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sFTIR, Raman, and SERS Imaging of Fungal Cells 151

spectroscopy (and spatial scale) to others. Other imaging tools that
we presently employ include the sFTIR and Raman methods described
above, several more conventional fluorescence staining, AFM, SEM,
TEM, x-ray fluorescence, high-spatial resolution SIMS, as well as the
tools of molecular genetics.
Experimental design must be formed around the questions being
posed and the methodologies chosen. When considering the desired
detection limits in complex biological systems, from desiccated fun-
gal samples described here, to the possibilities of in vivo imaging dis-
cussed elsewhere, single molecule detection may be only one of
several goals. In the case of fungal analysis with SERS, for example,
we must clarify what competes for binding to the most sensitive sites,
which compounds might have a more intense signature, and which
might interfere. There is now a considerable body of literature on the
36
gross analysis and classification of biomaterials from bacteria and
47
83
yeasts to cancerous tumors. In the case of fungal identification, it
has been shown that different species have different spectroscopic
characteristics on which classifications may be based in a statistically
84
robust manner. Our investigations are directed toward understand-
ing fungal lifestyle and the biochemical changes that occur during
growth cycles. Spatially resolved molecular information is the goal in
this work; single molecule detection may well be unimportant.
Multiple copies of the same proteins are likely released, albeit at low
levels, to support nutrient acquisition and adhesion activities. Charac-
terization of normal development may not require identification of
specific molecules as much as it will require reproducibility of signals.
When the latter are achieved, alterations in signals caused by stress,
genetic alterations, or antifungal treatments can be more easily detected.
The combined application of multiple spectroscopic tools, properly cor-
related, will continue to be a most fruitful direction of investigation.

Acknowledgments
We are pleased to acknowledge Natural Science and Engineering
Research Council (NSERC) Discovery Grant awards to KMG and
SGWK, and operating grant to KMG from Canadian Institutes of Health
Research (CIHR), and a CIHR-Regional Partnership Program grant to
SGWK. Equipment grants were supported by NSERC, Western Eco-
nomic Diversification Canada, and the University of Manitoba. This
work is based in part upon research conducted at the Synchrotron Radi-
ation Center, University of Wisconsin-Madison, which is supported by
the National Science Foundation under Award No. DMR-0537588. The
technical assistance of Dr. Robert Julian (SRC), Randy Smith and Dr. Lisa
Miller (NSLS) is gratefully acknowledged. Fusarium was provided by
Dr. R. J. Rodriguez (USGS, and University of Washington at Seattle) and
Dr. R. S. Redman (University of Washington at Seattle). Magnaporthe