Chapter 3 – Making Light Work in Biology  69

commercial SPR chip that can be removed and replaced as required). At a certain angle of

incidence to the surface, the sensing beam reflects slightly less back into the sample due

to a resonance effect via the generation of oscillations in the electrons at the metal surface

interface, surface plasmons. This measured drop in reflected intensity is a function of the

absolute amount of the adsorbed material on the metal surface from the sample, and so

DPI and SPR are essentially complementary. Both yield information on the stoichiometry

and binding kinetics of biological samples. These can be used in investigating, for example,

how cell membrane receptors function; if the surface is first coated with purified receptor

proteins and the sample chamber contains a ligand thought to bind to the receptor, then

both DPI and SPR can be used to measure the strength of this binding, and subsequent

unbinding, and to estimate the relative numbers of ligand molecules that bind for every

receptor protein (Figure 3.1d).

3.2.6  PHOTOTHERMAL SPECTROSCOPY

There are a group of related photothermal spectroscopy techniques that, although perhaps

less popular now than toward the end of the last century due to improvements in sensi­

tivity of other optical spectroscopy methods, are still very sensitive methods that operate

by measuring the optical absorption of a sample as a function of its thermal properties.

Photothermal spectroscopy is still in use to quantify the kinetics of biochemical reactions,

which are initiated by light, for example, by direct photochemical reactions or by environ­

mental changes induced by light such as changes in the cellular pH. The time scales of these

processes typically span a broad time range from 10⁻¹² to 10⁻³ s that are hard to obtain by

using other spectroscopy methods.

Incident light that is not scattered, absorbed, or converted into fluorescence emission

in optical spectroscopy is largely converted to heat in the sample. Therefore, the amount

of temperature rise in an optical absorption measurement is a characteristic of the sample

and a useful parameter in comparing different biological materials. Photothermal deflec­

tion spectroscopy can quantify the changes in a sample’s refractive index upon heating. It

uses a laser beam probe on an optically thin sample and is useful in instances of highly

absorbing biomolecule samples in solution, which have too low transmission signal to be

measured accurately. Photothermal diffraction can also be used to characterize a biological

material, which utilizes the interference pattern produced by multiple laser sources in the

sample to generate a diffraction grating whose aperture spacing varies with the thermal

properties of the sample.

In laser-​induced optoacoustic spectroscopy, a ~10⁻⁹ s VIS light laser pulse incident on

a sample in solution results in the generation of an acoustic pressure wave. The time evo­

lution of the pressure pulse can be followed by high-​bandwidth piezoelectric transducers,

typically over a time scale of ~10⁻⁵ s, which can be related back to time-​resolved binding

and conformational changes in the biomolecules of sample. The technique is a tool of choice

for monitoring time-​resolved charge transfer interactions between different amino acids in

a protein since there is no existing alternative spectroscopic technique with the sensitivity

to do so.

3.3  LIGHT MICROSCOPY: THE BASICS

Light microscopy in some ways has gone full circle since its modern development in the

late seventeenth and early eighteenth centuries by pioneers such as Robert Hooke (Hooke,

1665; but see Fara, 2009 for a modern discussion) and Antonj van Leeuwenhoek (see van

Leeuwenhoek, 1702). In these early days of modern microscopy, different whole organisms

were viewed under the microscope. With technical advances in light microscopy, and in the

methods used for sample preparation, the trend over the subsequent three centuries was to

focus on smaller and smaller length scale features.

KEY BIOLOGICAL

APPLICATIONS: BASIC

OPTICAL SPECTROSCOPY

Quantifying molecular and

cell concentrations in vitro;

Determining biomolecular

secondary structures in vitro;

Measuring molecular con­

formational changes from

bulk solutions; Characterizing

chemical bond types from bulk

solutions.