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Single molecule spectroscopy of VFPs is experimentally still challenging
because of the low photostability of VFPs, but the insights that can be gained
have immense potential, and make the challenge well worth the effort. Spectrally
resolved single molecule spectroscopy yielding full emission spectra from individ-
ual VFPs has proven to be an excellent method to access the amazing spectral
heterogeneity of VFPs. Single molecule emission spectroscopy has added a lot to
understanding the emission properties of VFPs by allowing the identification of
hidden subensembles, giving valuable information about the possible molecular
origins of these subensembles, by shedding light on the transitions and dynamics
between these subensembles and by allowing quantitative insights into the spectral
diffusion and its origins in VFPs. To date, single molecule analysis of VFPs has
focused on the most commonly used proteins from the GFP and the DsRed group of
proteins, but the rapidly growing number of VFPs, and in particular the photo-
activatable or photoswitchable proteins that underpin optical super-resolution
methods, holds further promise for exciting insights into the spectral heterogeneity
of these complex emitters.
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