Chapter 4 – Making Light Work Harder in Biology  153

coherent Raman spectroscopy methods. This has significant advantages for this emerging

field of chemical imaging; this is an example of a biophysical imaging technique used to

investigate the chemical environment of biological samples, and improving the time reso­

lution enables better insight into the underlying dynamics of this chemistry.

4.7.4  COHERENT RAMAN SPECTROSCOPY METHODS

Two related Raman techniques that specifically use coherent light sources are coherent anti-​

Stokes Raman scattering (CARS) and coherent Stokes Raman scattering (CSRS, pronounced

“scissors”). However, CSRS is rarely favored over CARS in practice because its scattering

output is at higher wavelengths than the incident light and is therefore more likely to be

contaminated by autofluorescence emissions. CARS, in particular, has promising applications

for in vivo imaging. Both CARS and Raman spectroscopy use the same Raman active vibra­

tion modes of molecular bonds, though the enhancement of the CARS signal compared to

conventional Raman spectroscopy is manifested in an improvement in sampling time by a

factor of ~10⁵. In other words, CARS can be operated as a real-​time video-​rate technique.

CARS is a third-​order nonlinear optical effect, which uses three lasers, one to pump at

frequency ωpump, a Stokes laser of frequency ωStokes, and a probe laser at frequency ωprobe. All

these interact with the sample to produce a coherent light output with anti-​Stokes shifted

frequency of (ωpump +​ ωprobe − ωStokes). When the frequency difference between the pump and

the Stokes lasers (i.e., ωpump − ωStokes) coincides with the Raman resonance frequency of a

specific vibrational mode of a molecular bond in the sample, there is a significant enhance­

ment of the output. Molecules such as lipids and fatty acids, in particular, have strong Raman

resonances. When combined with microscopic laser scanning, 2D images of in vivo samples

can be reconstructed to reveal high-​resolution details for cellular structures that have high

fat content, a good example being the fatty myelin sheath, which acts as a dielectric insulator

around nerve fibers, with some CARS imaging systems now capable of video-​rate time reso­

lution. This technology not only gives access to dynamic imaging of biochemical components

in cell populations that are difficult to image using other techniques but like conventional

Raman spectroscopy is also a label-​free approach with consequent advantages of reduced

impairment to biological functions compared to technologies that require components to be

specifically labeled.

4.7.5  BRILLOUIN LIGHT SCATTERING

When light is transmitted through a sample that contains periodic variation in refractive

index, Brillouin light scattering (BLS) can occur. In a biological sample, the most likely cause

of Brillouin inelastic scattering is the interaction between acoustic oscillation of the sample

matter, in the form of phonons, and the incident light. Phonons can establish standing waves

in a sample, which causes the periodic changes to the molecular structure correlated over

an extended region of the sample, resulting in periodicity in refractive index over the length

scale of phonon wavelengths.

Similar to Raman scattering, an incident photon of light can gain energy from a phonon

(Stokes process) or lose energy in creating a phonon in the material (anti-​Stokes process),

resulting in a small Brillouin shift to the scattered light’s wavelength. The shift can be quanti­

fied by a Brillouin spectrometer, which works on principles similar to a Fabry–​Pérot interfer­

ometer (or etalon), as discussed in Chapter 3. Although not a unique fingerprint in the

way that a Raman spectrum is, a Brillouin spectrum can render structural details of optically

transparent biological materials, for example, to investigate the different optical materials in

an eye. The largest technical drawbacks with Brillouin spectroscopy are the slowness of sam­

pling and the often limited spatial information, in that most devices perform essentially point

measurements, which require several minutes to acquire. However, recent developments

have coupled Brillouin spectroscopy to confocal imaging to generate a Brillouin microscope,

with improvements in parallelizing of scatter signal sampling to permit sampling rates of