Phase estimation from transmitted-light DIC images using rotational diversity

Differential-Interference-Contrast (DIC) 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 t..

Determination of Direction-Independent Optical Path-Length Distribution of Cells Using Rotational-Diversity Transmitted-Light Differential Interference Contrast (DIC) Images

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) com..