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In addition, a long lived dark state found for many GFP group proteins can be
depopulated by illumination with light around 400 nm [ 58 , 62 , 65 ]. This switching
is induced by a change of the protonation state of the GFP chromophore. The
protonated, neutral chromophore is essentially nonemitting due to its low quantum
efficiency and blue-shifted absorbance, while illumination with light of around
400 nm results in deprotonation of the chromophore and thereby the reconstitution
of the efficiently emitting chromophore [ 58 , 62 , 65 , 66 ]. This switching between
different states induced by light and the possibility to observe the emission from
single fluorescent proteins has played an important role in the development of
super-resolution microscopy techniques [ 1 - 7 ].
3.3 Spectrally Resolved Single Molecule Detection
Intensity trajectories give fascinating insights into the details of single emitters, but
for photophysically complex systems such as the VFPs, it is often necessary to
sample additional parameters. The emission spectrum of a single molecule is charac-
teristic of the molecule, and direct observation of the spectra and changes in the
spectral shape and position is a powerful means to gain further insights into the
photophysics of complex emitters. Techniques to record full emission spectra from
single molecules with high spectral resolution have been established [ 67 - 70 ].
The emission spectrum is a robust parameter since it does not suffer from
changes by reorientation of the analyzed molecule with respect to the excitation
light and detection, which change the detected emission intensity. Also spectra can
be used to effectively discriminate between target molecules and unwanted but
unavoidable contaminations. Further, emission spectra can be analyzed in great
detail, enabling access to parameters such as the emission maximum position, the
spectral shape, the spacing of the vibronic progression, or the intensity ratio
between different transitions of the vibronic progression.
Spectrally resolved analysis of the emission from single fluorophores unfolds its
full potential when series of emission spectra from a statistically relevant number of
single emitters are measured (Fig. 3 ). The identification of rare spectral forms
becomes directly accessible since the influence from other, dominating forms is
simply removed by looking at one emitter at a time. Recording the evolution of the
emission spectrum over time in spectral series makes possible the identification of
connected spectral species by direct observation of transitions of the single emitter
between these forms. These transitions between spectrally different forms originate
from rather large-scale modifications of the chromophore, such as conformational
reorganizations or chemical modifications of the chromophore itself. Besides these
pronounced changes in spectral signature, subtle changes in the spectral position
and shape can also be observed. These smaller spectral variations are termed
“spectral diffusion” and are attributed to fluctuations in the nanoenvironment of
the emitter that defines the exact photophysical properties of the emitter [ 71 ].
Precisely these capabilities of spectrally resolved single molecule spectroscopy
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