Page 155 - Vibrational Spectroscopic Imaging for Biomedical Applications

sFTIR, Raman, and SERS Imaging of Fungal Cells 131

The frequency of each vibration depends on the unique mode of vibration:
the atomic masses being displaced and the strength of the connecting
bonds. In IR spectroscopy, the intensity of the peak depends on the
magnitude of the change in the permanent molecular dipole moment
accompanying the vibration. A beam of light containing the entire mid-IR
radiation band is passed through (or reflected off) a sample. The transmit-
ted (or reflected) light is sent to a detector and displayed as a spectrum
(graph) of the amount of light absorbed at each frequency (energy). The
mathematics underlying the FT approach is extensively documented in
28
the literature, and described in many of the chapters in this book. Its
counterpart, Raman spectroscopy, provides information on the same
molecular vibrations, and is similarly well documented. 29,30
Raman spectroscopy is complementary to IR. Monochromatic laser
light is inelastically scattered off the sample. Some of the scattered light
is of lower frequency than that of the laser; the missing energy has been
deposited in the molecule, in the form of vibrational energy. The physical
process of excitation is different from IR; the resultant spectra are
different. In Raman scattering, the intensity, or differential scattering
cross section, recorded for each vibration depends on the induced change
in the molecular polarizability accompanying each molecular vibration.
Different modes may be excited by IR and Raman; where the same
modes are excited, frequencies are the same but relative intensities differ.
The use of vibrational spectroscopy in the study of biological
materials was suggested early in the history of the technique and the
first experiments on tissues were performed over 60 years ago. 31–33
Widespread use in the biological sciences began with the advent of the
interferometer-based instruments (FTIR) in the 1970s, until it became a
recognized standard tool for the analysis of model systems in vitro, as
well as of components in biotissues in situ. 34–38 For fungi, in situ refers
to samples of cells or tissues that have been grown on the imaging
substrate or taken from natural systems, and preserved without
chemicals. A key aspect is that characteristic absorption peaks from all
tissue components (proteins, lipids, carbohydrates, and nucleic acids)
may be detected in a single spectrum.
Coupled to a microscope, IR and Raman spectra may be obtained
from spot sizes of 1 to 100 μm diameter, permitting subcellular analysis
of biological tissues. 39–41 Both techniques may be used to determine
molecular component localization: relative amounts and structure of
tissue constituents can be ascertained in situ, without staining. The two
main considerations are (1) spectral differences and (2) spatial resolu-
tion. Relevant factors in the success of spectral analysis include:

• Degree to which the components differ from each other
chemically (different functional groups, significant confor-
mational differences).
• Presence of strong absorbance peaks arising from these
chemical differences.