Digital Holographic Microscopy (DHM) for Measuring Biophysical Parameters of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 81

phase signal changes mainly reflect intracellular refractive index variations [37,41] .
Consequently, within this framework, the phase signal will be predominantly sensitive to any
mechanism modifying the concentration of intracellular compounds, including transmembrane
water movements, which accompany various ionic fluxes through the plasma membrane for
osmotic reasons. Concretely, an entry of water will dilute the intracellular content, producing
a decrease in the phase signal. In contrast, an outflow of water will concentrate the
intracellular content, resulting, in this case, in an increase in the phase signal. This
interpretation was confirmed by the decoupling procedure which effectively showed that a
drop of phase was indeed related to a cellular swelling and vice versa [41] .

This ion water relationship was first illustrated using multimodality microscopy combining

DHM with epifluorescence microscopy and allowing to simultaneously monitor the dynamics
of intracellular ionic homeostasis and the DHM quantitative phase signal. The multimodal
ability of this set-up was illustrated by imaging the early stage of neuronal response induced
by glutamate, known to produce a cellular swelling [154] . Thanks to the decoupling
procedure. It has been possible to measure simultaneously the dynamics of intracellular
calcium, the concomitant transmembrane water movements and the corresponding neuronal
volume changes, occurring at the early stage of the glutamate-mediated activity [37] .

Following the demonstration that the phase signal can be used to monitor water movements
linked to ionic fluxes, DHM showed that glutamate produces the following three distinct
optical responses in mouse primary cortical neurons in culture, predominantly mediated by
NMDA receptors: biphasic, reversible decrease (RD), and irreversible decrease (ID)
responses. The shape and amplitude of the optical signal were not associated with a
particular cellular phenotype but reflected the physiopathological status of neurons linked to
the degree of NMDA activity [155] . Thus, the biphasic, RD, and ID responses indicated,
respectively, a low level, a high level, and an “excitotoxic” level of NMDA activation.
Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or
potassium-coupled chloride movement, strongly modified the phase shift, suggesting an
involvement of two neuronal cotransporters, NKCC1 (Na Cl), in the

genesis of the optical signal. This observation is of particular interest since it shows that
DHM is the first imaging technique able to monitor dynamically and in situ the activity of
these cotransporters during physiological and/or pathological neuronal conditions ( Figure 5.4 ).

K Cl) and KCC2 (K

5.3.6 Neuronal Cell Death

Glutamate, when present in excess, can provoke excitotoxic effects potentially triggering
cell death mechanisms typically through calcium saturation. The cell then takes one of the
many death pathways provoked by various mechanisms such as apoptosis or necrosis. In
this context, it could be shown that glutamate-mediated excitotoxicity can induce various
cell death pathways depending, for example, on the intracellular calcium uptake.