Chapter 4 – Making Light Work Harder in Biology  151

advantage over fluorescence detection in not requiring the addition of an artificial label to

the biomolecule.

The Raman effect can be utilized in biophysics techniques across several regions of the

electromagnetic spectrum (including x-​rays, see Chapter 5), but most typically, a near IR

laser (wavelength ~1 μm) is used as the source for generating the incident photons. The shift

in NIR Raman scattered energy for biomolecules is typically measured in the range ~200–​

3500 cm⁻¹. Lower energy scattering effects in principle occur in the range ~10–​200 cm⁻¹;

however, the signal from these typically gets swamped by that due to Rayleigh scattering.

A Raman spectrometer consists of a laser, which illuminates the sample, with scattered

signals detected at 90° from the incident beam (Figure 4.4d). A notch rejection filter, which

attenuates the incident laser in excess of 10⁶ over a narrow bandwidth of a few nanometers,

eliminates the bulk of the elastic Rayleigh scattering component, leaving the inelastic Raman

scattered light. This is imaged by a lens and then spatially split into different color components

using a diffraction grating, which is projected onto a CCD detector array such that different

pixel positions correspond to different wavenumber shift values. Thus, the distribution of

pixel intensities corresponds to the Raman spectrum.

In principle, Raman spectroscopy has some similarities to IR spectroscopy discussed in

Chapter 3. However, there are key differences to IR spectroscopy, for example, the Raman

effect is scattering as opposed to absorption, and also although the Raman effect can cause

a change in electrical polarizability in a given chemical bond, it does not rely on exciting a

different bond vibrational mode, which has a distinctly different electrical dipole moment.

Key biomolecule features that generate prominent Raman scattering signatures include many

of the bonds present in nucleic acids, proteins, lipids, and many sugars. The weakness of

the Raman scatter signal can be enhanced by introducing small Raman tags into specific

molecular locations in a sample, for example, alkyne groups, which give a strong Raman

scatter signal, though this arguably works against the primary advantage of conventional

Raman spectroscopy over fluorescence-​based techniques in being label-​free.

4.7.2  RESONANCE RAMAN SPECTROSCOPY

When the incident laser wavelength is close to the energy required to excite an electronic

transition in the sample, then Raman resonance can occur. This can be especially useful in

enhancing the normally weak Raman scattering effect. The most common method of Raman

resonance enhancement as a biophysical tool involves surface-​enhanced Raman spectros­

copy (SERS), which can achieve molecular level sensitivity in biological samples in vitro (see

Kneipp et al., 1997).

With SERS, the sample is placed in an aqueous colloid of gold or silver nanoparticles,

typically a few tens of nanometers in diameter. Incident light can induce surface plasmons

in the metallic particles in much the same way as they do in surface plasmon resonance (see

Chapter 3). In the vicinity of the surface, the photon electric field E is enhanced by a factor

~E⁴. This enhancement effect depends sensitively on the size and shape of the nanoparticles.

For spherical particles, the enhancement factor falls by 50% over a length scale of a few

nanometers.

Heuristic power-​law dependence is often used to model this behavior:

(4.41)

I z

I

R

R

z

a

( ) = ( )

+

(

)

0

where I(z) is the Raman scatter intensity at a distance z from surface of a spherical particle of

radius R. Although different experimental studies suggest that the parameter a varies broadly

in the range ~3–​6, with ~4.6 being given consensus by many.

A typical enhancement in measurement sensitivity, however, is >10⁵, with values up to

~10¹⁴ being reported. Therefore, if a biomolecule is bound to the surface of a nanoparticle,