Spectral Versatility of Fluorescent Proteins Observed on the Single Molecule Level - Fluorescent Proteins: From Understanding to Design - page 214

3 Ultrasensitive Spectroscopy at the Single Molecule Level
3.1 Technical Principles of Single Molecule Studies
To access and resolve the photophysical complexity of VFPs as described above,
single molecule spectroscopy is a very powerful method of choice. Standard
ensemble spectroscopy yields an average of a given observable for a large number
of presumably identical molecules that are sampled in parallel. By applying single
molecule spectroscopy - hence analyzing one molecule at a time - this averaging
effect is removed and variations of parameters characteristic of the individual single
molecule become visible. Sampling statistically relevant numbers of single mole-
cules gives access not only to the observable averaged over a large number of
molecules, but also to histograms of the observable that describe the distribution of
the respective parameter. The width and shape of such distributions can be analyzed
to gain even deeper insight into the analyzed systems. Especially for complex
systems where a multitude of different interactions between the emitting chromo-
phore and its direct environment are possible, the distributions of parameters
contain a wealth of information not accessible by conventional ensemble spectros-
copy. As outlined above, for VFPs, such heterogeneity arises due to differing
chromophores or chromophore conformations, or to variations in the specific
chromophore nanoenvironment.
Furthermore, single molecule detection allows the observation of a single
molecule over time, thereby giving access to dynamical changes, either sponta-
neous or arising from photophysics or photochemistry, without any need for
synchronization.
For the practical realization of single molecule fluorescence spectroscopy, a
number of approaches are by now well established [ 34 , 55 - 57 ]. In short, to realize
single molecule detection the sampled volume is reduced until there is only one
target molecule in the observation volume left at a time. The minimum sample
volume in optical microscopy and spectroscopy is defined by the optical diffraction
limit, although recently some techniques have been demonstrated to circumvent
this limit [ 3 ]. However, the sampled volume is in any case much larger than the size
of the target molecule. It is hence necessary to have the target molecule embedded
in a surrounding that is nonfluorescent to realize the condition to have only one
target molecule in the detection volume at a time. This is realized by working with
exceedingly high dilutions of target molecules in a nonfluorescent matrix or solvent
in the observation volume at any given time.
For applications discussed here that are targeted at the characterization of
emitters, the most commonly used technique is confocal raster-scanning micros-
copy. To localize the single emitters, the sample is raster-scanned, and for further
analysis the localized emitters are selectively positioned in the observation volume.
To detect the photons emitted by the single molecule highly sensitive avalanche
photo diodes (APDs) or highly sensitive intensified or back illuminated and cooled
CCD cameras are used. APDs can be used either to study the evolution of the single