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) 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.