150 4.7  Tools Using the Inelastic Scattering of Light

technique (see Chapter 3), which demonstrates the clear problem with attempting to detect

the scatter signal from small biomolecules directly. However, the interference term 2R|s|sin ϕ

only scales with V and so is far less sensitive to changes in scatterer size, and the detection of

this term is the physical basis of iSCAT.

An iSCAT microscope setup is similar to a standard confocal microscope, in terms of

generating a confocal laser illumination volume, which is laterally scanned across the

sample, though instead of detecting fluorescence emissions, the interference term intensity is

extracted by combining a quarter wave plate with a polarizing beamsplitter. This utilizes the

phase difference between the interference term with respect to the incident illumination and

rotates this phase to enable highly efficient reflection of just this component at the polarizing

beamsplitter, which is directed not through a pinhole as for the case of traditional confocal

microscopy but rather onto a fast CCD camera, such as a CMOS camera.

An enormous advantage of iSCAT, and similar interferometric imaging methods, over

fluorescence imaging is speed. Fluorescence imaging can achieve a significant imaging con­

trast but to do so ultimately requires a sufficiently large sampling time window to collect

fluorescence emission photons. Fluorophores, as we have seen, are ultimately limited by the

number of photons that they can emit before irreversible photobleaching. Interferometric

scattering is not limited in this manner; in fact, the background signal in iSCAT scales with

~√N from Poisson sampling statistics, where N is the number of scattered photons detected;

therefore, since the signal scales with ~N, then the imaging contrast, which is a measure of

the signal-​to-​noise ratio, itself scales with √N. That is, a larger contrast is achievable by simply

increasing the power of laser illumination. There is no photon-​related physical limit, rather a

biological one in increased sample damage at high laser powers.

4.7  TOOLS USING THE INELASTIC SCATTERING OF LIGHT

Scattering of light, as with all electromagnetic or matter waves, through biological matter is

primarily due to linear optical processes of two types, either elastic or inelastic. Rayleigh and

Mie scattering (see Chapter 3) are both elastic processes in which the emergent scattered

photon has the same wavelength as the incident photon. One of the key inelastic processes

with regard to biophysical techniques is Raman scattering. This results in the incident photon

either losing energy prior to scattering (Stokes scattering) or gaining energy (anti-​Stokes

scattering). For most biophysical applications, this energy shift is due to vibrational and rota­

tional energy changes in a scattering molecule in the biological sample (Figure 4.4c), though

in principle the Raman effect can also be due to interaction between the incident light and to

a variety of quasiparticles in the system, for example, acoustic matter waves (phonons). There

are also other useful inelastic light scattering processes that can also be applied to biophysical

techniques.

4.7.1  RAMAN SPECTROSCOPY

Raman scattering is actually one of the major sources of bleed-​through noise in fluorescence

imaging experiments, which comes mainly from anti-​Stokes scattering of the incident exci­

tation light from water molecules. A Raman peak position is normally described in terms

of wavenumbers (2π/​λ with typical units of cm⁻¹), and in water, this is generally ~3400 cm⁻¹

lower/​higher than the equivalent excitation photon wavenumber depending on whether the

peak is Stokes or anti-​Stokes (typically higher/​lower wavelengths by ~20 nm for visible light

excitation).

Some dim fluorophores can have comparable Raman scatter amplitudes to the fluores­

cence emission peak itself (i.e., this Raman peak is then the limiting noise factor). However, in

general, the Raman signal is much smaller than the fluorescence emission signal from typical

fluorophores, and only 1 in ~10⁶ incident photons will be scattered by the Raman effect. But

a Raman spectrum, although weak, is a unique signature of a biomolecule with a big potential

KEY BIOLOGICAL

APPLICATIONS: ELASTIC

SCATTERING TOOLS

Estimating molecular shapes and

concentrations in vitro; label-​free

monitoring of biomolecules.