Principles of Fluorescence Spectroscopy

774 SINGLE-MOLECULE DETECTION
Figure 23.29. Emission spectra and intensity decay for a mixture of
donor-alone (D) and donor–acceptor (D-A) pairs, and acceptor alone,
as seen in an ensemble-averaged measurement.
Observations on single molecules can resolve such heterogeneity
and bypass ensemble averaging. Suppose the
donor-labeled protein is immobilized on a glass surface
(Figure 23.30) and that single protein molecules could be
separately observed. Then the signal due to any single molecule
would be due to either a donor-alone molecule (left)
or a protein molecule that contains bound acceptor (right).
Figure 23.30. Emission spectra and intensity decays for single-molecule
measurements for a mixture of donor-alone and donor–acceptor
pairs immobilized on a surface.
Figure 23.31. Schematic of enzyme-catalyzed hydrolysis of a substrate–phosphate
(S–Pi) as seen by SMD.
Figure 23.32. Single-molecule RET of a ribozyme molecule. The
lower panels show the time-dependent donor (green) and acceptor
(red) intensity and RET efficiency. Reprinted with permission from
[56], [58].