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3.2  Basic UV-VIS-IR Absorption, Emission, and Elastic Light Scattering Methods

single-​molecule light microscopy methods originally developed for in vitro contexts has been

applied now to living cells.

3.2  BASIC UV-​VIS-​IR ABSORPTION, EMISSION,

AND ELASTIC LIGHT SCATTERING METHODS

Before we discuss the single-​molecule light microscopy approaches, there are a number of

basic spectroscopy techniques that are applied to bulk in vitro samples, which not only pri­

marily utilize VIS light but also extend into UV and IR. Some of these may appear mundane

at first sight, but in fact they hold the key to generating many preliminary attempts at robust

physical quantification in the biosciences.

3.2.1  SPECTROPHOTOMETRY

In essence, a spectrophotometer (or spectrometer) is a device containing a photodetector

to monitor the transmittance (or conversely the reflectance) of light through a sample as

a function of wavelength. Instruments can have a typical wavelength range from the long

UV (~200–​400 nm) through to the VIS (~400–​700 nm) up into the mid and far IR (~700–​

20,000 nm) generated from one or more broadband sources in combination with wave­

length filters and/​or monochromators. A monochromator uses either optical dispersion or

diffraction in combination with mechanical rotation to select different wavelengths of inci­

dent light. Light is then directed through a solvated sample that is either held in a sample

cuvette or sandwiched between transparent mounting plates. They are generally made from

glass or plastic for VIS, sodium chloride for IR, or quartz for UV to minimize plate/​cuvette

absorption at these respective wavelengths. Incident light can be scanned over a range of

wavelengths through the sample to generate a characteristic light absorption spectral

response.

Scanning IR spectrophotometers exclusively scan IR wavelengths. A common version of

this is the Fourier transform infrared (FTIR) spectrometer, which, instead of selecting one

probe wavelength at any one time as with the scanning spectrophotometer, utilizes several

in one go to generate a polychromatic interference pattern from the sample, which has some

advantage in terms of SNR and spectral resolution.

The absorption signal can then be inverse Fourier transformed to yield the IR absorp­

tion spectrum. Such spectra can be especially useful for identifying different organic chem­

ical motifs in samples, since the vibrational stretch energy of the different covalent bonds

found in biomolecules corresponds to IR wavelengths and will be indicated by measurable

absorption peaks in the spectrum. The equivalent angular frequency for IR absorption, ω, can

be used to estimate the mean stiffness of a covalent bond (a useful parameter in molecular

dynamics simulations, see Chapter 8), by modeling it as a simple harmonic oscillator of two

masses m1 and m2 (representing the masses of the atoms either end of the bond) joined by a

spring of stiffness kr:

(3.1)

kr = µω²

where μ is the reduced mass given by

(3.2)

µ =

+

m m

m

m

1

2

1

2