Holographic Microscopy Techniques for Multifocus Phase Imaging of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 93

This chapter focuses on multifocus quantitative phase imaging of living cells with digital
holographic microscopy (DHM). Holographic interferometric metrology is well established
in industrial nondestructive testing and quality control [4 6] . In combination with
microscopy, digital holography provides label-free, quantitative phase imaging [7 12] . The
reconstruction of digitally captured holograms is performed numerically. Thus, in
comparison to other phase contrast methods [13,14] , related interferometry-based
techniques [15 18] , and optical coherence tomography or optical coherence microscopy
[19 24] , DHM provides quantitative phase contrast imaging with subsequent numerical
refocusing (multifocus imaging) from a single recorded hologram.
In an overview, principles of DHM that have been found applicable for quantitative live
cells imaging are presented. First, two experimental arrangements for the recording of
digital off-axis and self-interference holograms are explained which are suitable for the
modular integration of DHM into common research microscopes. Afterward, the numerical
processing of digitally captured off-axis holograms by spatial phase shifting techniques and
the further evaluation of the reconstructed quantitative phase images are explained. This
includes a description of (subsequently) numerical autofocusing and the determination of
the integral refractive index of suspended cells. Then, the application of DHM for
quantitative cell imaging is illustrated by representative results. It is shown that DHM can
be used for monitoring of toxin-mediated changes of the cell morphology and cell
thickness. Furthermore, the analysis of vacuole formation and the imaging of apoptotic and
necrotic effects are illustrated. Finally, the usage of DHM for quantitative monitoring of
cell division and the tracking of cells in a three-dimensional (3D) environment are
demonstrated. The results demonstrate that DHM represents a helpful tool to achieve novel
insights in toxicity testing and cell dynamics.
6.2 DHM Setups for Live Cell Imaging
Various approaches for the implementation of DHM have been developed (for an overview,
see Refs. [25,26] and references therein). For imaging of living cells in a biomedical
laboratory environment, the combination of robust and flexible DHM setups with common
research microscopy systems is advantageous. In this way, the usage of the method and the
combination with other optical and nonoptical imaging methods for live cell imaging are
simplified. Thus, in this section, two different principles for quantitative phase imaging
with DHM are presented that are suitable for the integration into various inverted
microscopes. In order to ensure a vibration insensitive data acquisition, off-axis
arrangements in Mach Zehnder and Michelson interferometer configuration are chosen,
which enable a single-shot acquisition of digital holograms.