Page 261 - Vibrational Spectroscopic Imaging for Biomedical Applications

Raman Micr oscopy for Biomedical Applications 237

A photomicrograph and a Raman image of mucosa in a colon tis-
sue section are displayed in Fig. 8.6b and c, respectively. The color
code indicates the segmentation of the Raman spectra by k-means
cluster analysis: mucus beyond the top tissue margin (pink/magenta/
violet), connective tissue of submucosa (red) and epithelium (blue/
cyan/black). The averaged Raman spectra of the cyan and black clus-
ters are overlaid in Fig. 8.6a. Details and further Raman spectra have
21
previously been published. A difference spectrum was calculated to
inspect the spectral changes. Whereas the protein and lipid bands
that are labeled in the Raman spectra did not change significantly, all
positive difference bands can be assigned to DNA. A Raman spec-
trum of calf thymus DNA was inserted for comparison. The labeled
bands in the reference spectrum can be assigned to thymine (669,
−1
−1
747, 781, 1368, 1670 cm ), guanine (680, 1482, 1575 cm ), adenine
−1
−1
(726, 1303, 1336, 1482, 1575 cm ), cytosine (781, 1253 cm ), the phos-
−1
phate backbone (833, 1094 cm ), and CH groups of the backbone
2
−1
(1420 cm ). Note that some bands contain spectral contributions from
−1
two nucleotides. Negative difference bands at 833 and 875 cm were
correlated with the expression of secretion products in glands. The
−1
833 cm band overlaps with the DNA backbone band.
A disadvantage of Raman imaging in the serial registration mode is
longer exposure times compared with FTIR imaging in the parallel reg-
istration mode using focal plane array (FPA) detectors. Collecting 39 ×
71 = 2769 Raman spectra at a step size of 10 μm took more than 24 hours
using the Raman system Envia (Renishaw, UK). Collecting eight FTIR
images of 64 × 64 = 4096 FTIR spectra per image could be finished within
2
15 minutes. Each image covered an area of 170 × 170 μm as indicated by
the boxes in Fig. 8.6b. Even shorter exposure times can be obtained
using coherent anti-Stokes Raman scattering (CARS) and rapid laser
scanning microscopes. CARS microscopy is a nonlinear variant of
Raman spectroscopy. Details of theoretical and technical consideration
of CARS microscopy are outside the scope of this contribution and can
be found in Chap. 11 and elsewhere. 22–24 Its advantages include (1) signal
enhancement of several orders of magnitude for single vibrational
modes, (2) coherent emission in a direction determined by phase
matching which allows efficient signal detection, (3) narrow spectral
bandwidth which allows registration without monochromators, and
(4) absence of an autofluorescence background as the signal is recorded
on the anti-Stokes side of the excitation light. CARS images of colon tis-
sue sections could be obtained from approximately 600 × 600 μm 2
25
regions within 5 seconds. CARS images were compared with Raman
images. The information content of the both techniques was found to be
complementary. Whereas variations within tissue sections could be
visualized more rapidly using CARS microscopy than Raman micros-
copy, identification of tissue types and characterization of variances
between different tissue sections can be achieved better in Raman
images because the information from an extended spectral interval is
simultaneously recorded.