Digital Holographic Microscopy (DHM) for Measuring Biophysical Parameters of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 72

smooth topographic measurements for phase recovery [108] and generalized for use in digital
holographic microscopy (DHM) with amplitude and phase recovery [98] . The main
characteristic of this approach is its capability of recovering the complex object wave through
only one acquisition, thus greatly reducing the influence of vibrations. However, as the
diffraction terms are spatially encoded in the hologram, this one shot capability comes
potentially at the cost of usable bandwidth. In addition, the frequency modulation, induced by
the angle between the reference and the object waves, has to guarantee the separability of the
information contained in the different diffraction terms that are encoded in the hologram
while carrying a frequency compatible with the sampling capacity of digital detectors.
Despite the fact that digital detectors have a sampling capability significantly lower than
photosensitive plates, their use in off-axis digital holography microscopy does not represent a
drawback. Rather, the microscope objective (MO) introduced in the interferometer allows the
wavefield to adapt to the sampling capacity of the camera: the lateral components of the
wave vector k x or y are divided by the magnification factor M of the MO, therefore permitting
the wavefield to be adequately sampled by the electronic camera and to obtain a
reconstructed quantitative phase image with a diffraction-limited lateral resolution [34,98] .

As mentioned earlier, holographic measurements often rely on recording the object wave in a
nonimaging plane such as in lensless holography. Consequently, it is necessary to propagate
the recovered object wave resulting from the filtered spectrum of the hologram, to retrieve a
focus image. Depending on the distance between the recording position and the focus plane,
different approximations are commonly used for numerical implementation of the object
wave propagation [98,109 111] . The digital propagation can be considered as one of the

major advantages provided by DH, as it enables off-line autofocusing [30] , as well as extends
the depth of focus, enabling the reconstruction of different focal planes from a single
hologram [112] . In addition, the demodulation of the filtered spectrum, resulting from the off-
axis geometry, as well as aberration compensation can be efficiently associated with the
numerical calculation of the object wave propagation [98,113 115] . A priori, any type of

aberration, including apparent spherical aberration resulting from the mismatch between the
reference and the object waves, can be numerically compensated enabling optimization of
imaging capabilities and making the use of simplified interferometric set-ups possible. In
summary, the development of digital means has greatly changed the research field in
holography leading to the development of various simplified interferometric configurations
and numerical reconstruction procedures well suited to explore many different fields, whose
scope extends beyond that of this chapter.

5.2.4 Cell Imaging and Quantitative Phase Signal Interpretation

In the field of cell imaging, transparent cells are probed by quantitatively measuring the
phase retardation that they induce on the transmitted wave front. This phase signal is related
to the biophysical cell parameter: