Page 161 - Vibrational Spectroscopic Imaging for Biomedical Applications

sFTIR, Raman, and SERS Imaging of Fungal Cells 137

Other options available on current IR bench instruments are
approaching spatial and spectral quality from synchrotron sources.
An instrument with a focal plane array (FPA) detector would still
require extensive coaddition of scans in order to achieve sufficient
signal, but the array collects spectra from an entire map area (e.g., for
nominal 5.5 μm pixel, a 64 × 64 array would cover about 350 × 350
2
μm ) in a single scan. Thus, a 2-3 hour scan would produce excellent
S/N and retain spatial resolution of about 7 μm. One problem here is
that each individual detector in the array is slightly different, and
while one attempts to obtain similar signal at each detector, it is not
possible to calibrate the detectors to guarantee uniform absorbance
characteristics. A system in which an infrared spectrometer and
microscope equipped with an FPA detector has just being commis-
sioned at the Synchrotron Radiation Center, University of Wisconsin at
Madison (see Chap. 2).

5.5 Raman Spectroscopy of Fungi
55
Raman spectroscopy was first shown by C. V. Raman. In this
phenomenon, molecular vibrations are excited through inelastic
scattering of incident radiation. The magnitude of the scattering
cross section for a vibrational mode, counterpart to IR band inten-
sity, depends on the magnitude of the change in the molecular polar-
izability tensor α during a vibration, where α is a measure of the
magnitude of the dipole moment μ induced in a molecule when an
electric field ξ is applied:

μ = αξ (5.1)

The derivative of the polarizability with respect to some normal
mode of vibration q is denoted ∂α/∂q. Note that Eq. (5.1), when α is
expressed in Å , is isomorphic to that defining the dipole moment
3
induced in a perfectly conducting sphere by an external field:
3
μ = r E (5.2)

This latter relationship is highly important for SERS of nanoparti-
cles (see Sec. 5.6). Raman scattering from molecules provides a
molecular fingerprint for every molecule, hence just as for FTIR,
Raman offers tremendous potential value for sensitive analyte recog-
nition. The vibrational spectra obtained with Raman can, in principle,
be interpreted in terms of molecular composition and in some case,
changes in molecular environment. The process of Raman scattering is
well understood and is described in many standard texts. 29,30 Accord-
56
ing to polarizability theory, the differential Raman scattering cross
section of a fundamental vibrational band (∂σ/∂Ω, where Ω = solid