<title>Quantitative interferometric imaging using a conventional differential interference contrast microscope</title>

Abstract

Interference- and phase-contrast microscopy are techniques developed to visualize transparent objects without the incorporation of stains, allowing observations on living cells. The development of CCD cameras and image analysis technology in the last decades permits quantitative analysis of interferometric data, obtained by interference microscopy. A method is presented to determine the optical pathlength at each position in digitized microscopic images. Unstained cells and artificial test objects were observed under a standard, commercially available, Nomarski differential interference contrast (DIC) microscope, using monochromatic light. A cooled CCD camera was used to record images. Image processing software was used to convert the measured intensities to phase differences. A wiener filter based on the typical lateral shift introduced by the DIC microscope, was applied to reconstruct the original optical pathlength distribution. This system, based upon a conventional DIC microscope, rather than upon a purpose build interferometric scanning system, allows easier determination of optical pathlength differences. After image acquisition and determination of optical pathlength distribution, analysis of cell parameters can be performed using standard image processing software. Dry mass- or DNA content, for instance, can be estimated by integrating the optical pathlength over the cellular surface.