Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM)

Abstract

We present a single molecule method for counting proteins within a diffraction-limited area when using photoactivated localization microscopy. The intrinsic blinking of photoactivatable fluorescent proteins mEos2 and Dendra2 leads to an overcounting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe blinking and show that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra2 a better photoactivated localization microscopy tag than mEos2 for molecular counting. The simultaneous activation of multiple molecules is another source of error, but it leads to molecular undercounting instead. We propose a photoactivation scheme that maximally separates the activation of different molecules, thus helping to overcome undercounting. We also present a method that quantifies the total counting error and minimizes it by balancing over-and undercounting. This unique method establishes that Dendra2 is better for counting purposes than mEos2, allowing us to count in vitro up to 200 molecules in a diffraction-limited spot with a bias smaller than 2% and an uncertainty less than 6% within 10 min. Finally, we demonstrate that this counting method can be applied to protein quantification in vivo by counting the bacterial flagellar motor protein FliM fused to Dendra2.super-resolution optical microscopy | single molecule counting | fluorescence blinking K nowing the state of oligomerization of molecules can help to establish their structural organization in space and ultimately unravel their function. Electron microscopy, cryo-EM, and crystallography are the methods of choice to elucidate macromolecular structures at intermediate and high resolution, respectively, but these techniques often involve elaborate sample preparation or require isolating the protein complexes from their natural environment. Optical microscopy with fluorescent proteins provides a much less invasive alternative and allows quantification of proteins with single molecule sensitivity (1, 2). However, the resolution of conventional microscopy is diffraction-limited to approximately 250 nm, a dimension much larger than the size of protein complexes.In photoactivated localization microscopy (PALM), multiple fluorescent molecules spatially closer than the diffraction limit can be resolved by separating their contributions in time (3). PALM can, in principle, be used to count single molecules located within a diffraction-limited spot in the image. Ideally, any irreversibly photoactivatable fluorescent protein (PA-FP) can be used as a tag to count, for example, the number of proteins of a particular kind inside a cell, by simply tallying the number of emission bursts (4-6). In practice, however, the potential counting error incurred by the fluorescent blinking of PA-FPs in their photoactivated form (3, 6, 7) has been a major obstacle to the use of PALM for molecular counting. Although a semiempirical approach to correct the artifact due to the mEos2...

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Single molecule counting-PALM is used to determine protein stoichiometry when single step photobleaching is not feasible (such as high density of proteins) [11][12][13][14] . Fluorescent proteins are photoactivated one at a time, detected and subsequently photobleached (Fig.…”

“…Imaging schemes have been proposed for controlled photoactivation of fluorescent proteins 11,13 . Furthermore, the cumulative number of fluorescent proteins detected in each frame has been used to determine whether the fluorescent proteins have been exhaustively imaged 11 .…”

“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

“…For example, probes with many switching cycles lead to smoother images due to repeated sampling of the same structure 28 . The irreversible probes are preferable for protein stoichiometry measurements using single molecule counting with methods like photoactivated localization microcopy (PALM) 13,14 , because in principle each probe is detected and counted only once. However, even the irreversible probes can undergo multiple re-activation events 29,30 .…”

“…Therefore, methods have been developed to reduce over-counting artifacts. These include pair correlation analysis (PC-PALM) 15,31 or methods that characterize fluorescence off-times and group localizations together based on a characteristic dark time 13,30 . After accounting for re-activation, the remaining localizations are a measure of the number of detected fluorescent probes.…”