Page 169 - Vibrational Spectroscopic Imaging for Biomedical Applications

sFTIR, Raman, and SERS Imaging of Fungal Cells 145

evidence for the detection of single molecules with SERS has very
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recently been published. The elegant proof involved detection of a
strong Raman scatterer (a dye: Rhodamine 6G) attached to silver
aggregate nanoparticles. By ensuring that the number of silver parti-
cles greatly exceeded the number of dye molecules, and using two
isotopomers which had distinguishable spectra, they were able to
demonstrate anticorrelation, that is, the spectra were, in the main,
either one isotopomer or the other, but almost never both. The
enhancement was aided by the match between the exciting laser line
and an electronic transition of the dye molecule, thus the phenome-
non was actually SERRS. Discussion continues regarding the possi-
bility of single molecule detection, but the fact of signal enhancement
is indisputable, as is the fact that it can be utilized. Ways and means
of utilization include tethering chromophores to be used as selective
tags, injecting gold nanodots, etc., as discussed in the numerous
recent reviews and articles. For fungi, it should be possible to prepare
growth media that contains nanoparticles that may interact with the
developing hyphae; however, such experiments must be approached
with caution, to ensure that abnormal physiological responses are not
provoked.

5.6.1 Substrates: The Key to SERS Imaging
In order to obtain further spectroscopic information on fungal devel-
opment, we have begun a study with SERS imaging that parallels the
sFTIR and Raman imaging to date. Nanodots are colloidal particles
with a range of diameters; it is now thought that the greatest enhance-
ment may be associated with the small crevices formed within
nanodot aggregates. Rather than embark on the development of in-
house substrates, for convenience, we elected to use the commer-
cially available Klarite substrates (D3 Technologies Ltd., Glasgow).
While nanodots offer much promise for other applications, we have
found these regularly nanopatterned substrates provide significant
enhancement for the rapid imaging of developing hyphae and pos-
sibly of fungal exudates. Other types of regularly patterned substrate
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are being developed. Many challenges remain; our recent results are
described below.
5.6.2 SERS: Applications for Fungi
All fungi secrete compounds during growth; these molecules have
roles in substrate adhesion and/or nutrient acquisition. Spores and
germination structures of plant pathogens may attach to hydropho-
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bic leaf surfaces by specialized adhesives. Excellent images of hyphal
adhesive spore tips and exudates are given in Ref. 78. Much current
research is directed toward understanding the fungal “secretome,” on
the assumption that most interesting molecules are proteins, but
spatial resolution and appropriate tools for molecular identification
remain challenging issues.