Page 90 - Vibrational Spectroscopic Imaging for Biomedical Applications

66 Cha pte r T h ree

tissue can be reduced by using an appropriate cryogen for the initial
snap-freezing of the resected tissue specimen. Higher freezing rates
are achieved using propane or mixtures of propane and isopentane in
26
preference to isopentane. Also, since ice crystals develop between a
26
temperature range of 0 to −140ºC it is advantageous to maintain
cryo-preserved tissues at temperatures lower than –140ºC during
microtomy and freeze-drying. Finally, the size of the specimen also
dictates the extent of ice crystal damage; in liquid-nitrogen-cooled
3
propane, it has been reported that guinea pig liver of 0.5 mm is the
critical size that separates complete crystallization from partial vitri-
26
27
fication. Nevertheless, Stone et al. have demonstrated on a num-
ber of different tissue types that freeze-thawed tissue, without PBS,
can be used to differentiate different pathologies with greater than
90 percent sensitivity and specificity. More recently, the same
method of sample preparation was used to demonstrate the bio-
chemical basis for tumour progression of prostate and bladder can-
cers by determining the relative amounts of a number of tissue
28
constituents. This was carried out by obtaining basis Raman spectra
of pure standards and correlating these with tissue spectra derived
from each disease state using ordinary least-squares analysis. The
biological explanations for these constituent fluxes through the dif-
ferent pathological states could be associated with known tumour
behavior. Thus, freeze-thawed tissue warmed to room temperature is
not only diagnostically useful but may also provide relative quantifi-
cations and qualitative biomolecular characterization of the sample.
For FTIR investigations, snap-frozen tissue has been analyzed fol-
lowing thawing and subsequent air-drying to avoid interference from
water bands. 29–32 However, this dehydration process results in unde-
sirable perturbations to cellular chemistry, as outlined in Sec. 3.1,
therefore, some researchers have used cryosections dried under a
stream of nitrogen gas to reduced oxidative and surface tension
effects. 33,34 Both protocols have successfully been applied to the spec-
tral classification of tissue pathologies 29–34 and have been shown to
generate detailed biospectroscopic maps that localize tumour lesions
34
33
within oral and brain tissues. Additionally, using an univariate
32
analytical approach to process tissue maps, Wiens et al. reported the
use of dried snap-frozen sections to investigate the early appearance
and development of scar tissue using FTIR signals corresponding to
lipids, sugars, phosphates as well as collagen and its fibril orientation.
Chemically Fixed Tissue
Compared to fresh tissue, Raman spectra of formalin-fixed tissue 19
following 24-hour fixation, exhibit a significantly reduced intensity of
−1
the amide I peak as well as the appearance of a peak at 1490 cm . The
reduced intensity of the amide I peak is attributed to the formation of
tertiary amides (and loss of secondary amide), resulting from the
reaction of methylene glycol (in formalin) cross-linking the nitrogen