Page 98 - Vibrational Spectroscopic Imaging for Biomedical Applications

74 Cha pte r T h ree

42
trimmers are the most abundant polymers. The aldehyde groups of
glutaraldehyde react with the amino groups of proteins to form imines
in an irreversible reaction. The commonly used postfixative to glutaral-
dehyde is OsO , which preserves unsaturated lipids by the formation of
4
42
cyclic esters and is also an irreversible reaction. With the exception of a
−1
weak peak at 960 cm , it also does not give rise to absorption bands
within the spectra region of interest in the mid-IR range, where most
biomolecules absorb. CPD-glutaraldehyde-OsO -fixed cells preserve
4
fine structure as observed in electron microscopy studies. The cells were
found to be negative to trypan blue, indicating good preservation of
plasma membrane lipid molecules. SR-FTIR images of cells fixed using
this method (optical image in Fig. 3.6a) are shown in Fig. 3.6b and 3.6c.
The neat FTIR spectrum of glutaraldehyde is shown in Fig. 3.6d and a
spectrum obtained from the SR-FTIR image of the cell is also presented.
The localizations of phosphate and lipid ester ν (C=O) signals were
s
consistent with SR-FTIR maps of formalin-fixed cells (see Fig. 3.5).
Although no significant spectral markers from glutaraldehyde were
detected in the SR-FTIR spectrum of these cells, compared to forma-
lin-fixed cells (see Fig. 3.5d), a reduction in the intensity of peaks
−1
between 1500 and 1000 cm was observed and the lipid ester
ν (C=O) signal appeared as a less resolved shoulder on the amide I
s
band (Fig. 3.6d).
Unfixed cells were also investigated following preparation using
7
a protocol outlined by Tobin et al. in which cells were removed from
culture medium, rinsed in PBS, and dried under centrifugation. The
cells stained positive for trypan blue indicating loss of membrane
integrity. SR-FTIR images of these cells revealed the expected local-
ization of high-phosphate intensity within the nucleus; however, in
other parts of the cell, phosphates were homogenously distributed in
intensity. This was attributed to phosphates from PBS retained on the
38
surface of the dried cells. Interestingly, intense lipid ester ν (C=O)
s
signal also localized to the nucleus and decreased in intensity as a
series of concentric rings between the nucleus and its periphery. This
was unlike the lipid ester ν (C=O) distributions observed for forma-
s
lin (Fig. 3.5c) or CPD-glutaraldehyde-OsO -fixed cells (Fig. 3.6b and
4
3.6c) in which this signal was distributed with high-intensity sur-
rounding the nucleus.
43
In a recent study by Gazi et al. chemical fixation was investi-
gated for the preparation of adipocytes for FTIR analysis. Adipo-
cytes are specialised for the synthesis and storage of fatty acids
(FAs) as triacylglycerides (TAGs) as well as for FA mobilization
through lipolysis. Figure 3.7a shows the appearance of adipocytes
in growth medium and illustrates the presence of numerous lipid
droplets contained within their cytoplasm. Figure 3.7b shows adi-
pocytes, prepared for FTIR analysis, following fixation in 4 percent
formalin (in PBS), a brief water-rinse to remove residue salts and
air-drying at ambient conditions. Although this fixation protocol