Page 117 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 93

of specific biomolecules using emerging bioanalytical approaches
will shape the tissue repositories of the future. These developments
will also impact biomedical vibrational spectroscopy, since this tech-
nology can play an important role in determining the biochemical
basis underpinning disease progression. Nevertheless, it is apparent
that existing tissue banks have proven adequate for FTIR and Raman
studies of tissue pathologies, providing high-classification power.
This is despite the fact that spectral artifacts exist as a result of tissue
processing or section postprocessing. These spectral artifacts can be
due to protein depolymerisation or a change in the lipid to protein
ratio for dried cryosections or the case of deparaffinized specimens,
due to residual paraffin, coagulation of proteins and loss of lipids.
Some of these artifacts can be minimized. Protein depolymeriza-
tion of freeze-dried/thawed cryosections can be reduced by careful
attention to the cryogen used for initial tissue snap-freezing as well as
cryomicrotomy and freeze-drying environmental temperatures.
Other artifacts such as residual paraffin can now be confidently
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removed in the light of work carried out by Faolain et al. It appears
that deparaffinization using hexane for >24 hours is an appropriate
method for this purpose and has wider implications in immunohisto-
chemical pathology. However, this protocol can be time limiting and
so less rigorous protocols may be sufficient where spectroscopic mark-
ers for pathological assessment do not overlap with paraffin signals.
12
The early work of Fox et al. investigating the binding time of
formalin to tissue, may be of significance to those vibrational spec-
troscopists presently using formalin-fixed cells in imaging or bio-
mechanistic studies, since the effects of formalin-binding time on cell
spectra have not been assessed. Chemical fixation with formalin has
been shown to produce spectral images of cells, where Raman or
FTIR signals of various biomolecules localize to subcellular compart-
ments that are expected to give rise to these signals. Tailored chemical
fixation protocols for vibrational spectroscopy may, however, be nec-
essary in some instances. For example, in Sec. 3.3.1, the lipid compo-
nent of adipocytes is volatile in air and requires fixation with OsO ,
4
which itself does not absorb in the mid-IR region. Coupled to para-
formaldehyde, which would influence the FTIR spectrum to a lower
degree than to the relatively larger molecular weight polymers of
glutaraldehyde, one can obtain a well-preserved sample for spectro-
scopic analysis. The lengthy procedure of OsO paraformaldehyde
4
with critical-point-drying is appropriate where experiments are cap-
turing a cellular event at time frames that are far apart. However, for
shorter time frames (intervals of 15 minutes), faster fixation methods
are required and formalin has so far been proven to be adequate.
Interestingly, it has also been demonstrated that air-dried cells fol-
lowing exposure to a pharmacological drug or stimulus can also pro-
duce spectral changes that may be associated with response to the
condition administered. This is assuming that the underlying stress