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Phase imaging digital holography for biological microscopy

Phase imaging digital holography for biological microscopy

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

作者： Kim, MK Parshall, D Univ S Florida Dept Phys Tampa FL 33620 USA

ISBN: (纸本)0819452327

The digital holographic microscopy is being developed, to offer a number of significant advantages and capabilities in phase imaging. The direct availability of both the amplitude and phase information offers a range of versatile processing techniques that can be applied to the complex field data, including the phase imaging which is particularly straightforward in digital holography. One of the techniques we are developing addresses the problem of 2-pi phase discontinuity in the phase image, where most of the conventional phase unwrapping algorithms require subjective intervention for multi-lambda discontinuity. In the present system, we generate two phase maps by digital holography using two different wavelengths. Numerical superposition of the two phase maps results in a new phase map whose effective wavelength is inversely proportional to the difference of the two wavelengths. The axial range can in principle be arbitrarily large compared to the wavelength, while maintaining axial resolution to a fraction of wavelength. We apply the phase imaging digital holography to a number of systems, including the imaging of thin films and biological cells. Preliminary results from these experiments are presented and future development and applications are discussed.

关键词： phase imaging digital holography phase unwrapping microscopy multi-wavelength imaging

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Wide-Field microscopy using Microcamera Arrays

Wide-Field Microscopy using Microcamera Arrays

引用

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

作者： Marks, Daniel L. Youn, Seo Ho Son, Hui S. Kim, Jungsang Brady, David J. Duke Univ Dept Elect & Comp Engn Durham NC 27708 USA

ISBN: (纸本)9780819493583

A microcamera is a relay lens paired with image sensors. Microcameras are grouped into arrays to relay overlapping views of a single large surface to the sensors to form a continuous synthetic image. The imaged surface may be curved or irregular as each camera may independently be dynamically focused to a different depth. Microcamera arrays are akin to microprocessors in supercomputers in that both join individual processors by an optoelectronic routing fabric to increase capacity and performance. A microcamera may image ten or more megapixels and grouped into an array of several hundred, as has already been demonstrated by the DARPA AWARE Wide-Field program with multiscale gigapixel photography. We adapt gigapixel microcamera array architectures to wide-field microscopy of irregularly shaped surfaces to greatly increase area imaging over 1000 square millimeters at resolutions of 3 microns or better in a single snapshot. The system includes a novel relay design, a sensor electronics package, and a FPGA-based networking fabric. Biomedical applications of this include screening for skin lesions, wide-field and resolution-agile microsurgical imaging, and microscopic cytometry of millions of cells performed in situ.

关键词： Wide-field microscopy camera array image processing agile resolution

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Optimization of spinning disk confocal microscopy: Synchronization with the ultra-sensitive EMCCD

Optimization of spinning disk confocal microscopy: Synchroni...

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

作者： Chong, FK Coates, CG Denvir, DJ McHale, N Thornbury, K Hollywood, M Andor Technol Ltd Belfast BT12 7AL Antrim North Ireland

ISBN: (纸本)0819452327

The advent of Electron Multiplying Charge Coupled Device (EMCCD) technology and it's ability to overcome previous hurdles in low-light fluorescence microscopy, such as phototoxicity to live cells, photobleaching of fluorophores and exposure time restrictions, has resulted in a significant resurgence of interest in use of confocal spinning disk techniques for live cell microscopy. Here provide an understanding of, and technical solutions to, the issues of synchronization that have previously marred the coupling of fast CCD camera technology to confocal spinning disk arrangements. We examine the challenges arising from both old and new models of the Nipkow spinning disk confocal unit and suggest solutions throughout based on a sound comprehension of both (a) relative scan/exposure times;(b) relative orientation of the coupled devices;(c) optimisation of EMCCD clocking parameters.

关键词： CCD EMCCD Nipkow confocal spinning disk confocal microscopy live cell microscopy electron multiplying CCD single photon sensitivity

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Design and performance evaluation of reflection confocal microscopy using acousto-optical deflector and slit detector

Design and performance evaluation of reflection confocal mic...

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

作者： Lee, SW Kang, DK Yoo, HK Gweon, DG Lee, SW Kim, KS Korea Adv Inst Sci & Technol Dept Mech Engn Taejon South Korea

ISBN: (纸本)0819452327

We describe the design and the implementation of reflection confocal scanning microscopy (CSM) using an acousto-optical deflector (AOD) for the fast horizontal scan and a galvanometer mirror (GM) for the slow vertical scan. In the beam scanning system it is important to maintain the lateral and the axial performance during scanning operation. We propose a simple method to design a scanning system using the finite ray tracing and the diffraction theory. We define a cost function which contains the effect of aberrations on the performance of microscopy. We construct the designed system and evaluate its performance. The OSLO simulation shows that the performances of CSM are not changed with deflection angle. So we conclude that the beam scanning system is properly designed. In addition, we propose an image formation method and show images obtained with the system.

关键词： confocal microscopy beam scanning system acousto-optical deflector slit detector finite ray tracing relay optics image formation

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Bright field segmentation tomography (BFST) for use as surface identification in stereomicroscopy

Bright field segmentation tomography (BFST) for use as surfa...

引用

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

作者： Thiesse, J Namati, E de Ryk, J Hoffman, E McLennan, G Univ Iowa Iowa City IA 52242 USA

ISBN: (纸本)0819452327

Stereomicroscopy is an important method for use in image acquisition because it provides a 3D image of an object when other microscopic techniques can only provide the image in 2D. One challenge with this type of imaging is determining the top surface of a sample that has otherwise indistinguishable surface and planar characteristics. We have developed a system that creates a form of oblique illumination and in conjunction with image processing, the top surface can be viewed. The use of BFST consists of the Leica MZ16A stereomicroscope with a unique attached lighting source, and is part of the Large image Microscope Array (LIMA) [1]. The lighting source consists of eight light emitting diodes (LED's) that are separated by 45-degree angles. Each LED in this system illuminates with a 20-degree viewing angle once per cycle with a shadow over the rest of the sample. Subsequently, eight segmented images are taken per cycle. After the images are captured they are stacked through image addition to achieve the full field of view, and the surface is then easily identified. image processing techniques, such as skeletonization, can be used for further enhancement and measurement. With the use of BFST, advances can be made in detecting surface features from metals to tissue samples, such as in the analytical assessment of pulmonary emphysema using mean linear intercept.

关键词： stereomicroscopy illumination surface identification image processing

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Transmission confocal microscopy: making it a reality

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

作者： Arnison, MR Fekete, PW Serrano, M Nguyen, PTA Romagnoli, R Guan, L O'Byrne, JW Cogswell, CJ Univ Sydney Sch Phys Dept Phys Opt Sydney NSW 2006 Australia

ISBN: (纸本)0819427004

Imaging thick specimens in 3D transmission confocal modes presents two key problems. The first problem is variable aberrations introduced by changes in refractive index. The second problem is revealed when visualising acquired data, where thick 3D datasets are difficult to interpret. In this paper we present our emerging solutions to these problems. Aberrations can be classified as simple tip-tilt deflection of the beam, or more complicated higher order aberrations. We discuss our results which demonstrate successful on-the-fly detection and correction for tip-tilt For detecting higher order aberrations, we have chosen to investigate the wavefront curvature sensing technique. The second problem of rendering thick 3D datasets can be solved by extracting features of interest from the background. Simple intensity threshholding is not sufficient for complex biological specimens, and image processing in only two dimensions neglects any three dimensional structure. Use of Kohonen's self-organising map neural network in 3D results in clear segmentation of features for sample chromosome specimens.

关键词： 3D microscopy confocal transmission DIC adaptive optics neural network image processing

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Intensity-based segmentation and visualization of cells in 3D microscopic images using the GPU

Intensity-based segmentation and visualization of cells in 3...

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

作者： Kang, Mi-Sun Lee, Jeong-Eom Jeon, Woong-Ki Choi, Heung-Kook Kim, Myoung-Hee Ewha Womans Univ Dept Comp Sci & Engn Seoul 120750 South Korea Inje Univ UHRC Sch Comp Engn Gyeong 621749 South Korea

ISBN: (纸本)9780819493583

3D microscopy images contain abundant astronomical data, rendering 3D microscopy image processing time-consuming and laborious on a central processing unit (CPU). To solve these problems, many people crop a region of interest (ROI) of the input image to a small size. Although this reduces cost and time, there are drawbacks at the image processing level, e. g., the selected ROI strongly depends on the user and there is a loss in original image information. To mitigate these problems, we developed a 3D microscopy image processing tool on a graphics processing unit (GPU). Our tool provides efficient and various automatic thresholding methods to achieve intensity-based segmentation of 3D microscopy images. Users can select the algorithm to be applied. Further, the image processing tool provides visualization of segmented volume data and can set the scale, transportation, etc. using a keyboard and mouse. However, the 3D objects visualized fast still need to be analyzed to obtain information for biologists. To analyze 3D microscopic images, we need quantitative data of the images. Therefore, we label the segmented 3D objects within all 3D microscopic images and obtain quantitative information on each labeled object. This information can use the classification feature. A user can select the object to be analyzed. Our tool allows the selected object to be displayed on a new window, and hence, more details of the object can be observed. Finally, we validate the effectiveness of our tool by comparing the CPU and GPU processing times by matching the specification and configuration.

关键词： GPU CUDA 3D microscopic image processing visualization

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Extended depth-of-field microscopy

Extended depth-of-field microscopy

引用

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

作者： Smith, Christopher Botcherby, Edward Booth, Martin Juskaitis, Rimas Wilson, Tony Univ Oxford Dept Engn Sci Oxford OX1 3PJ England

ISBN: (纸本)9780819479662

In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a near-isotropic scan rate. This means that the focal spot can be scanned at high speed along any direction in the specimen, without introducing systematic aberrations. We present experimental point spread function measurements for this system using an Olympus 1.4 NA 60X oil immersion lens that demonstrates an axial range of operation greater than 70 mu m. We give details of a novel actuator device used to displace the focusing element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present an application of this system in liquid crystal research to image the dynamic response of a nematic device during switching. Information about the director field at different levels in the device can be inferred from images acquired with a temporal resolution of 2.5 ms.

关键词： optical refocusing two-photon microscopy

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Gabor wavelet transform for dynamic analysis in digital holographic microscopy

Gabor wavelet transform for dynamic analysis in digital holo...

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

作者： Zhong, Jingang Weng, Jiawen Hu, Cuiying Jinan Univ Inst Optoelect Engn Guangzhou 510632 Guangdong Peoples R China

ISBN: (纸本)9780819479662

Real time dynamic analysis of micro-object is one of the significant advantages of digital holographic microscopy, but the process of the spatial filtering limits the application of dynamic and automatic analysis in digital holographic microscopy. In this paper, a numerical reconstruction technique by means of Gabor wavelet transform (GWT) is presented for real time dynamic analysis. Appling the GWT to the hologram, the quantitative phase information of the micro-object is obtained automatically without the process of the spatial filtering. An experiment with a sequence of holograms of an animalcule is proposed for the dynamic and automatic analysis by employing the GWT method.

关键词： Digital holographic microscopy dynamic analysis wavelet transform full-field quantitative phase imaging

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Optical characterization of microfabricated solid immersion lenses

Optical characterization of microfabricated solid immersion ...

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

作者： Fletcher, DA Crozier, KB Goodson, KE Quate, CF Kino, GS Stanford Univ Ginzton Lab Stanford CA 94305 USA

ISBN: (纸本)081943535X

Circumventing the optical diffraction limit is important for data storage, photolithography, and imaging. The Solid Immersion Lens (SIL), in which light is focussed through a high refractive-index lens held close to a sample, offers a method for improving resolution. Microfabrication of SILs enables the use of lenses as small as a few wavelengths in diameter. When these are integrated onto cantilevers, the SIL can be scanned over topography with control of lens-sample separation. We have extended Mie theory to investigate aberration in small lenses and the effect of lens size on a converging beam. We also report progress on the fabrication and first results from micromachined silicon SILs.

关键词： microlens microfabrication solid immersion lens scanning probe microscopy

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