Sample-less calibration of the differential interference contrast microscope

Abstract

Analysis of image formation in a differential interference contrast (DIC) microscope and retrieval of the specimen's properties require calibration of its key parameters, viz. shear and bias. We present a method of measuring the shear and the bias of a DIC microscope from the interference fringes produced in the back focal plane of the objective. Previous approaches, which use calibrated specimens such as polystyrene or fluorescent beads, provide rather approximate measurements of shear or require a complex optical setup. The method presented is accurate, relies on simple image analysis, and does not require a specimen. We provide a succinct and accurate description of properties of Nomarski prisms to explain the rationale behind the method. © 2010 Optical Society of America.