Page 109 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 85

with values taken from a region of high-protein intensity to a region
of low-protein intensity and bisecting the cells.
Could the low-protein signal at the cell be due to local proteolysis
of the Matrigel matrix? Time-lapse video microscopy provided valu-
able insight into this speculation. The final frame of the time-lapse
video and the brightfield image of the same area after fixation, relo-
cated for FTIR microspectroscopic imaging, are shown in Fig. 3.12b(i)
and (ii), respectively. These optical images demonstrate that the
morphology of the cells (elongated and rounded), when in culture, is
suitably retained by the formalin fixation procedure. In Figure 3.12a,
the video frame captured at the start of the time-lapse recording
shows that the cells at internal locations on Matrigel display a
rounded morphology. The cell marked with a red arrowhead was
stationary throughout the course of time-lapse recording and retained
its rounded appearance. It is reasonable to assume that the low-
protein intensity at this cellular location [Fig. 3.12b(iii)] would be
indicative of local proteolysis or mechanical degradation of the Matri-
gel. However, between 5 hours and the point of termination of the
time-lapse study (22 hours, 22 minutes), the cell marked with the green
arrowhead migrated, several times, toward and away from the cells
marked with the blue and white arrowheads. Since these cells (green,
blue, and white arrowhead) were motile throughout the time-lapse
recording, it is unlikely that there was local digestion of the Matrigel
just prior to termination of the time-lapse recording via MMPs pro-
duced by the prostate cancer cells. Moreover, if proteolytic digestion
was a dominant mechanism by which the green, blue, and white
arrowhead cells transversed over the Matrigel, then one would expect
low-protein signals to arise from the entire path occupied by these
cells. It was concluded from this, that light-scattering artefacts influ-
enced the protein intensity maps of these cells on Matrigel, giving the
illusion of protein degradation at the cell locations. 55
A model was produced, which showed that a switch from a higher
than background signal to a lower than background signal will occur
at a given thickness or concentration of protein within the Matrigel layer.
Importantly, the model includes light that is directly back-scattered into
the microscope collection optics. These findings implicate fundamen-
tally on research in the field of FTIR spectroscopy concerning cells on
two-dimensional matrices.

3.3.4 Preparation of Living Cells for FTIR and
Raman Studies

FTIR Studies
A number of studies concerning the analysis of living cells by FTIR
have been performed with synchrotron radiation sources. 56–58 An
56
early study by Holman et al. reported spectral changes in HepG2