Digital Holographic Microscopy (DHM) for Measuring Biophysical Parameters of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 67

CHAPTER 5
Digital Holographic Microscopy (DHM)
for Measuring Biophysical Parameters of
Living Cells

Benjamin Rappaz 1 , Christian Depeursinge 2 , Pierre Marquet 1 , 3

1

Laboratory of Neuroenergetics and Cellular Dynamics, Brain and Mind Institute, Ecole Polytechnique
F´d´rale de Lausanne (EPFL), Lausanne, Switzerland 2 Microvision and Microdiagnostic Group, STI,
Ecole Polytechnique F´d´rale de Lausanne (EPFL), Lausanne, Switzerland 3 D´partement de
Psychiatrie-CHUV, Centre des Neurosciences Psychiatriques, Site de Cery, Prilly, Switzerland

Editor: Natan T. Shaked

5.1 Introduction

Recording noninvasive high-resolution 3D images of living cells in real time remains a
technical challenge. Most biological cells are transparent, i.e., they differ only slightly from
their surroundings in terms of optical properties (including absorbance, reflectance, etc.)
which precludes the generation of adequate contrast required to obtain high-resolution video
microscopy images.

Consequently, developing methods to access the phase information for imaging purposes
has been investigated for a long time. In practice, phase contrast (PhC), initially proposed
by Zernike and presented in detail in Chapter 1, and Nomarski's differential interference
contrast (DIC), discussed in detail in Chapter 2, are two widely used PhC-generating
techniques available for high-resolution light microscopy. In contrast to fluorescence
technique, PhC and DIC are noninvasive and allow the visualization of transparent
specimens, particularly the fine subcellular structural organization, without using any
staining contrast agent. However, despite their high sensitivity, PhC and DIC do not allow
the direct and quantitative measurement of phase shift or optical path length. Consequently,
the interpretation of minute DIC or PhC signal variations in terms of quantitative
modification of specific biophysical cell parameters including cell volume remains difficult.
More recently, efforts have been made to employ DIC as well as PhC as a quantitative