Chemistry Reference
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and intrinsic
fluorescence from other molecular species in the sample. The concen-
tration of the sample must be diluted so that, on average, less than one molecule is
within this optical detection volume at a time. Under these conditions, individual
molecules can be observed.
There are two general methods of performing single-molecule spectroscopy. In the
first, a small point detection volume is de
ned using confocal microscopy. Typical
sizes for the detection volume are just below a femtoliter. The
fluorescence is
observed using high sensitivity single photon detectors. In the second, a wide, thin
volume of solution is illuminated at a water
glass interface. Both formats are
commonly used. As we will see, the critical issue for deciding on the excitation
format is whether the process under observation is irreversible, and for how long the
process needs to be observed (Figure 9.4).
-
9.3.3.1 Optical Isolation of a Single Point
Confocal
For single molecule spectroscopy at a single point, a confocal excitation
-
detection format is used. A laser excitation is focused using a high numerical aperture
(N.A.) microscope objective to a diffraction limited spot (size is
/N.A.). In order to
achieve the best focus, the laser beam must be collimated with a Gaussian beam
shape. Spatial
filtering using a pinhole or single mode
fiber optic is used to clean up
the beam. Fluorescent molecules within the focus are ef
ciently excited from the
ground state S
0
to the excited state S
1
. The
fluorescence produced by transitions from
S
1
to S
0
may be emitted in all directions. For a high N.A. objective, a large fraction (up
to 30%) of the
fluorescence is collected by the same objective that focuses the laser
beam. This
fluorescence travels the same optical path as the focused laser beam, but
in reverse. In order to separate these paths, a dichroic mirror (DM) is used. The DM
re
ects wavelength of the laser excitation, but transmits the wavelengths of the
fluorescence. Here, we take advantage of the Stokes shift described above, which
causes a separation between the excitation and emission bands of
uorophores.
Although the excitation of
fluorophores is most ef
cient at the focus of the laser
beam, there is also a large amount of excitation of
uorescence outside the focus. The
resulting
l
fluorescence from outside the focus is also collected by the objective, and
creates a large background. In order to carry out single-molecule spectroscopy, optical
3
Figure 9.4 (Continued )
can be obtained either with illumination through
the objective (C) or by coupling the laser through
a prism (D), both methods having their
advantages and inconveniences (for details,
see [30]). In epifluorescence (E), a laser beam
focused at the back focal plane of the objective or
a standard arc lamp source is used to illuminate
the whole sample depth, possibly generating
additional background signal. A wide-field
detector (camera) is used in all three cases,
allowing the recording of several single molecule
signals in parallel, although with a potentially
reduced time resolution than that achievable
with point detectors. The image on the RHS
represents the case of a dual-color experiment,
where both spectral channels are imaged
simultaneously on the same camera (signals
from the samemolecule are connected by dotted
line). Individual intensity trajectories can be
extracted from films, resulting in similar
information as that obtained with the confocal
geometry. Reproduced with permission from [8].
