<title>Quantitative phase contrast microscopy of living cells by numerical reconstruction of digital holograms</title>

Abstract

ABSTRACT We describe a new method ofoptical microscopy which allows simultaneously intensity and quantitative phase contrastimaging. The method is based on a new holographic imaging technique which involves a CCD camera as recording device anda numerical method for the reconstruction. An application to phase contrast imaging of living astrocytes is presented.Keywords: Holography, CCD camera, Phase contrast microscopy, Astrocytes, Cellular microscopy. 1. INTRODUCTION Optical phase contrast microscopy is widely used in biology for cellular imaging. Although, they are efficient forimaging purposes, standards techniques, such as the Zernicke or the Nomarski methods, do not provide phase contrast imageswhich map quantitatively the exact distribution of the optical path length at the surface of the specimen and these techniquescannot be directly used for applications in optical metrology such as surface profilometry or refractive index measurements.With Zernicke microscopy the obtained images suffer from two optical artifacts; the so-called hallo and shading-offeffects1 which enhance the specimen visibility but make correct quantitative measurements impracticable. With Nomarskimicroscopy,2 also called Differential Interference Contrast (DIC) microscopy, the contrast depends on the gradient of a phasevariation and precise quantitative measurement are possible but require the use of sophisticated techniques for the signalacquisition and processing.3For applications in optical metrology, interference microscopy techniques4 have been developed. Most of them arebased on phase-shifting inteferometry (PSI) and require the recording of four interferograms for appropriate phase steps (900)of the reference wave. On the basis of these four acquired interferograms, an amplitude contrast image and a phase contrastimage of the specimen can be obtained. In comparison with the Zernicke or the Nomarski methods, the obtained phase contrastis absolute and quantitative, meaning that the obtained phase distribution is equal, modulo 2m, to the distribution of the optical