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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
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