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Raman Imaging for Biomedical Applications in Clinics   271


        suppression of light at the laser wavelength (preferably by 8 or more
        orders of magnitude) with a very high transmission of Raman signal,
        starting at wavelengths close to the laser line.
            The first filters that were used were edge filters, which transmit
        low-wavelength side from excitation, Stokes Raman lines. Later holo-
        graphic notch filters were made to suppress stray light and very effec-
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        tively filter the Rayleigh line (10 ). They have a very high laser line
        rejection which enables measurements of bands close to the laser line,
        but they are sensitive to temperature and aging. The same high laser
        line rejection ratio can be achieved with a chevron-type filter, which is
        a combination of two dielectric high-pass filters, between which the
        light is reflected multiple times—in every pass the most of the signal
        reflects and the laser line is absorbed. 17,25  For global illumination, when
        the whole field of view is illuminated and the whole image is collected
        in a narrow spectral interval, commonly used filter types include
        dielectric, acousto-optic, and liquid-crystal tunable filters (LCTFs).
        Using a high-quality refractive microscope objective with LCTF-based
        Raman imaging systems provides sufficient magnification, so the reso-
        lution becomes diffraction limited (around 250 nm for 514 nm laser).
                                            −1
        Nowadays narrowband LCTFs (5 to 8 cm ) are available that allow
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        high-spectral-resolution imaging. However, for recording the whole
        spectrum, this type of imaging is very inefficient, since most generated
        Raman photons will be rejected by the filter. Therefore this technique
        seems only useful for band or band-ratio imaging.

        9.2.4 Spectrometer
        The standard configuration for a dispersive spectrometer is either a
        triple monochromator or a single monochromator in combination
        with high-quality laser line suppression filters. In a triple monochro-
        mator the first two stages are used for the elimination of the Rayleigh
        scattered light, whereas the third monochromator, the so-called spec-
        trograph, disperses the collected Raman radiation onto a multichan-
        nel detector or a CCD camera. Then the Raman spectra can be
        recorded very close to the Rayleigh line (within a few wavenumbers),
        but the transmittance of such systems is low. Much higher collection
        efficiency can be obtained with a single grating monochromator in
        combination with a notch or chevron-type filter.

        9.2.5 CCD
        The detectors that are now used are multichannel detectors, either
        intensified diode arrays but in most cases CCDs. A standard silicon
        CCD is sensitive from 400 to 1000 nm and has a quantum efficiency
        (QE) in the NIR of around 40 percent. To make it more sensitive for
        NIR signal, deep depletion silicon is used. Nowadays back-thinned
        (or back-illuminated) deep depletion CCDs are used with a QE up to
        80 percent. Cooling of the CCD (down to −70°C) helps to reduce the
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