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Sample Pr eparation of Cells and T issue 65

Intensity (a.u.) (b) 1493 cm –1 –1 1637 cm –1

1002 cm –1 1447 cm
(a)

400 600 800 1000 1200 1400 1600 1800
–1
Wavenumber (cm )
FIGURE 3.1 Raman spectra of (a) fresh tissue compared to (b) frozen tissue
section. (Reproduced from Ref. 19 with the permission from Elsevier Limited.)

from the placenta (Fig. 3.1). Here, the authors did find significant dif-
ferences in the spectra, realized through a reduction in the intensities of
−1
−1
bands at 1002 cm (C⎯C aromatic ring stretching), 1447 cm (CH
2
−1
bending mode of proteins and lipids), and 1637 cm (amide I band of
proteins) in the frozen tissue compared with fresh tissue. Additionally,
−1
frozen tissue exhibited a new peak at 1493 cm , which was not found
to be OCT contamination but was attributed to an artifact of the freez-
19
ing process. Faolin et al. suggested that this artifact was due to depo-
lymerisation of the actin cytoskeleton, resulting in an increased
+
contribution of the NH deformation mode.
3
19
It is important to note that in the study by Faolain et al., the
comparison of fresh and frozen tissue was carried out with prior
mounting onto MirrIR plates in which the frozen tissue had been
thawed before analysis. Hence, depolymerization of proteins can also
result from postthawing of the frozen tissue, whereby the undesir-
able transition of vitreous ice into ice crystals could effect the integ-
rity of the cytoskeletal proteins. It is well known within the structural
cell biology community that this can be prevented by the application
20
of freeze-drying. However, Shim and Wilson’s investigations sug-
gest that the spectral changes in protein vibrational modes, caused
by heat-induced denaturing of thawed frozen tissue, can be circum-
vented by thawing in PBS (maintained at room temperature). Another
molecular change associated with tissue thawing and dehydration
was found to include a change in the relative intensities of the amide
I and methyl bending modes. 20
At this juncture, it is important to note that the extent of protein depo-
lymerization that has been observed in Raman spectra of freeze-dried