The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 3

nuanced properties of each dye class will enable the effective use of existing
probes and the development of new fluorogenic molecules.

2. FLUORESCENCE AND FLUOROPHORES

Fluorescence is the emission of a photon by an excited-state molecule
following the absorption of light. The phenomenon of fluorescence from the
small molecule quinine was first described in 1845 by Herschel 10 and further
elucidated in 1852 by Stokes. 11 Fluorescence remained a novelty for nearly a
century until the first commercial fluorometers 12 and fluorescent micro-
scopes 13 appeared in the 1950s. Since that time, fluorescence has evolved from
a curiosity to a crucial scientific tool. Fluorescent measurements are highly
sensitive, allowing measurement and visualization of fluorescent compounds
against a background of a myriad of nonfluorescent molecules. Fluorescent
materials enable biological imaging, high-throughput screening, genome se-
quencing, and many other useful technologies. 1-9
Key properties of fluorophores include the absorption maximum ( l max ),

the emission maximum ( l em ), the extinction coefficient ( e ), and the fluores-

cence quantum yield ( em is termed

the “Stokes shift” in homage to Stokes. 11 The extinction coefficient, or mo-
lar absorptivity, is a measure of the probability of light absorption by the dye.
The quantum yield, or quantum efficiency, is the ratio of the number of
photons emitted to the number of photons absorbed. The relative brightness
of fluorophores can be determined by comparing values of

F ). The difference between l max and l

, which
takes into account both the photons absorbed and the efficiency of the fluo-
rescence process. 5 For use in biological experiments, other properties of
fluorophores become important, such as solubility, tendency for aggrega-
tion, photobleaching rates, and sensitivity to environments. In this chapter,
we move from the blue to the red region of the electromagnetic spectrum,
detailing the

e F

of the major classes of dyes, and discussing
the chemical strategies used for controlling fluorescence.

l max , l em , e , and F

3. MODES OF FLUORESCENCE MODULATION

Chemistry provides diverse methods to control the fluorescence in-
tensity of small-molecule dyes. Figure 1.1 presents schematic representation
of these different modes of modulation and corresponding examples of fluo-
rogenic compounds. The most straightforward method to control fluores-
cence is to install a blocking group onto the dye that suppresses or