Chapter 3 – Making Light Work in Biology  83

(processes involving direct emission of photon radiation) and nonradiative decay time, tnon-​rad

(processes not involving the direct emission of photon radiation, such as molecular orbital

resonance effects). In general, if there are a total of n fluorescence decay mechanisms, then

(3.32)

t

k

b

i

n

i

=

=

∑

1

1

where ki is the rate constant of the ith fluorescence decay mechanism. Photobleaching of each

single fluorophore molecule is a stochastic Poisson process such that its photoactive life­

time is an exponential distribution of mean time tb. The principal cause of irreversible photo­

bleaching of a fluorophore is light-​dependent free radical formation in the surrounding water

solvent, especially from molecular oxygen (under normal conditions, the concentration of

dissolved oxygen in biological media is relatively high at ~0.5 mM, unless efforts are made

to remove it). Free radicals are highly reactive chemicals containing an unpaired electron,

which can combine with a fluorophore to destroy its ability to fluoresce. Many fluorophores

also exhibit reversible photobleaching (or blinking), often under conditions of high excitation

intensity, in which the excited state is transiently quenched to generate a stochastic dark “off”

state as well as the bright “on” state.

Blinking is also known as fluorescence intermittency and is related to the competition

between radiative and nonradiative relaxation pathways for the excited electron state (i.e.,

an excited state electron can return to its ground state via more than just a single energy

transition pathway). The blinking phenomenon is exhibited by many fluorophores, especially

semiconductor-​based systems such as quantum dots, and also organic dyes and fluorescent

proteins (FPs) (see the following sections in this chapter). Blinking often appears to obey a

power-​law distribution of on and off times with dark states in some systems lasting for tens

of seconds, which is enormous on the quantum time scale, but remarkably a dark blinker will

recover its fluorescence state after such a huge dark period and start emitting once again. The

underlying specific physical mechanisms for blinking are largely unresolved but appear to be

very specific for the fluorophore type.

3.5.5  ORGANIC DYE FLUOROPHORES

There are a large range of different organic dyes, for example, cyanines and xanthenes, whose

chemical structures facilitate electron delocalization through a so-​called π-​electron system.

A π bond is a covalent molecular orbital formed from the overlap of two p atomic orbitals;

multiple π bonds in close proximity in a molecular structure can form a pool of spatially

extended, delocalized electron density over a portion of the molecule through orbital reson­

ance. This enables a large portion of the molecule to operate as an efficient electric dipole.

Historically, such dyes were first used to specifically label single biomolecules using

immunofluorescence. Here, a primary antibody binds with high specificity to the biomol­

ecule of interest, while a secondary antibody, which is chemically labeled with one or more

fluorophores, then binds to the primary antibody (Figure 3.4b). The main issues with this

technique concern the size of the probe and how to deliver it into a cell. The effective size

of the whole reporter probe is ~20 nm, since each antibody has an effective viscous drag

radius (the Stokes radius) of ~10 nm, which is an order of magnitude larger than some of

the biomolecules being labeled. This can impair their biological functions. Second, intro­

ducing the antibody labels into living tissue is often difficult without significantly impairing

the physiological functions, for example, permeabilizing the tissue using harsh detergents.

With this caveat, this can result in very informative fluorescence images in vivo.

Fluorescence in situ hybridization (FISH) is a valuable labeling technique using organic

dyes for probing specific regions of nucleic acids. A probe consists of a ~10 nucleotide base

sequence either of singled-​stranded DNA or RNA, which binds to a specific sequence of

nucleic acid from a cell extract or a thin, fixed (i.e., dead) tissue sample via complementary

base pairing following suitable incubation protocols normally >10 h, sometimes a few days.