BioPHYSICAL chemistry

In single-molecule spectroscopy, a key concept is that a single molecule
can be repetitively cycled between the ground and excited states, yield-
ing a large enough number of photons to be measured by averaging for
about 1 ms. In particular, fluorescence is an ideal measurement since the
system can be poised such that only molecules of interest fluoresce and
background contributions can be minimized. Molecules can either be
set to low concentrations and detected when they diffuse into the probe
volume or spatially trapped at a specific location (Figure 14.19). In this case,
the fluorescence is detected in bursts and is limited by the diffusion time
of the molecule. Alternatively, the molecule can be spatially restricted by
being tethered to a smooth surface, or trapped in a matrix or optical trap.
Spatial restriction allows for extended measurements but can be limited
by loss of the signal due to over-excitation of the molecule. Excitation is
usually performed with a laser as its high intensity provides a high rate
of excitation and consequently a measurable rate of emitted photons at
the detector. In these measurements, two dyes are used to allow FRET
measurements between an open state, with the two markers far apart, and
a closed state, with the two markers close together. The single-molecule
measurements can be interpreted in terms of the number of observations
in different molecular configurations, corresponding to different FRET

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0

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(a)(i) (ii) (iii)

(b)(i) (ii) (iii)

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Events

FRET efficiency

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Intensity Events

FRET efficiency

Time

Intensity

Time

Dwell time in the closed state (s)

Figure 14.19Illustration of the two basic configurations for observation of single-molecule
fluorescence. (a) Molecules are allowed to freely diffuse and burst of fluorescence results in FRET
efficiencies of 0.3 and 0.9, reflecting molecules in open and closed conformations, and (b)
molecules are restricted in space allowing a time profile of the changes between the open and
closed states. Modified from Mollova (2003).