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sFTIR, Raman, and SERS Imaging of Fungal Cells   135


        bands in this region. The variation in the CH profile from A. nidulans
        through Neurospora to Rhizopus may be attributed to both membranes
        and walls. This same characteristic feature is also responsible for the
        increasing slope of the baseline, arising from scattering artifacts
        associated with the thick, rounded cell structure (see spectral
        anomalies,  below). More recently, we have been using a different
        preparation, whereby the hyphae are allowed to thaw for 10 seconds
        at room temperature prior to the drying step. This permits the cells to
        collapse slightly, reducing the scatter, but is not long enough for
        enzyme-induced degradation.
                                           51
            Prior to the acquisition of sFTIR data,  it had been thought that,
        within a saprotrophic hypha, the biochemical content would be
        mainly concentrated toward the growing, organelle-rich, metabolically
        active tips, since much of the cytoplasm actively migrates to keep up
        with the extending tip, whereas vacuoles predominate in basal
               10
        regions.  Our sFTIR studies have shown that vacuolate regions are
        far richer in total biochemical content than had been anticipated. The
        spectra in Fig. 5.3 not only illustrate the impressive differences in
        sugar wall composition and structure that exist between different
        species, they also illustrate that Aspergillus hyphae differ biochemically
        150 μm from the tip, although atomic force microscopy used for imag-
        ing and elastic modulus shows that Aspergillus cell wall surfaces have
        matured by 3 μm from the tip. 52
            The sFTIR spectra have also proved useful in demonstrating drastic
        differences in cell wall composition in an A. nidulans temperature-
        sensitive mutant,  hypA1, that had previously been inferred from
        transmission electron microscopy. 18,51,53  Visible light micrographs of
        the hypA1 mutant (Fig. 5.4a) grown at permissive and restrictive
        temperatures illustrate the morphological differences provoked by
        growth at slightly elevated temperatures. The sFTIR spectra show
        significant elevation of absorption across the carbohydrate fingerprint
        region. The bottom spectrum, showing profiles of hypA1 tips grown
        at 28°C, is actually the sum of spectra for five tips that had grown
        together. Even so, the intensity of the amide I band is <0.1 absorbance
        unit, much less that that of a single tip grown at 42°C (~0.15 unit). The
        S/N in the sugar region is only about 3:1, and is heavily affected by
        diffraction limitations. The restrictive phenotype growth can be seen
        for the hypA1 mutant even at 39°C (Fig. 5.4c, top) compared to a wild
        type A. nidulans strain in which long hyphae grow far across the slide.
        The samples in Fig. 5.4c were grown on the same slide, and thus
        experienced identical growing conditions.
            The greater hyphal diameter of some species presents a different
        kind of challenge; rounded cell walls can produce huge scattering
        artifacts, as seen in some much poorer spectra shown in Fig. 5.5 . Sim-
        ilar problems have been identified in the FTIR spectra of cell types,
        wherein the Mie scatter from the nuclei produced comparable spec-
        tral problems. 54
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