Page 104 - Vibrational Spectroscopic Imaging for Biomedical Applications

80 Cha pte r T h ree

from the exchange of media to serum-free Roswell Park Memorial
Institute (RPMI) media at the zero minute time-point. Conversely, cells
incubated with D -PA showed an initial rise in endogenous lipids.
31
Since the incubation media (RPMI) contains no FAs, this increase in
lipid content must be due to de novo biosynthesis. This initial rise in
endogenous lipid signal was followed by a fall, attributed to metabolic
breakdown into adenosine triphosphate (ATP), which is a major prod-
uct of lipid metabolism. This notion is supported by a phosphate spike
at 30 minutes accompanied by a significant shift in the amide I fre-
quency, indicating protein phosphorylation.
The time-efficient formalin fixation method not only suitably pre-
served biomolecular composition so that lipid metabolism and protein
phosphorylation could be measured, but also preserved the subcellu-
lar localizations of biomolecules for imaging studies. Figure 3.9b shows
an optical photomicrograph of PC-3 cells on MirrIR substrate, follow-
ing exposure to 50μM D -PA for 24 hours. This area was analyzed by
31
imaging FTIR microspectroscopy and the resulting distribution of the
integrated intensity of the phosphate diester [v (PO ); (1274 to 1181
as 2
−1
cm )] peak area is shown. As expected, for cells 1 and 2 in the optical
image, it can be seen that the most intense phosphate signals localise at
the nucleus. Whereas, the most intense v (CD ) signal localized
as+s 2+3
at the cytoplasm, suggesting that the subcellular localization of D -
31
PA or its metabolites is predominately in the cytoplasm.
Another FTIR-based dose-response study had been undertaken
where prior to spectroscopic examination, drug induced cells had been
44
removed from culture media, washed in PBS and air-dried. This
study reports spectroscopic changes (ratio of peaks) that could be asso-
ciated with exposure of the cells to increasing doses of the chemothera-
peutic drug. An additional bioanalytical modality was combined with
FTIR to demonstrate correlations of spectroscopic changes with cell
sensitivity to the drug using the MTT [(3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] assay. Thus, there is an evidence to
suggest spectroscopic changes associated with drug exposure can be
determined and this is in fact dominant over metabolite perturbations
resulting from autolysis during the drying process.

3.3.3 Growth Medium and Substrate Effects on
Spectroscopic Examination of Cells

Growth Medium Influences
A number of studies have investigated the use of FTIR or Raman
microspectroscopies as diagnostic tools to differentiate and classify
cell lines, in vitro, based on their pathological state 45–49 or resistance
46
to drugs. Interestingly, we find that some researchers have grown
their different cell lines in the same culture media, 46–48 whereas others
have used different media for each cell type. 49,50 The European Col-
lection of Cell Cultures (ECACC) provides standard protocols for the