Digital Holographic Microscopy (DHM) for Measuring Biophysical Parameters of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 75

5.3.1 Extended Depth of Focus and 3D Tracking

In addition to visualization applications, other groups have taken advantage of the ability
of digital propagation to apply autofocusing [30,122,123] as well as extended depth of
focus [112,124,125] , hence opening the ability to track particles and cells in 3D [126,127]
and offering an alternative to the shallow depth of field of conventional microscopy
which hampers 3D tracking of cells in their environment with a high temporal
resolution. Sheng et al. [128] used DHM to track predatory dinoflagellates in 3D and
reveal prey-induced changes in their swimming behavior in dense suspensions. Using
this technique, they were also able to analyze their response to various stimuli and
toxins [129] .

Three-dimensional tracking with DHM was also extensively used in hematology where the
measurement of blood flow with high spatial and temporal resolutions in a volume is a
challenge in biomedical research fields. Choi and Lee [14] , with a cinematographic
holography technique, continuously tracked individual red blood cells (RBCs) in a
microtube flow to characterize their trajectories as well as their 3D velocity profiles.
Similarly, Sun et al. [45] studied the rapid movement of RBCs in the blood stream of live
Xenopus tadpoles. Both studies took advantage of the extended depth of focusing offered by
holography to track RBCs in 3D after offline reconstruction of multiple focal planes with
high spatial and temporal resolution.

5.3.2 Dry Mass and Cell Cycle

In addition to its noninvasive capability for visualizing and 3D tracking, DHM offers other
advantages when considering the direct interpretation (without requiring any decoupling
procedure) of the phase signal for biological applications. The measured quantitative phase
shift induced by the observed cell on the transmitted light wave front is proportional to the
intracellular refractive index, which largely depends on protein content. Therefore, it can be
directly interpreted to monitor protein production, thanks to a relation established more than
50 years ago by Barer [4,130] within the framework of interference microscopy. The phase
shift induced by a cell is related to its dry mass (DM) by the following equation (converted
to the International System of Units):

ð

10 λ 10 λ

DM 5

S c Δϕ

d s 5

πα ΔϕS c

(5.4)

2 πα 2

where Δϕ is the mean phase shift induced by the whole cell, λ is the wavelength of the

illuminating light source, S c is the projected cell surface and is a constant known as the

specific refraction increment (in cubic meters per kilogram) related to the intracellular

α