Biomedical Engineering Reference
In-Depth Information
For illumination in transmission the thickness of cells
d
cell
in cell culture
medium with a homogenous refractive index
n
medium
can be determined by
measuring the optical path length change ∆
ϕ
cell
of the cells to the surrounding
medium:
d
cell
(
x, y, z
0
)=
λ
∆
ϕ
cell
(
x, y, z
0
)
2
π
1
n
cell
− n
medium
,
(9.12)
with the integral refractive index
n
cell
and the wave length
λ
of the applied
laser light. For fully adherently grown cells, the parameter
d
cell
is estimated
in first order to describe the shape of single cells [34, 38].
Figure 9.15 illustrates the evaluation process of digital recorded holo-
grams. Figure 9.15a,b shows a digital hologram obtained from a living human
pancreas carcinoma cell (Patu8988T) with an inverse microscope arrange-
ment in transmission mode (
40 microscope lens, NA = 0.65) and the re-
constructed holographic amplitude image that corresponds to a microscopic
bright field image at coherent laser light illumination. Figure 9.15c depicts the
simultaneously reconstructed quantitative phase contrast image modulo 2
π
.
The unwrapped data without 2
π
ambiguity, representing the optical path
length changes that are affected by the sample in comparison to the sur-
rounding medium due to the thickness and the integral refractive index, are
shown in Fig. 9.15d. Figure 9.15e depicts a pseudo 3D plot of the data in
Fig. 9.15d. Figure 9.15f shows the cell thickness along the marked dashed line
in Fig. 9.15d, which is determined by application of (9.12) with
n
cell
=1
.
38
and
n
medium
=1
.
337.
×
Fig. 9.15.
Example for evaluation of digital holograms. (
a
) Digital hologram of a
human pancreas carcinoma cell (Patu8988T); (
b
) Reconstructed holographic am-
plitude image; (
c
) Quantitative phase contrast image (mod 2
π
); (
d
) Unwrapped
phase distribution; (
e
) Pseudo 3D plot of the unwrapped phase image in gray level
representation; (
f
) Calculated cell thickness along the dashed white line in (
d
)
