Digital Holographic Microscopy (DHM) for Measuring Biophysical Parameters of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 74

( 2 ) 5 n m,2 ), to achieve separate measurements of the intracellular refractive

index and the cell morphology in real time [40] .

λ 1 ) 6¼ n m ( λ

5.3 Biological Applications

DHM provides new ways for noninvasive contactless and label-free quantitative PhC
recording to enable the visualization of transparent living cells. In combination with the
unique possibilities presented by digital means (real-time imaging, extended depth of focus,
etc.) several original applications have been performed in the field of cell imaging.
However, although providing unique information about cell morphology and intracellular
content, the quantitative phase signal, due to its dual origin, could remain difficult to
interpret in terms of biological processes. Consequently, in this part we will present a
selection of relevant new applications of digital holography in the field of cell biology with
a special emphasis on the interpretation of the phase signal. For specific applications, a
description of which important biophysical parameters can be extracted from the phase
signal will be provided as well as how they can contribute to bring a new understanding of
specific biological mechanisms.

Straightforward applications with DHM exploit the ability of the technique to generate
contrast from transparent specimens in order to image them and hence offer a quantitative
alternative to PhC or DIC. For instance, M ¨ lder et al. [36] compared a DHM to the
conventional manual cell counting method using a hemocytometer and showed that
digital holography can be used in noninvasive automatic cell counting as precisely as
conventional manual cell counting, thereby offering a faster and easier alternative.
Applications in reproduction research include the development of a holographic set-up to
improve the visualization and detection of cow spermatozoa [118] or to compare the
quantitative phase signal of human sperm heads in normozoospermia (normal sperm)
and oligoasthenozoospermia (reduced sperm motility and low count) [119] . Hence,
proposing that quantitative evaluation of the phase shift recorded by DHM could
provide new information on the exact structure and composition of the sperm head
could be useful for clinical practice. Other recent developments involved combining
digital holography with a total internal reflection set-up [120] , allowing QPM of
cell substrate interfaces and quantifying focal adhesions at the cellular membrane.

Similarly, Pache et al. [121] studied the cellular modifications of the actin cytoskeleton
in microgravity using a DHM. Such results were previously obtainable only with the
use of labeled probes in conjunction with conventional fluorescence microscopy,
with all the classically described limitations in terms of bias, bleaching, and temporal
resolution.