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Determination of direction-independent optical path-length distribution of cells using rotational-diversity transmitted-light differential interference contrast (dic) images

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Conference on 3-Dimensional and Multidimensional microscopy - Image Acquisition and Processing V

作者： Preza, C van Munster, EB Aten, JA Snyder, DL Rosenberger, FU Washington Univ Inst Biomed Comp St Louis MO 63130 USA

ISBN: (纸本)0819427004

Two different phase-estimation methods that have been developed for the computation of the optical path-length (OPL) distribution of a specimen from dic images are compared in this paper. The first method is based on the Wiener filter approach and uses only a single dic image for the determination of the OPL distribution. The second phase-estimation method is based on the conjugate-gradient optimization method and estimates the OPL distribution using rotational-diversity dic images;i.e. multiple dic images obtained by rotating the specimen. For this study, 24 different dic images of a single bovine spermatozoa head were acquired by rotating the cell by approximately 15 degrees between images. The images were registered and aligned using fiducial marks, and then processed with both methods. Results obtained with the filtering method were found to be dependent on the orientation of the cell with respect to the shear direction. Comparison of the integrated optical path length (IOPL) computed with the the filtering method and the rotational-diversity method using two, four and eight dic images at different rotation angles showed that the IOPL estimated with the rotational-diversity method is less dependent on the rotation angle, even when only two images separated by 90-degree cell rotation are used for the phase estimation. Our results show that the use of rotational-diversity images in the determination of the OPL distribution is very beneficial because it overcomes the directional dependence of dic imaging.

关键词： computational nomarski dic microscopy phase estimation optical path length

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Phase estimation from transmitted-light dic images using rotational diversity

Phase estimation from transmitted-light DIC images using rot...

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Conference on Image Reconstruction and Restoration II

作者： Preza, C Snyder, DL Rosenberger, FU Markham, J Conchello, JA Washington Univ. (United States)

ISBN: (纸本)0819425923

Differential-Interference-Contrast (DIG) microscopy is a powerful technique for the visualization of unstained transparent specimens, thereby allowing in vivo observations. Quantitative interpretation of dic images is difficult because the measured intensity is nonlinearly related to the gradient of a specimen's optical-path-length distribution along the shear direction. The recent development of reconstruction methods for dic microscopy permits the calculation of a specimen's optical-path-length distribution or phase function and provides a new measurement technique for biological applications. In this paper we present a summary of our work on quantitative imaging with a dic microscope. The focus of our efforts has been in two areas: 1, model development and testing for 3D dic imaging;and 2, development of a phase-estimation method based on this model. Our method estimates a specimen's phase function using rotational-diversity dic images, i.e. multiple dic images obtained by rotating the specimen. Test objects were viewed with a conventional dic microscope using monochromatic light, and images were recorded using a cooled CCD camera. Comparison of the images to model predictions show good qualitative and quantitative agreement. Results obtained from testing the phase-estimation method with 2D simulations and with measured dic images demonstrate that an estimate of an object's phase function can be obtained even from a single dic image and that the estimated phase becomes quantitatively better as the number of rotational-diversity dic images increases.

关键词： computational nomarski dic microscopy imaging theory phase estimation image reconstruction 3D microscopy

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