Calibrating Differential Interference Contrast Microscopy with dual-focus Fluorescence Correlation Spectroscopy

Abstract

We present a novel calibration technique for determining the shear distance of a Nomarski Differential Interference Contrast prism, which is used in Differential Interference Contrast microscopy as well as for the recently developed dual-focus fluorescence correlation spectroscopy. In both applications, an exact knowledge of the shear distance induced by the Nomarski prism is important for a quantitative data evaluation. In Differential Interference Contrast microscopy, the shear distance determines the spatial resolution of imaging, in dual-focus fluorescence correlation spectroscopy, it represents the extrinsic length scale for determining diffusion coefficients. The presented calibration technique is itself based on a combination of fluorescence correlation spectroscopy and dynamic light scattering. The method is easy to implement and allows for determining the shear distance with nanometer accuracy.