Page 173 - Vibrational Spectroscopic Imaging for Biomedical Applications

sFTIR, Raman, and SERS Imaging of Fungal Cells 149

adjacent to a Klarite substrate and hyphae were allowed to grow
overnight in a moist chamber at 28ºC. After freezing and drying, the
sample is ready for spectroscopic analysis.
The region shown in Fig. 5.13a contains a hypha that has grown
out several millimeters from the agar block and is free of any other
material. Several spores from the mature base of the colony have fallen
onto the substrate close to the growing hypha, and under the humid
growing conditions, have just germinated. Clumps of spores are more
likely to germinate in nutrient-free conditions than are isolated spores.
Short germ tubes can be seen emerging from several of these spores.
A Raman map of the area was recorded with the 785 nm edge set at
5.0 percent power, 1200 L/mm grating, exposure time 4.00 seconds for
an entire column or about 200 ms/point. The SERS enhancement is
immediately obvious from the processed image (Fig. 5.13b) that shows
−1
the intensity of the peak at 1050 cm , characteristic of these hyphae
51
under normal Raman spectroscopy. Four spectra representative of the
variety observed in this map are shown in Fig. 5.13c. The weakest
intensity (purple) is associated with the hypha growing across the
−1
smooth gold surface. The 1050 cm peak is not visible; the fact that this
region even registers in the mapping image is due to the slight increase
in baseline from the natural fluorescence of the biomaterial. The next
strongest signal (green) comes from the mature hypha on the SERS
−1
patterned surface. Here the 1050 cm peak is clearly apparent.
The signal enhancement factor provided by the SERS active substrate
cannot be properly quantified from this map, since we have no signal at
all from the smooth gold for comparison in this map, but it is clearly sev-
eral orders of magnitude. An estimate is obtained by comparison between
the green spectrum from Fig. 5.13 with a Raman spectrum from a hypha
on the smooth gold surface, illustrated in Fig. 5.14. The Raman spectrum
was acquired with 640 seconds of exposure at 0.1 percent laser power,
compared to 0.045 seconds exposure at 0.5 percent laser power and
the same optics for the SERS spectrum. From the signal to noise for these two
spectra, scaled for power and exposure time, we get a rough estimate of 4000
as the SERS enhancement factor.
Some additional peaks appear in spectra from regions containing
the germinating spores (blue, red) in Fig. 5.13. We interpret these
strong spectral peaks as arising from the minute quantities of exu-
date produced as hyphae extend, from spores as they begin the ger-
mination process or from adhesion materials associated with the
spore. Such molecules would likely be able to occupy SERS hot
spot sites within the pyramidal wells or at the edges of the wells,
where the enhanced electric field will provide the expected SERS
magnification. Again, neither the molecular identity of these com-
pounds nor the quantity can be established at this point; thus the
true SERS enhancement factor cannot be obtained. Likewise, we
can only speculate about the possibility that we are detecting spectra
from single molecules, or even few molecules.