Biomedical Engineering Reference
In-Depth Information
(A)
(B)
Zero-order
wave
Reference wave
k r
Real image
( 1 order)
Illumination
wave r *
z
θ
θ
d i
k o
Hologram
d o
Virtual image
(+1 order)
Recording plane
Figure 5.1
Principle of off-axis (A) recording and (B) reconstruction in DHM. A hologram resulting from the
interference of the reference and object waves is recorded on the camera (in the recording plane)
with a small incident angle,
. For the reconstruction process, the hologram is digitally re-illuminated
and the zero order is filtered to obtain a complete representation of the wave front (both in terms of
intensity and phase) originally emitted by the object. Source: Modified from Ref. [6] .
θ
The observations of quasi-transparent specimens, including living cells, are usually
performed in a transmission implementation providing a quantitative phase signal through
the specimen proportional to the integrated refractive index along each optical path length.
Thus, quantitative phase signals, depending on both the thickness and the refractive index,
provide unique information about the morphology and content of the observed specimen
that can be regarded as a powerful endogenous contrast agent. On the other hand, using
quantitative phase signals can be difficult to interpret underlying biological processes due to
its dual dependence.
In Section 5.2 , we present the principle of classical holography as well as the recent
developments of digital holography as a quantitative phase technique aiming at
exploring cell structure and dynamics. In addition, we address the issues of interpreting
the quantitative phase signal. We present some recent technical developments that allow
us to measure the refractive index and the morphology of living cells. In Section 5.3 ,
we present a series of biological applications to illustrate how specific cell
biophysical parameters, such as absolute volume, morphology shape, transmembrane
water permeability, and membrane fluctuations at the nanometric scale are derived
from the quantitative phase signal and applied to obtain a better understanding of cell
dynamics.
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