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

extinction coefficient ε (often cited in non-​SI units of M⁻¹ cm⁻¹) and the molar concentration

of the fluorophore cm by

(3.6)

σ λ

ε λ

( ) = ^{( )c}

C

m

Therefore, the Beer–​Lambert law for a fluorescent sample can be rewritten as

(3.7)

I z

I

c

z

m

( ) = ( )

−

( )

(

)

0 exp

ε λ

The key physical process in bulk fluorimetry is single-​photon excitation, that is, the processes

by which energy from one photon of light is absorbed and ultimately emitted as a photon of

lower energy. The process is easier to understand when depicted as a Jablonski diagram for

the various energy level transitions involved (Figure 3.1b). First, the photon absorbed by the

electron shells of an atom of a fluorophore (a fluorescent dye) causes an electronic transition

to a higher energy state, a process that takes typically ~10⁻¹⁵ s. Vibrational relaxation due to

internal conversion (in essence, excitation of an electron to a higher energy state results in a

redistribution of charge in the molecular orbitals resulting in electrostatically driven oscilla­

tory motion of the positively charged nucleus) relative movements can then occur over typ­

ically 10⁻¹² to 10⁻¹⁰ s, resulting in an electronic energy loss. Fluorescence emission then can

occur following an electronic energy transition back to the ground state over ca. 10⁻⁹ to 10⁻⁶

s, resulting in the emission of a photon of light of lower energy (and hence longer wavelength)

than the excitation light due to the vibrational losses.

In principle, an alternative electronic transition involves the first excited triplet state

energy level reached from the excited state via intersystem crossing in a classically forbidden

transition from a net spin zero to a spin one state. This occurs over longer time scales than the

fluorescence transition, ca. 10⁻³–​100 s, and results in emission of a lower-​energy phosphores­

cence photon. This process can cause discrete photon bunching over these time scales, which

is not generally observed for typical data acquisition time scales greater than a millisecond as

it is averaged out. However, there are other fluorescence techniques using advanced micros­

copy in which detection is performed over much faster time scales, such as fluorescence

lifetime imaging microscopy (FLIM) discussed later in this chapter, for which this effect is

relevant.

The electronic energy level transitions of ground-​state electron excitation to excited

state, and from excited state back to ground state, are vertical transitions on the Jablonski

diagram. This is due to the quantum mechanical Franck–​Condon principle that implies

that the atomic nucleus does not move during these two opposite electronic transitions

and so the vibration energy levels of the excited state resemble those of the ground state.

This has implications for the symmetry between the excitation and emission spectra of a

fluorophore.

But for in vitro fluorimetry, a cuvette of a sample is excited into fluorescence often using

a broadband light source such as a mercury or xenon arc lamp with fluorescence emission

measured through a suitable wavelength bandwidth filter at 90° to the light source to min­

imize detection of incident excitation light. Fluorescence may either be emitted from a fluor­

escent dye that is attached to a biological molecule in the sample, which therefore acts as a

“reporter.” However, there are also naturally fluorescent components in biological material,

which have a relatively small signal but which can be measurable for in vitro experiments,

which often include purified components at greater concentrations that occur in their native

cellular environment.

For example, tryptophan fluorescence involves measuring the native fluorescence of the

aromatic amino acid tryptophan (see Chapter 2). Tryptophan is very hydrophobic and thus

is often buried at the center of folded proteins far away from surrounding water molecules.

On exposure to water, its fluorescence properties change, which can be used as a metric for