Internal rulers to assess fluorescent protein photoactivation efficiency.

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 laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405 nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. © 2017 International Society for Advancement of Cytometry.