Page 92 - Vibrational Spectroscopic Imaging for Biomedical Applications

68 Cha pte r T h ree

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Plashco et al. conclude that evaluation of protein structure in this
tissue should be limited to snap-frozen, or formalin-fixed tissues
where there were no observable shifts in the amide I and II peaks. The
19
latter does not conform to observations made by Faolain et al. in for-
malin-fixed tissue spectra. However, this difference may be due to the
different types of tissue used in each study, mineralized tissue in Pleshko
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19
et al. study and nonmineralized tissue in Faolain et al. study.
3.2.3 The Effects of Xylene on Fixed Tissue and
Deparaffinization of Paraffin-Embedded Tissue
As mentioned in Sec. 3.2.1, fixed tissue is immersed in xylene prior to
19
impregnation with paraffin wax. Faolain et al. have shown that
Raman spectra of formalin-fixed tissue exposed to xylene produces a
number of strong peaks, associated with its aromatic structure, at
−1
−1
620 cm (C⎯C twist of aromatic rings), 1002 cm (C⎯C stretching of
−1
aromatic rings), 1032 cm (C⎯C skeletal stretch of aromatic rings),
−1
601 cm (C⎯C in plane bending of aromatic rings), and 1203 cm −1
19
(C⎯C H stretching mode of aromatic rings). As mentioned in the
6 5
section “Chemically Fixed Tissue,” formalin fixation reduces the
intensity of the amide I band. Interestingly, upon xylene exposure,
the amide I band reappears with appreciable intensity. Faolain et al.
suggest that the cross-linking of proteins by methylene glycol is
reversed upon xylene treatment so that the amide I band reverts back
to the secondary amide. As expected, the FTIR spectrum of xylene
treated formalin-fixed tissue demonstrated a loss of the lipid ester
−1
(C=O) band at 1740 cm , due to significant removals of cellular lipids.
Presently in the fields of FTIR and Raman spectroscopy, there is
lack of consensus with regard to a standard protocol for deparaf-
finization of paraffin-embedded sections and several approaches
18
have been used. For example, Fernandez et al. deparaffinized their
prostate tissue sections by immersing in hexane at 40ºC with continu-
ous stirring for 48 hours. During this period, the vessel was emptied
every 3 to 4 hours, rinsed thoroughly with acetone followed by hex-
ane and after thorough drying, refilled with fresh hexane to promote
dissolution of paraffin embedded in the tissue. The disappearance of
−1
a peak at 1462 cm in the FTIR spectrum was used to ensure com-
plete deparaffinization.
35
Sahu et al. deparaffinized samples of colon tissue using xylene
and alcohol. The researches washed their 10-μm paraffin-embedded
sections with xylol for 10 minutes (three changes) with mild shaking.
Following this, the slide was washed with 70 percent alcohol for 12
hours. To evaluate the efficacy of this procedure, FTIR spectra from the
tissue were collected at each stage of the deparaffinization process—
before deparaffinization, at each xylol washing step (following air-dry-
ing) and following alcohol treatment. The authors report that
following two washes with xylol, a third xylol wash did not produce