Page 260 - Vibrational Spectroscopic Imaging for Biomedical Applications

236 Cha pte r Ei g h t

model of gliomas. A classification model based on infrared spectros-
copy has already been developed and validated using magnetic reso-
nance tomography and histopathology. 19
8.2.3 Human Colon Tissue
Raman spectroscopy was applied to investigate epithelial cancers of
the gastrointestinal tract, including colon, esophageal, and stomach
disease because of its relative accessibility for the in vivo application
as an adjunct to current diagnostics using endoscopic techniques. A
review has detailed the application of optical techniques to colon
20
cancer. Human colon tissue was also selected for a comparative
Raman and FTIR imaging study. A prerequisite was that the sample
thickness and the substrate material were properly chosen. Ten-
micrometer tissue sections absorb approximately 90 percent of mid-
infrared radiation which yields an absorbance equals 1. Substrates
made of pure calciumfluoride (CaF , vacuum ultraviolet grade) are
2
transparent for mid-infrared radiation with wavenumbers above
−1
900 cm . In addition, they show an extremely low Raman back-
−1
ground except for a single band near 322 cm . Furthermore, they
−3
have a low solubility in water (1.5 × 10 g per :100 g H O). Although
2
quite expensive (approximately :100 per slide) CaF is a better choice
2
for biological samples than other alkaline halogenated salts such as
NaCl, KBr, or BaF that are more soluble in water. Less expensive
2
alternatives are glass slides that are coated with a thin metal film.
Selecting an appropriate thickness, these slides are transparent
enough for visible light microscopy, they reflect mid-infrared radia-
tion, and they suppress spectral contributions from the glass sub-
strate that are very intense relative to the Raman signals of tissue
using 785 nm excitation. Raman spectra can be detected in the rela-
−1
tive wavenumber range (= Raman shift) from 200 to 3550 cm using
785 nm excitation. In particular, the low-wavenumber range is
extended for samples on CaF substrates compared with FTIR
2
spectroscopy. The low-wavenumber limit in modern Raman systems
is determined by the transmission properties of the Notch filter which
suppresses the elastic Rayleigh scattering. A number of informative
vibrations occur in the wavenumber interval between 400 and
−1
−1
1030 cm . They include well resolved bands of cholesterol (700 cm ),
−1
hydrophilic groups in lipids such as choline (717 cm ), amino acids
−1
bands of phenylalanine (621, 1004, 1030 cm ), tyrosine (643, 828,
−1
−1
853 cm ) and tryptophan (760, 880, 1011 cm ) and nucleic acids (see
next paragraph). The high-wavenumber limit using 785 nm is deter-
mined by the quantum efficiency of the silicon-based charge-coupled-
device (CCD) detector which drops toward zero for Raman shifts
−1
above 3400 cm (1070 nm). The Raman spectra in Fig. 8.2 were nor-
malized for the different detector sensitivity throughout the wave-
number interval. However, the normalization procedure also amplifies
−1
the noise above 3400 cm .