Chemistry Reference
In-Depth Information
FIGURE 12.1
(a) Confocal fluorescence microscopy setup. The excitation light is focused
via an objective lens into a diffraction limited spot. The fluorescence light from the spot is
collected by the same objective and passed through optical filters to remove excitation and
scattering light. A pinhole is inserted at an image plane to reject scattering/fluorescence light
that is not incident from the focal point. Finally, photons are detected by an APD. (b)Wide-field
fluorescence microscopy setup. A collimated laser beam is focused into the back-focal plane of
the objective to realize a uniform field of illumination. The emission is collected through the
same objective and, after passing through filters to remove excitation and scattering light,
imaged onto the CCD detector. Reproduced from Ref. [26] with permission of the Royal
Society of Chemistry.
glasses, optics and of course from the dark counts generated by the detector. Only a
limited number of fluorescent molecules possesses the (photo)physical properties
like molar absorption coefficient (
K
fl
)and
photostability necessary for single-molecule experiments. In general, the lower
limit of the
e
), quantum yield of fluorescence (
e
K
fl
product, at the applied excitation wavelength, is set at
20,000M
1
cm
1
. However, as a result of autofluorescence, optical density, and
so on, these spectroscopic requirements are also a function of the specific sample
and wavelength used. It is fair to say that nowadays the two most widely used
fluorescence microscopy modes to detect and study single molecules are confocal
microscopy and wide-field microscopy (Figure 12.1). In confocal microscopy,
the objective lens focuses the excitation light to a diffraction limited spot. The
fluorescence generated by a single molecule residing in the confocal volume is
collected by the same objective lens. Before reaching the detector, the fluorescence
light passes appropriate filters and a pinhole, rejecting background fluorescence
and out of focus light, respectively. The emitted fluorescence can be measured by a
point detector, such as an avalanche photodiode (APD), which very efficiently
