Page 95 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 71

hexane was found to be a superior deparaffinization agent, removing
nearly all of the paraffin 18 hours posttreatment (Fig. 3.4d).
These important findings were found to have practical implica-
tions for immunohistochemical analysis. It was found that the inten-
sity of positive staining in tissue sections treated with hexane for 18
hours was 28 percent greater when compared to an adjacent section
treated with xylene for the same period. 37
37
In the light of these findings from Faolain et al. the protocol
18
used by Fernandez et al., where prostate tissue sections were treated
with hexane for 48 hours, provides the most efficient method of par-
affin removal used in an FTIR study to date. Infact, using this sample,
preparation protocol high levels of accuracy (≥90 percent) were
achieved for classifying a number of different tissue components for
FTIR chemomectric imaging of prostate tissue microarrays. Never-
36
theless, it has been shown by Gazi et al. that although a less rigorous
method of deparaffinization was used to process malignant prostate
−1
tissue sections, the spectral region between 1481 and 999 cm could
be used to discriminate and classify the different pathological grades
of prostate cancer as well as to provide statistically significant distinc-
tion between tumors localized to the prostate gland from those show-
ing extracapsular penetration. Moreover, the time-efficient method of
less rigorous deparaffinization is suitable for other FTIR-based diag-
nostic parameters that do not include spectral regions that overlap
with paraffin signals, such as the peak area ratio of the 1030-cm −1
−1
(glycogen):1080 cm (phosphate) bands, which differentiated malig-
nant from benign prostate tissues in imaging studies. 9
It may be argued that for FTIR studies, the removal of paraffin is
not necessary at all as discrete frequency ranges corresponding to the
lipid hydrocarbon modes are only affected. However, visualisation of
the unstained tissue’s anatomical features is severely hampered if the
35
paraffin is not removed. Sahu et al. report that colonic crypts in a
10-μm paraffin-embedded tissue section appeared as circular entities
when viewed under light microscopy. Moreover, even between adja-
cent microtomed sections, tissue components can vary significantly,
which in turn prevents the positioning of the IR beam upon a specific
tissue location by comparison with an H&E section.

3.3 Cell Preparation

3.3.1 Chemical Fixation for FTIR and Raman Imaging
As mentioned in Sec. 3.1, to avoid potential confounding variables
from autolytic processes initiated by cells during air-drying, it is
important that the cells are appropriately fixed to maintain localiza-
38
tions of biomolecular species. To this end, Gazi et al. studied the use
of several chemical fixation methods for biospectroscopically mapping