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Raman Imaging for Biomedical Applications in Clinics   273


            Point and line Raman mapping, involve a laser spot or a laser line
        sample illumination. In point mapping, a laser spot is scanned in two
        spatial dimensions (x and y) with small step increments and a spectrum
        is collected at each pixel. The laser can be focused to a diffraction-limited
        spot size, and at such a high-spatial-resolution, spectra of subcellular
        organelles can be obtained with features dominated by only a single bio-
        chemical component like DNA or lipids. These point-to-point measure-
        ments may exhibit large spectral variation, and collection of hundreds or
        thousands of spectra is required to completely characterize a cellular sys-
        tem. Since a complete Raman spectrum is associated with each pixel,
        such maps can be used to generate detailed chemical images revealing
        the distribution of different molecular components in a cell. For many
        tissue-imaging applications such a high-spatial resolution is not required.
        If a lower-spatial resolution in the image plane is desired while main-
        taining the confocality, the laser spot or the stage can be moved during
        measurement, thus averaging out over the illuminated area.
            Line mapping is an extension of point mapping that makes full
        use of a two-dimensional image sensor such as a CCD. By illuminat-
        ing the sample with a line using a cylindrical lens or scanning mirror(s),
        the whole line can be imaged on the CCD- the spatial data are regis-
        tered on the detector parallel to the entrance slit of the spectrometer,
        while the spectral information is dispersed perpendicularly.
            In the direct (also called global or widefield) imaging approach, all
        spatial points of the Raman image at a specific wavenumber (range)
        are imaged simultaneously on the detector. In contrast to point map-
        ping approaches, direct Raman imaging methods employ global or
        widefield sample illumination. The Raman scattered light from the
        sample is collected, filtered, and magnified onto a two-dimensional
        CCD detector. Commonly used filter types include dielectric, acousto-
        optic, and liquid-crystal tunable filters (LCTFs). Using high-quality
        refractive microscope objective with LCTF-based Raman imaging sys-
        tems provides sufficient magnification, the resolution can be diffrac-
        tion limited (around 250 nm for 514 nm laser, that is d = 1.22λ/NA). 27
            An interesting comparison between the different Raman imaging
                                            28
        techniques was made by Schlucker et al. ; they implemented the
        three different Raman imaging modalities (point mapping, line map-
        ping, and direct global imaging) within a single instrumental config-
        uration that shares a similar optical path and the same microscope
        objective and CCD detector. As a well-defined test sample for use
        with all three techniques, they employed a scanning electron micros-
        copy (SEM) standard consisting of a grid of 5-μm squares of silicon.
        All imaging was done with 514 nm excitation. A detailed experimen-
        tal comparison was made of the various Raman imaging methodolo-
        gies with a sample-limited excitation power, in which the advantages
        and limitations of each method based on their spectral SNR, spatial
        resolution, and data acquisition times were considered. In Table 9.1
        the parameters and results are summarized.
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