Biomedical Engineering Reference
In-Depth Information
21-35 DIV
Biph.
RD
ID
Figure 5.4
Application of glutamate triggers three main types of phase shifts. Averaged traces of the three
characteristic optical signals induced by glutamate application (30
M, 30 s, arrowhead) and
recorded from 21 to 35 DIV (mature) neurons. These phase signals are classified into three
categories: a biphasic response (Biph., n
μ
137), a reversible decrease of the phase signal (RD,
5
n
80), and an irreversible decrease of the phase signal (ID, n
35). Scale bar: horizontal, 2 min;
vertical, 5 . Source: Modified from Ref. [155] .
5
5
In a recent study, Pavillon et al. [37] correlated the calcium dynamics with phase
measurements. Furthermore, we assessed the cell viability at the end of the experiment with
propidium iodide (PI), a fluorescent dye relying on the loss of membrane permeability
following necrotic mechanisms.
Most importantly, the neuronal cell volume regulation, accurately monitored using the high
sensitivity of DHM quantitative phase signal, has permitted us to predict within a time
frame of tens of minutes, whether a subsequent neuronal death would occur [156]
( Figure 5.5 ).
5.3.7 Future Perspectives
As illustrated through these various applications, DHM allows developing a QPM
specifically well adapted to study cell structure and dynamics. In practice, low energy
levels and short exposure time are required to prevent any photo-damage even during long
experiments. In addition, DHM, as a label-free technique, does not require any solution
change or insertion of dye, providing efficient conditions for high-throughput screening.
Although the quantitative phase signal provides unique information about cell morphology
and content with a high sensitivity, its interpretation in terms of biophysical parameters in
analyzing specific biological mechanisms remains an issue. Within this framework,
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