Page 150 - Vibrational Spectroscopic Imaging for Biomedical Applications

126 Cha pte r F i v e

imaging modalities; there are some very good books and numer-
ous recent review articles to which we refer the interested reader. 1–7
In the 80 years since Raman spectroscopy was first demonstrated,
perhaps nothing has received as much fanfare as the potential
applications of SERS and surface enhanced resonance Raman spec-
troscopy (SERRS) for highly sensitive detection down to single
molecule level, thus rivaling fluorescence while providing better
molecular identification. This chapter presents a brief overview of
some of the latest theories, methods, and applications (see Sec. 5.6).
This should in no way be taken to imply that the method is now
reliable and robust. However, the promise remains bright, and new
strategies to achieve the goals are appearing at an ever-increasing
rate.
We devote a portion of the chapter to fungi, wonderful subjects to
illustrate the power, the promise and the challenges in these techniques.
Fungi are exemplary experimental model systems due to their tractable
genetics, and their significant impacts on biotechnology as well as on
human and plant health. High-spatial resolution chemical analyses are
essential to correlate information from other experimental methods
with cell genotype and phenotype. Fungal hyphae (tubular cells that
form most species) vary tremendously in composition and function
over micrometer (μm) spatial scales, so analytical methods such as
NMR and mass spectrometry are impractical for understanding fungal
biology. FTIR microscopy is an excellent analytical methodology for
chemical characterization at high-spatial resolution; however, FTIR
analysis of fungal cells is challenging because information on cell walls,
through which the hyphae interact with their environment, relates
mostly to carbohydrate composition. The most distinctive carbohydrate
signatures are at the longer infrared wavelengths (circa 10 μm), where
the diffraction limit is 5 to 10 μm at best. Fungal cells are typically
rounded and 3 to 10 μm wide (though hundreds of microns long), so in
addition to diffraction issues, spectra are prone to scattering artifacts.
Also, fungal cells are supported internally by water pressure, so they
have relatively low biomass and the signal from individual spores or
from hyphal tips is weak. With a synchrotron source to offset these
issues, FTIR can assess subtle and cell type specific biochemical differ-
ences in fungal cells. 8,9
Our ongoing research is focused mainly on imaging single fun-
gal cells, with the biological goal of elucidating the biochemistry of
fungal lifestyles and the spectroscopic goal of advancing the technol-
ogy. For the spectroscopists in the audience, we will present some
background information on fungi; for the fungal biologists who may
be interested in applying these spectroscopic tools to their own
research, we present a basic explanation of the fundamentals;
finally we present an overview of the ways in which we have been
applying these tools in our work. The research presents a curious