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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