Biomedical Engineering Reference
In-Depth Information
2
π
λ
ðn
c
2
n
m
Þd
Φ
5
(5.1)
where
d
is the cellular thickness,
n
c
is the intracellular refractive index averaged over the
optical path length through the specimen, and
n
m
is the refractive index of the surroun
di
ng
medium. Consequently, the information concerning the intracellular content related to
n
c
is
intrinsically mixed with the morphological information related to the thickness
d
. Due to this
dual dependence, the phase signal remains difficult to interpret. As an illustration, a simple
hypotonic shock induc
e
s a phase signal decrease
[41]
difficult to interpret as cellular swelling
but consistent with an
n
c
decrease from a dilution of the intracellular content by an osmotic
water influx. Ac
co
rdingly, some strategies have been developed to separately measure the
morphology and
n
c
. Kemper et al.
[20]
and Lue et al.
[116]
measured the intracellular
refractive index by trapping cells between two cover glasses separated by a known distance.
These approaches, preventing cell movements, preclude the possibility of exploring cell
dynamic processes. We have developed another approach to separately measure the
parameters
n
c
and
d
from the phase signal
, based on a modification of the extracellular
refractive index
n
m
. Basically, this method consists of performing a slight alteration of the
extracellular refractive index
n
m
and recording two holograms corresponding to the two
different values of
n
m
, allowing the reconstruction of two quantitative phase images (
Φ
Φ
1
,
Φ
2
)
described by the following system of two equations for each pixel
i
:
2
π
λ
1
ðn
c
;
i
2
n
m
;
1
Þd
i
Φ
1
;
i
5
(5.2)
2
π
λ
2
ðn
c
;
i
2
n
m
;
2
Φ
Þd
i
5
(5.3)
2
;
i
where (
λ
1
,
λ
2
) are the wavelengths of the light source.
By solving this system of two equations, we obtain
n
c
;
i
and
d
i
for each pixel
i
.Wehave
considered two different approaches to modify
n
m
: the first approach requires
sequentially perfusing a standard cell perfusion solution, and a second solution with a
different refractive index but with the same osmolarity (to avoid cell volume variation)
to record the two corresponding holograms, at a single wavelength, (
λ
5
λ
2
). The
refractive index of the second solution is increased by replacing mannitol (a hydrophilic
sugar present in the standard perfusion solution) with equal molarity of the hydrophilic
molecule Nycodenz, a small molecule known for its high capability to modify the
refractive index
[41]
. Nevertheless, this approach, due to the solution exchange time,
precludes the possibility of monitoring dynamic changes of cell morphology and
n
c
occurring during fast biological processes. To overcome these drawbacks, we have
developed a dual-wavelength (
1
2
)DHM
[117]
, which exploits the dispersion of the
extracellular medium, enhanced by the utilization of an extracellular dye (
n
m,1
5
n
m
λ
6¼ λ
1
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