Principles of Fluorescence Spectroscopy

758 SINGLE-MOLECULE DETECTION
Figure 23.1. Comparison of single-molecule and ensemble emission
spectra for a molecular beacon.
tion. If the observed single-molecular beacon were open the
single-molecule spectrum would have the intensity expected
for a single fluorophore. If the observed molecular beacon
were closed, it would display a lower intensity or would
not be observable. Intermediate intensities would be
observed if the beacon was partially quenched. If the molecular
beacon folds and unfolds on the timescale of the
measurements then the signal would fluctuate. This example
shows how observation of single molecules can reveal
information about the conformation of biomolecules which
is not available when observing many molecules.
Another opportunity using SMD is to study reaction
kinetics without synchronization of the reaction. Consider
an enzyme catalyzed reaction where the substrate is nonfluorescent
and the product is fluorescent. The reaction is usually
started by addition of the substrate (Figure 23.2, top
panel) and the progress of the reaction followed by the
change in fluorescence intensity. These data would not
reveal any intermediate steps in the reaction. Now consider
an experimental design where a single enzyme molecule is
bound to a surface. In this case the enzyme (E) is labeled
with a fluorophore (F) and the substrate contains a
quencher. When the substrate (S–Q) binds to the enzyme
Figure 23.2. Comparison of ensemble and single-molecule enzyme kinetics. The enzyme is labeled with a fluorophore (F). The substrate contains a
group (Q) that quenches the fluorophore.