Quantitative Retardance Imaging using Quadri-Wave Lateral Shearing Interferometry (QWLSI)

Abstract

We describe the use of quantitative phase imaging to enhance specifically the contrast of ordered components like fibers and membranes inside biological samples. Phase contrast imaging can be considered as a powerful method for the label-free and long-time duration imaging of semi-transparent biological samples. Recent techniques give access to a quantitative measurement of optical thickness. Changes in the refractive index inside the samples can be the main contrast sources in living cells. It is known that some biological structures inside cells are optically anisotropic and thus scatter light differently depending on the illumination light polarization. This property is widely used in polarized light microscopy to reveal ordered structures without staining or labeling but most techniques are purely qualitative or hard to implement when one wants to obtain quantitative measurements on living specimens. Measurement of phase shifts introduced with different incident polarization angles, would give access to quantitative values of both linear retardance and orientation of optical axes. We propose here to use QWLSI to measure a set of polarization-dependent phase shifts, in order to reveal collagen fibers local structure and some components like actin stress fibers in living cells samples. The high-resolution wave front sensor is mounted on a non-modified transmission microscope to measure characteristic optical path difference of the sample [1]. The very simple setup is composed by a single rotating polarizer placed in the illumination light path before the sample that leads to record one quantitative phase image for each excitation polarization angle. The set of those images, recorded in few seconds so as to deal with living samples, is numerically computed to obtain what we call Quantitative Retardance Images which represent the local retardance value of the sample and its optical axis local orientation.