Towards clinico-pathological application of Raman spectroscopy

Raman Spectroscopic Characterization of Porcine Brain Tissue Using a Single Fiber-Optic Probe
show that, compared to the gray matter, the white matter has relatively stronger signal contributions of
sphingomyelin, cholesterol, and galactocerebroside but weaker signal contributions from protein (BSA),
phosphatidylcholine, and DNA. G: Fit-coefficients resulting from the fit of the average spectrum of corpus
callosum with the averaged spectrum of nucleus caudatus caput plus the same set of reference spectra (see
legend of panel F). Gray bars represent uncertainty interval of fit-coefficients. The fit-coefficients indicate
that the corpus callosum has stronger signal contributions from lipids (i.e., sphingomyelin, cholesterol, and
galactocerebroside) in comparison with the nucleus caudatus caput, which is characterized by stronger
protein, phosphatidylcholine, and DNA signal contributions. H: Fit-coefficients resulting from the fit of
the average spectrum of capsula interna and with the averaged spectrum of nucleus caudatus caput plus
the same set of reference spectra (see legend of panel F). Gray bars represent uncertainty interval of fitcoefficients.
The fit-coefficients indicate that the capsula interna compared to the nucleus caudatus caput
has stronger signal contributions from lipids. Nucleus caudatus caput is characterized by stronger protein,
phosphatidylcholine and DNA signal contributions.
vibrations 2500 – 2600 cm -1 , and NH-stretching vibrations between 3100 and 3500 cm -1 . 38,39
Because almost all spectral signatures from lipids and proteins appear in the 2750 – 3100
cm-1 interval, this range was chosen for analysis of the Raman spectra.
Raman spectra from commercially available pure compounds are shown in Figure 1. Note
that, although quite similar in appearance, the spectra of galactocerebroside (b), phosphatidylcholine
(c), and sphingomyelin (d) are linearly independent (see Materials and Methods).
The adjoining brain structures that could easily be identified by visual inspection of cross
sections were separated for 100% by HCA based on the averaged Raman spectra obtained
from all seven pig brains used in these experiments. In all cases, white matter (cerebrum
and cerebellum) and other structures consisting of myelinated fibers such as corpus callosum,
capsula interna, crus cerebri, fimbria hippocampi, and tractus opticus, were clearly
distinguished from cortical gray matter and from basal nuclei and subcortical gray areas
(e.g., nucleus caudatus, putamen, thalamic nuclei, substantia nigra, corpus geniculatum
laterale, and hippocampus). Although histomorphologically similar, the structures such as
white matter of cerebrum and white matter of cerebellum were distinguished from each
other in all brains investigated by HCA. Similarly, the averaged spectra of fimbria hippocampi
and of tractus opticus formed separate clusters for all cases. Moreover, a number
of adjoining nuclei could also be 100% separated from each other for all brains by HCA (e.g.,
thalamic nuclei and nucleus caudatus caput).
Examples of the results of Raman experiments on the cross sections from the frontal part of
brain for all seven pigs are shown in Figure 2. Panel A shows the cut surface of a cross section.
From the cortex towards the basis of the brain, a number of structures is highlighted including
gray matter (labeled “gray”), white matter (labeled “white”), corpus callosum (labeled
“cc”), nucleus caudatus caput (labeled “ncc”), capsula interna (labeled “ci”), and putamen
(labeled “p”). HCA performed on averaged spectra of gray and white matter for all pig brains
resulted in a clear separation of gray and white matter, as shown in (B). Panels C - E show
the averaged Raman spectra of adjoining brain structures: white and gray matter (C), corpus
callosum and nucleus caudatus caput (D), and capsula interna and nucleus caudatus caput
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