Page 291 - Vibrational Spectroscopic Imaging for Biomedical Applications

266 Cha pte r Ni ne

IR absorption has been very popular in the early periods of biomedi-
cal vibrational imaging due to its relatively high-signal strength, but
Raman spectroscopy is currently becoming the method of choice
because a higher-spatial resolution can be achieved, smaller sensitiv-
ity to artifacts and presence of water, and possibility of application in
vivo. Technological improvements in the last two decades have helped
to overcome the sensitivity problem of Raman spectroscopy and have
made the technique broadly applicable in biomedical research. 2
Because of the heterogeneous nature of cells and tissues, single
point Raman microspectroscopy cannot adequately describe the
chemical microstructure of cells and tissues. Also spatial information
about molecular concentrations is needed. By measuring Raman spec-
tra as a function of the position within the sample, a three-dimensional
(x, y, and λ), or even four-dimensional dataset (x, y, z, and λ) can be
obtained. Because Raman spectroscopy is an optical technique, the
spatial resolution is determined by the diffraction limit.
From such a multidimensional spectral dataset, one can generate
many Raman images, depending on which part of the spectral infor-
mation is used for generating the contrast in the image. Studying the
detailed molecular composition and dynamics of complex organized
systems such as cells and tissues using Raman spectroscopic imag-
ing is becoming increasingly popular. There is no need for dyes or
labels, and one can look at different biochemical aspects of the sam-
ple simultaneously. 3,4
Raman imaging has high potential as a clinical diagnostic tech-
nique that offers hematologists and pathologists spatially resolved
5–9
molecular information on their patient material. It can also be used
by surgeons to determine whether the resection margins of excised
tumors are free of cancerous cells. However, such applications of the
technology are hampered by the time it currently takes to obtain a
Raman image with a sufficient resolution and spectral quality. 10,11
In this chapter an overview is given of the current state of the art
in instrumentation and methodologies for biomedical Raman imag-
ing. We have limited the technical overview to dispersive Raman
instrumentation (leaving out FT Raman spectroscopy and techniques
like CARS and SERS) as this measurement technique is most effective
in collecting Raman photons and therefore in our view most suited
for Raman imaging of biomedical samples, which is mostly signal
intensity limited. Potential areas of application of Raman imaging in
(bio) medical research and diagnosis are discussed and illustrated
with examples from the recent literature. Lastly, current limitations in
(clinical) application of the technique, and perspectives to overcome
these limitations are reviewed.

9.1.1 History
Observing the wonderful blue opalescence of the Mediterranean
Sea during a voyage to Europe in the summer of 1921, gave Sir