Minimizing scattering-induced phase errors in differential interference contrast microscopy

Abstract

Significance: Differential interference contrast (DIC) microscopes allow noninvasive in vivo observation of transparent microstructures in tissue without the use of fluorescent dyes or genetic modification. We show how to modify a DIC microscope to measure the sample phase distribution accurately and in real-time even deep inside sample tissue. Aim: Our aim is to improve the DIC microscope's phase measurement to remove the phase bias that occurs in the presence of strong scattering. Approach: A quarter-wave plate was added in front of the polarization camera, allowing a modified phase calculation to incorporate all four polarization orientation angles (0 deg, 45 deg, 90 deg, and 135 deg) captured simultaneously by the polarization camera, followed by deconvolution. Results: We confirm that the proposed method reduces phase measurement error in the presence of scattering and demonstrate the method using in vivo imaging of a beating heart inside a medaka egg and the whole-body blood circulation in a young medaka fish. Conclusions: Modifying a polarization-camera DIC microscope with a quarter-wave plate allows users to image deep inside samples without phase bias due to scattering effects.