Fig. 11 (a) For all protein variants rare spectral forms with emission maximum positions between
~530 nm and 575 nm were observed. (b) Transitions between the predominant form and a rare
spectral form with emission maximum at 570 nm via a dark state provide evidence that the rare
forms are connected to the main forms [ 88 ] (Copyright Wiley-VCH Verlag GmbH & Co. KGaG.
Reproduced with permission)
It is essential to understand VFP emission properties in depth, since the applications
in which VFPs are used to quantitatively elucidate biological and biochemical
processes are becoming more and more complex. Single molecule emission spec-
troscopy is a powerful tool to access aspects of VFP photophysics, which are hidden
to the averaging of properties inherent to conventional ensemble spectroscopy.
In particular, techniques that record more parameters than simply the change of
emission intensity over time give detailed insights into the photophysics of VFPs.
These techniques even give access to the dynamics of single molecules when the
evolution of the respective parameter is followed in time.