantitative information about the sample. Thus, the method is simple, robust and low cost compared to PZT devices commonly employed for phase shifting.

关键词： Ronchi grating LEDs phase shifting three step phase shifting algorithm quantitative phase imaging

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Computational Model of Optical Scattering by Elastin in Lung

Computational Model of Optical Scattering by Elastin in Lung

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Swedish, Tristan B. Robinson, Joseph P. Silva, Maricris R. Gouldstone, Andrew Kaeli, David DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 360 Huntington Ave Boston MA 02115 USA Northeastern Univ CenSSIS Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA

ISBN: (纸本)9780819484413

Little is understood about the detailed micromechanical properties of lung in vivo. Attempts to improve imaging are hampered by heterogeneity of the tissue. One common ex vivo technique is optical coherence tomography (OCT). Simulated OCT with a Finite-Difference Time-Domain (FDTD) computer model elucidates the relationship between captured images and the physical geometry of the lung. Parallel computation and improved processing power make accurate coherent imaging models feasible. A previous FDTD model of pulsed laser wave propagation in the lung produced images that displayed many of the properties of experimental images. The model was improved with the addition of elastin and increased computational volume. Elastin plays an important role in the simulation because the combination of its fibrous structure and high index of refraction acts as an excellent scatterer of light. This strong scattering increases the signal reported by the simulated OCT scan in areas where elastin is most abundant, improving visualization of the structure as more light is reflected back from the heterogeneous elastin network. However, scattering by elastin decreases the depth of penetration and leads to images that are more difficult to interpret. Gaining a better understanding of how lung structures affect light propagation will lead to improved signal processing, instrumentation, and the development of new probing techniques. This image modeling technique can also be applied to other imaging modalities such as confocal and other laser scanning methods.

关键词： Optical Coherence Tomography (OCT) Confocal Reflectance microscopy (CRM) Computational microscopy Lung Alveolar Structure Elastin Finite Difference Time Domain (FDTD)

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image processing in 3-D standing-wave fluorescence microscopy

Image processing in 3-D standing-wave fluorescence microscop...

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

作者： Krishnamurthi, V Bailey, B Lanni, F CARNEGIE MELLON UNIV CTR LIGHT MICROSCOPE IMAGING & BIOTECHNOLDEPT PHYSBIOMED ENGN PROGRAMPITTSBURGHPA 15213

ISBN: (纸本)0819420298

Standing-wave fluorescence microscopy, a method which utilizes interference to create a periodic excitation pattern along the optical axis, has been shown to provide improved axial resolution in thin, fluorescently labeled specimens. In each plane of focus, a complete standing wave data set is obtained by acquiring an image at each of three distinct positions of the interference fringes. Thicker specimens require through-focus data consisting of three images per plane. In this report we describe the recovery of information from this data using 3D image processing. The effective optical transfer function (OTF) of the standing wave microscope consists of the conventional OTF and two sidebands which are copies of the conventional OTF shifted axially by the spatial frequency of the interference fringes. The large gaps between the central band and the sidebands lead to significant ringing in the 3D reconstruction if linear deconvolution methods are employed. The use of non-linear, constrained image processing techniques has been shown to allow accurate extrapolation outside the OTF band limit. We demonstrate the extent to which the sidebands enhance recovery of information in the gaps, and provide a comparison between deconvolution using inverse-filtering and maximum-likelihood estimation.

关键词： Composites Fermium Frequency modulation Optical transfer functions image processing Luminescence Microscopes microscopy 3D image processing Point spread functions

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Investigating the usage of point spread functions in point source and microsphere localization 13

Investigating the usage of point spread functions in point s...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXIII

作者： Chao, Jerry Ram, Sripad Ward, E. Sally Ober, Raimund J. Texas A&M Univ Dept Biomed Engn College Stn TX 77843 USA Texas A&M Hlth Sci Ctr Dept Mol & Cellular Med College Stn TX 77843 USA Univ Texas Dallas Dept Elect Engn Richardson TX 75080 USA Texas A&M Hlth Sci Ctr Dept Microbial Pathogenesis & Immunol College Stn TX 77843 USA

ISBN: (纸本)9781628419474

Using a point spread function (PSF) to localize a point-like object, such as a fluorescent molecule or microsphere, represents a common task in single molecule microscopy image data analysis. The localization may differ in purpose depending on the application or experiment, but a unifying theme is the importance of being able to closely recover the true location of the point-like object with high accuracy. We present two simulation studies, both relating to the performance of object localization via the maximum likelihood fitting of a PSF to the object's image. In the first study, we investigate the integration of the PSF over an image pixel, which represents a critical part of the localization algorithm. Specifically, we explore how the fineness of the integration affects how well a point source can be localized, and find the use of too coarse a step size to produce location estimates that are far from the true location, especially when the images are acquired at relatively low magnifications. We also propose a method for selecting an appropriate step size. In the second study, we investigate the suitability of the common practice of using a PSF to localize a microsphere, despite the mismatch between the microsphere's image and the fitted PSF. Using criteria based on the standard errors of the mean and variance, we find the method suitable for microspheres up to 1 mu m and 100 nm in diameter, when the localization is performed, respectively, with and without the simultaneous estimation of the width of the PSF.

关键词： Cramer-Rao lower bound Fisher information localization accuracy maximum likelihood estimation microsphere pixel integration point spread function single molecule microscopy

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Confocal photoluminescence microscopy in II-VI materials: annealing and degradation dynamics

Confocal photoluminescence microscopy in II-VI materials: an...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing VI

作者： McCabe, EM Jordan, C Fewer, DT Donegan, JF Taniguchi, S Hino, T Nakano, K Ishibashi, A Uusimaa, P Pessa, M Univ Dublin Trinity Coll Dublin 2 Ireland

ISBN: (纸本)0819430757

Confocal photoluminescence imaging is an important tool in the investigation of recombination in semiconductors and in the characterisation of material growth. This characterisation is particularly important for II-VI wide band-gap semiconductors where the potential for blue-green lasers is being explored currently. To achieve room-temperature cw operation of these lasers over the multi-thousand hours necessary for commercialisation, extremely low defect densities are required. The confocal microscope is used in this work to image photoluminescence from II-VI materials to characterise the defect formation and propagation within the quantum well region of the material. This imaging approach permits the degradation to be monitored in real time and over a large area in samples with low defect densities. The additional advantages of this set-up over a conventional microscope are, of course, the higher lateral resolution and narrow depth of field associated with a confocal microscope. While considerable effort has been focused on the degradation in these II-VI semiconductors, we have recently observed that annealing can occur simultaneously in the same sample when the material is exposed to intense optical excitation. images of annealing and degradation of a range of II-VI samples will be presented to highlight these observations.

关键词： confocal microscopy photoluminescence imaging semiconductors II-VI materials blue green lasers

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New Light Field Camera based on Physical Based Rendering Tracing

New Light Field Camera based on Physical Based Rendering Tra...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXI

作者： Chung, Ming-Han Chang, Shan-Ching Lee, Chih-Kung Natl Taiwan Univ Inst Appl Mech Taipei Taiwan

ISBN: (纸本)9780819498625

Even though light field technology was first invented more than 50 years ago, it did not gain popularity due to the limitation imposed by the computation technology. With the rapid advancement of computer technology over the last decade, the limitation has been uplifted and the light field technology quickly returns to the spotlight of the research stage. In this paper, PBRT (Physical Based Rendering Tracing) was introduced to overcome the limitation of using traditional optical simulation approach to study the light field camera technology. More specifically, traditional optical simulation approach can only present light energy distribution but typically lack the capability to present the pictures in realistic scenes. By using PBRT, which was developed to create virtual scenes, 4D light field information was obtained to conduct initial data analysis and calculation. This PBRT approach was also used to explore the light field data calculation potential in creating realistic photos. Furthermore, we integrated the optical experimental measurement results with PBRT in order to place the real measurement results into the virtually created scenes. In other words, our approach provided us with a way to establish a link of virtual scene with the real measurement results. Several images developed based on the above-mentioned approaches were analyzed and discussed to verify the pros and cons of the newly developed PBRT based light field camera technology. It will be shown that this newly developed light field camera approach can circumvent the loss of spatial resolution associated with adopting a micro-lens array in front of the image sensors. Detailed operational constraint, performance metrics, computation resources needed, etc. associated with this newly developed light field camera technique were presented in detail.

关键词： Physical Based Rendering Tracing PBRT Light field Ray tracing image processing

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Preprocessing method to correct illumination pattern in sinusoidal-based structured illumination microscopy 25

Preprocessing method to correct illumination pattern in sinu...

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conference on Three-dimensional and multidimensional microscopy: image acquisition and processing XXV

作者： Shabani, H. Doblas, A. Saavedra, G. Preza, C. Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA Univ Valencia Dept Opt E-46100 Burjassot Spain

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614840;9781510614833

The restored images in structured illumination microscopy (SIM) can be affected by residual fringes due to a mismatch between the illumination pattern and the sinusoidal model assumed by the restoration method. When a Fresnel biprism is used to generate a structured pattern, this pattern cannot be described by a pure sinusoidal function since it is distorted by an envelope due to the biprism's edge. In this contribution, we have investigated the effect of the envelope on the restored SIM images and propose a computational method in order to address it. The proposed approach to reduce the effect of the envelope consists of two parts. First, the envelope of the structured pattern, determined through calibration data, is removed from the raw SIM data via a preprocessing step. In the second step, a notch filter is applied to the images, which are restored using the well-known generalized Wiener filter, to filter any residual undesired fringes. The performance of our approach has been evaluated numerically by simulating the effect of the envelope on synthetic forward images of a 6-ium spherical bead generated using the real pattern and then restored using the SIM approach that is based on an ideal pure sinusoidal function before and after our proposed correction method. The simulation result shows 74% reduction in the contrast of the residual pattern when the proposed method is applied. Experimental results from a pollen grain sample also validate the proposed approach.

关键词： structured illumination microscopy sinusoidal pattern residual fringe Fresnel biprism

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