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Raman Micr oscopy for Biomedical Applications 241
spectral contributions of other constituents such as proteins and lip-
ids are partly masked by hemoglobin bands. Careful inspection of
spectrum (C) in Fig. 8.7a reveals that lipid-associated bands at 1062,
−1
1300, and 1440 cm increase in CCAM. Increased spectral contribu-
tions of lipids were more pronounced in infrared spectra of CCAM
which confirmed the Raman result. We conclude that each pathology
is characterized by a distinct biochemical composition which can be
probed by Raman and infrared spectroscopy. The power of the vibra-
tional spectroscopic fingerprint was demonstrated in one BPS patient
where the coexistence of CCAM was detected and confirmed by his-
28
topathology. This diagnosis was overseen during the first histo-
pathological inspection.
8.3 Raman Imaging of Cells
Raman imaging of cells can complement current techniques in cell
biology to study cellular and subcellular processes and structures or
to identify cell differentiation and cell type. Raman and infrared
microspectroscopic studies of individual cells were summarized. 31
Frequently used methods for single cell studies are electron micros-
copy, fluorescence microscopy, and autoradiography. However, com-
plicated preparations and manipulations have to be performed to
fulfill the specific requirements of these analytical methods before
they can be applied. Furthermore, the environments are sometimes
not physiological, e.g., the vacuum in experiments with electron
beams. Autoradiography and fluorescence depend on exogenous
fluorophores or other contrast enhancing agents because most bio-
molecules cannot directly be detected. A decision has to be made in
advance: Which property should be probed and which marker should
be used? Problems result from the limited stability, bleaching, and
restricted accessibility of external markers. In principle, Raman imag-
ing can overcome many restrictions. It combines molecular specific-
ity with diffraction limited spatial resolution in the sub-micrometer
range. Due to its nondestructivity using near-infrared wavelengths
for excitation, it can be applied under in vivo conditions without
staining or other markers because the Raman signals are based on
inherent vibrational properties of the cells’ biomolecules. All vibra-
tional signatures overlap giving complex spectra. High signal to noise
ratios are required to identify subtle spectral differences. Further-
more, Raman spectroscopy is a rapid technique because single Raman
spectra can be recorded within seconds. Raman signals can be
enhanced using special techniques such as resonance Raman spec-
troscopy, surface-enhanced Raman spectroscopy (SERS) and coher-
ent anti-Stokes Raman spectroscopy. All these techniques have
successfully been applied for single cell studies. Single cells are very
suitable objects for Raman spectroscopy because of the high concentra-
tions of biomolecules in their condensed volume. Protein concentration