By sampling the photon emission for a large number of excitations, the probability
distribution can be constructed. The time-dependent decay of an individual fluorophore
species follows an exponential distribution, and the time constant is thus termed the
lifetime of this fluorophore. Curve fitting of fluorescence decays enables the identifica-
tion of the number of species of fluorophores (within certain limits), and the calculation
of the lifetimes for these species. In this context, different species can be different
fluorophores or distinct conformations of the same fluorophore.12.4 Luminometry
In the preceding section, we mentioned the method of bioluminescence resonance
energy transfer (BRET) and its main workhorse, luciferase. Generally, fluorescence
phenomena depend on the input of energy in the form of electromagnetic radiation.
However, emission of electromagnetic radiation from a system can also be achieved
by prior excitation in the course of a chemical or enzymatic reaction. Such processes
are summarised asluminescence. Luminometry is not strictly speaking a spectro-
photometric technique, but is included here due to its importance in the life sciences.12.4.1 Principles
Luminometryis the technique used to measure luminescence, which is the emission
of electromagnetic radiation in the energy range of visible light as a result of a
reaction.Chemiluminescencearises from the relaxation of excited electrons transi-
tioning back to the ground state. The prior excitation occurs through a chemical
reaction that yields a fluorescent product. For instance, the reaction of luminol with
oxygen produces 3-aminophthalate which possesses a fluorescence spectrum that is
then observed as a chemiluminescence. In other words, the chemiluminescence spec-
trum is the same as the fluorescence spectrum of the product of the chemical reaction.
Bioluminescencedescribes the same phenomenon, only the reaction leading to a
fluorescent product is an enzymatic reaction. The most commonly used enzyme in this
context is certainly luciferase (see Section 15.3.2). The light is emitted by an intermedi-
ate complex of luciferase with the substrate (‘photoprotein’). The colour of the light
emitted depends on the source of the enzyme and varies between 560nm (greenish
yellow) and 620 nm (red) wavelengths. Bioluminescence is a highly sensitive method,
due to the high quantum yield of the underlying reaction. Some luciferase systems work
with almost 100% efficiency. For comparison, the incandescent light bulb loses about
90% of the input energy to heat.
Because luminescence does not depend on any optical excitation, problems with
autofluorescence in assays are eliminated.12.4.2 Instrumentation
Since no electromagnetic radiation is required as a source of energy for excitation, no
light source and monochromator are required. Luminometry can be performed with a507 12.4 Luminometry