Page 70 - Vibrational Spectroscopic Imaging for Biomedical Applications

46 Cha pte r T w o

cells from four separate flow chamber assemblies have been meas-
ured and analyzed (neglecting assemblies with problems). Out of
35 cells, 2 exhibited an initial F /F value of zero and were excluded
v m
from further analysis. The thick, solid black line in Fig. 2.13a corre-
sponds to the F /F average of the remaining 33 cells with the error
v m
bars indicating the standard deviation at each time point. The overall
average and standard deviation of all cells over all nine time points is
F /F = 0.40 ± 0.17, respectively. Since we performed the PAM fluo-
v m
rescence measurements on individual cells within the population the
relatively high standard deviation is likely due to the variability
among the cells. The standard deviation of the time development of
the average (thick, solid line) is 0.03 (or 7 percent). The dashed lines
show individual F /F measurements of five representative algal
v m
cells out of 33. As can be seen from the dashed lines, some cells ceased
their photosynthetic activity at the end of the experiment, some had a
constant activity and some even increased their activity during 260
minutes of the experiments. The thick, solid curve however confirms
that on average 33 observed cells could maintain a fairly constant
F /F (within 7 percent) over the entire experiment period.
v m
The average and natural variability (standard deviation) of F /F
v m
among the 33 cells is 0.43 ± 0.18, respectively, which is derived from
the measurements at the initial time point only (t = 15 minute.). This
assumes that the influence of the flow chamber is minimal at that
point in time. The cells whose F /F values over time remained
v m
within a standard deviation (42 percent) relative to the initial F /F
v m
were considered healthy. All other cells were considered unhealthy.
The black line in Fig. 2.13b represents the percentage of cells that
remain viable up to the corresponding point in time illustrating the
development of the number of healthy cells in the chamber with time.
It demonstrates that after the first hour the number of photosyntheti-
cally active cells stays virtually constant. The red bars in Fig. 2.13b
show a histogram of the number of dying cells in each time slot. The
elevated bar (second from the left) corresponds to cells that ceased
their photosynthetic activity within roughly 1 hour after the flow
chamber was filled. This might be due to cells that were stressed
before they were loaded into the flow chamber. After 260 minutes
61 percent (= 20 cells) of the initial 33 cells remain photosynthetically
active. These PAM measurements demonstrate the viability of algal
cells in the flow chamber for an extended period of time.
In this section, we presented a new flow chamber design for in
vivo mid-IR and visible measurements of biological cells. The use of
sub-micrometer-thick diamond as a window material has several
major advantages over conventional halide windows like ZnS. Nota-
bly, it exhibits lower optical aberrations and is transparent over an
extended spectral range in the mid-IR as well as in the visible. The
slim design of the flow chamber accommodates high-resolution/
numerical aperture microscope objectives, which typically have a