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Chrysanthe Preza

Vol. 17, No. 3 / March 2000 / J. Opt. Soc. Am. A

415

Rotational-diversity phase estimation from differentialinterference-contrast microscopy images

Chrysanthe Preza

Institute for Biomedical Computing, Washington University, 700 South Euclid Avenue, St. Louis, Missouri 63110 Received June 16, 1999; revised manuscript received October 21, 1999; accepted October 29, 1999 An iterative phase-estimation method for the calculation of a specimen’s phase function or optical-path-length (OPL) distribution from differential-interference-contrast (DIC) microscopy images is presented. The method minimizes the least-squares discrepancy measure by use of the conjugate-gradient technique to estimate the phase function from multiple DIC images acquired at different specimen rotations. The estimate is regularized with a quadratic smoothness penalty. Results from testing the method with simulations and measured DIC images show improvement in the estimated phase when at least two rotationally diverse DIC images instead of a single DIC image are used for the estimation. The OPL of a cell that is estimated from two DIC images was found to be much more reliable than the OPL computed from single DIC images (which had a coefficient of variation equal to 15.8%). © 2000 Optical Society of America [S0740-3232(00)02303-6] OCIS codes: 100.5070, 180.6900, 100.6890, 100.3020.

1. INTRODUCTION

Transmitted-light Nomarski differential-interferencecontrast (DIC) microscopy1,2 is widely used to image unstained transparent biological specimens both in motility analysis and for structural studies of biological specimens. DIC is extremely useful in such studies because it enhances specimen edges, gives an aesthetically satisfying bas-relief appearance, and avoids staining, which, in some cases, is toxic to living specimens and thus prohibitive for in vivo applications. DIC is particularly advantageous for time-lapse studies because sufficient light can be acquired in short...