82

3.5  Fluorescence Microscopy: The Basics

KEY POINT 3.2

An ideal fluorescent probe binds in a highly specific way to the biological component

under investigation, is small so as to cause a minimal steric interference with native

biological processes, has a high quantum yield, and is relatively photostable (i.e., min­

imal irreversible photobleaching and/​or reversible photoblinking, but note that some

techniques can utilize both to generate stoichiometric and structural information).

Currently, there is no single type of fluorescent probe that satisfies all such ideal cri­

teria, and so the choice of probe inevitably results in some level of compromise.

3.5.4  PHOTOBLEACHING OF FLUOROPHORES

Single-​photon excitation of a bulk population of photoactive fluorophores of concentration

C(t) at time t after the start of the photon absorption process follows the first-​order bleaching

kinetics, which is trivial to demonstrate and results in

(3.31)

C t

C

t

tb

( ) =

( )

−



^



^

0 exp

where tb is a characteristic photobleach time, equivalent to the lifetime of the fluorescence

excited state. This photobleach time is the sum of an equivalent radiative decay time, trad

FIGURE 3.4  Fluorophores in biophysics. (a) Bespoke (i.e., homebuilt) dual-​color fluorescence microscope used in imaging

two fluorophores of different color in a live cell simultaneously. (b) Fluorescently labeled secondary antibody binding to

a specific primary antibody. (c) Dependence of QD fluorescence emission wavelength on diameter. (d) Normalized exci­

tation (green) and emission (orange) spectra for a typical QD (peak emission 0.57 μm). (e) Example fluorescence intensity

time trace for a QD exhibiting stochastic photoblinking. (f) Structure of GFP showing beta strands (yellow), alpha helices

(magenta), and random coil regions (gray).