Internal rulers to assess fluorescent protein photoactivation efficiency

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Malte Renz, Christian Wunder

Abstract

Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405 nm light. Using a...Continue Reading

References

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Citations

Aug 14, 2020·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Ágnes Szabó, Peter Nagy
Jun 20, 2020·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Brian DanielsMalte Renz
Oct 26, 2020·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Attila Tárnok
Sep 23, 2020·Chembiochem : a European Journal of Chemical Biology·Dhiraj BhatiaLudger Johannes

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