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emission of the protein, but to merely confirm that indeed the targeted proteins and
not impurities were being studied. The single protein emission spectrum was
embedded in a study analyzing intensity trajectories of single GFP variants [ 73 ].
In a similar way, the single protein emission spectra from the GFP variant EGFP
and from DsRed were used to prove that the immobilization of the proteins in a film
of polyvinylalcohol for single molecule analysis did not change their photophysical
properties [ 74 ].
3.5 Spectral Diffusion: Sampling the Parameter Distribution
Classical bulk spectroscopy averages over all emitters in a sample and thereby
results in one value, e.g., the emission maximum position, for the ensemble. Clearly,
if there are deviations from the average value attributable to individual emitters,
these are not discernible within the broad distribution of the ensemble. Single
molecule spectroscopy, on the contrary, yields detailed insights into the spatial
and temporal heterogeneity of single emitters. Instead of one value for a specific
photophysical parameter, the full distribution of this parameter within the ensemble
becomes accessible. Thus, in the case of the emission maximum position, single
molecule spectroscopy gives access to the distribution of emission maximum posi-
tions of the individual emitters within the sampled ensemble rather than one value
for the peak of the emission. In a sample composed of molecules of identical
chemical forms, the different emitters can experience variations in their immediate
nanoenvironments. The heterogeneity of spectral properties of single molecules
originates exactly from this fact that any non-gas-phase chromophore interacts with
its immediate environment; it is this interaction between the chromophore and its
environment that determines the exact photophysical properties of the emitter.
These local variations in the nanoenvironment of a single chromophore lead to
spatial and temporal heterogeneity of the spectral properties of the chromophores.
These small variations in spectral properties induced by varying chromophore-envir-
onment interactions are termed “spectral diffusion” since the variations are typically
small and occur randomly [ 71 ]. Spectral diffusion generally shows a unimodal
distribution of the respective parameter around one peak value. The unimodal nature
of the distribution discriminates spectral diffusion from transitions between distinct
spectral forms, since the transition between two forms will show a bimodal distribu-
tion around two peak values. Each of the two spectral forms will be characterized by
a central value of the distribution (the most likely value of the respective observable),
and a distribution width that is characteristic of the spectral diffusion associated with
each form.
The heterogeneity of different single molecule emission parameters has been
shown for single emitters at cryogenic temperatures and at room temperature. For
polymer embedded dyes, variations in the shape as well as the exact spectral
position of the emission spectrum have been observed [ 69 ], although it was impos-
sible to conclusively describe the mechanisms underlying the observed variations
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