Introduction to Spectroscopy - Vibrational Spectroscopy for Tissue Analysis - page 6

(a) (b) (c)

Figure 1.4
Different Raman scattering processes. (a) Shows spontaneous Raman scattering produced by
scattered laser beam causing vibrations of chemical bonds; (b) A coherent Raman signal on
the anti-Stokes side of the spectrum is generated when two or more coherent laser beams with
different frequencies overlap spatially and temporally onto the specimen; (c) The Coherent
anti-Stokes Raman spectroscopy (CARS) signal is produced by the nonlinear mixing of the
ultrashort excitation pulses and is several order of magnitude stronger than the spontaneous
Raman signal. Molecules adsorbed on metallic nanostructures will experience a strong elec-
tromagnetic field in addition to chemical enhancement, due to the presence of plasmon reso-
nances near the surface of metals.

and represented as 1/cm. Raman peaks are spectrally narrow, and in many
cases can be associated with the vibration of a particular chemical bond (or
a single functional group) in the molecule [16,17,19,20,21]. The vibrations are
molecular bond specific allowing a biochemical fingerprint to be constructed
of the material [8].
Confocal Raman microspectroscopy (Figure 1.5) is the amalgamation of
traditional Raman spectroscopy and confocal microscopy, and allows the exam-
ination of Raman spectra from small volumes. Briefly, a temperature-stabilised
diode laser operating at 785 nm is expanded and introduced via a holographic
notch filter (HNF) into an inverted microscope and passed to the sample via
an objective. The backscattered Raman light is collected by the same objec-
tive and passed through the HNF. The Raman signal is then reflected by the
dichroic mirror and imaged onto a confocal aperture. Finally, the beam is
imaged onto the spectrograph [22].
It is important to note that depending on whether the bond length or angle
is changing, vibrations are subdivided into two classes:

• Stretching (symmetric and asymmetric)
• Bending (scissoring, rocking, wagging and twisting) (Figure 1.6)

In other words, there are six different vibrational modes for chemi-
cal bands. In symmetric and asymmetric stretching types, the lengths of