Page 97 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 73

of DNA. The nucleolus also gave rise to intense protein amide I signal
attributed to the densely packed histone proteins. Spectral subtraction of
the neat formalin spectrum from the FTIR spectrum of the formalin-fixed
cell resulted in negligible differences in the intensities of peaks across the
−1
frequency range 3000 to 1100 cm (Fig. 3.5d). This was performed follow-
ing normalization of the spectrum of formalin to the intensity value of the
−1
peak at 1000 cm in the formalin-fixed cell spectrum, since this frequency
gives rise to the most intense peak in the spectrum of formalin.
The formalin fixation protocol outlined above has successfully been
used to image unstained cells in the process of mitosis and cytokinesis
using both FTIR and Raman microspectroscopies. 39,40 Matthaus et al. 39
report Raman images showing the protein and phosphate scattering
intensities of cells in various stages of mitosis, which were used to probe
microtubules and the dense histone-packed chromatin as well as DNA
40
condensation. Gazi et al. reported SR-FTIR maps of a formalin-fixed
cell in the process of cytokinesis, where features such as the contractile
ring as well as organelle placement could be determined using the pro-
tein amide I and lipid ester [ν (C=O)] signals, respectively.
s
41
Krafft et al. used formalin fixation to obtain highly spatially
resolved Raman microspectroscopic maps of lung fibroblast cells
grown on quartz slides. These cells were analyzed at 4ºC in 10 mM
phosphate buffer with 1 mM sodium azide at neutral pH. Spectra from
these maps could be used to identify RNA and DNA, proteins, choles-
terol and phospholipids (phosphatidylcholine and phosphatidyletha-
nolamine). Reprocessed Raman cell maps, depicting the fit coefficients
for each of these biomolecules enabled an approximation of the com-
position of different subcellular structures: nucleus, cytoplasm, endo-
plasmic reticulum, vesicles, and the peripheral membrane.
38
Gazi et al. also studied cells that had been formalin fixed and sub-
sequently critical-point-dried (CPD). The CPD process involves several
steps. First, the intercellular water molecules (from saline) in the pre-
formalin-fixed cells must be displaced gradually with increasing con-
centrations of ethanol. The ethanol is then displaced by acetone, which
is miscible with liquid CO . The acetone within the cells is then dis-
2
placed by liquid CO , within a chamber. The chamber is heated with a
2
simultaneous rise in pressure as liquid CO enters the vapor phase. At
2
a specific temperature and pressure, the density of the vapor equals the
density of the liquid, the liquid–vapor boundary disappears, and the
surface tension is zero. Thus, this method reduces any residual distor-
tions that may occur in the prefixed cell as a result of air-drying. Since
the formalin-fixed CPD dried cells were exposed to significant lipid-
leaching reagents (ethanol and acetone), the cells were positive to try-
pan blue staining and the SR-FTIR spectrum of these cells demonstrated
loss of the lipid ester ν (C=O) peak.
s
38
A third fixation method was investigated by Gazi et al. in which
cells were fixed with glutaraldehyde and osmium tetroxide (OsO ) prior
4
to CPD. Glutaraldehyde polymerizes in solution, where dimmers and