Abstract
Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions. Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments. To this aim, we developed a data processing strategy based on the clustering optimization procedure proposed by Lee et al (PNAS 109, 17436–17441, 2012). Using simulations, we estimated the range of experimental parameters (molecular density, molecular orientation, background level, laser power, frametime) adequate for an accurate determination of the phototransformation yields. Under illumination at 561 nm in PBS buffer at pH 7.4, the photobleaching yield of Dendra2 fused to actin was measured to be (2.5±0.4)×10−5, whereas the blinking-off yield and thermally-activated blinking-on rate were measured to be (2.3±0.2)×10−5 and 11.7±0.5 s−1, respectively. These phototransformation yields differed from those measured in poly-vinyl alcohol (PVA) and were strongly affected by addition of the antifading agent 1,4-diazabicyclo[2.2.2]octane (DABCO). In the presence of DABCO, the photobleaching yield was reduced 2-fold, the blinking-off yield was decreased more than 3-fold, and the blinking-on rate was increased 2-fold. Therefore, DABCO largely improved Dendra2 photostability in fixed mammalian cells. These findings are consistent with redox-based bleaching and blinking mechanisms under (F)-PALM experimental conditions. Finally, the green-to-red photoconversion quantum yield of Dendra2 was estimated to be (1.4±0.6)×10−5 in cellulo under 405 nm illumination.
Highlights
Single-molecule localization fluorescence microscopy [1,2,3,4] has become a central tool to investigate cells with unprecedented spatial resolution
In the case of green-to-red photoconvertible fluorescent proteins (PCFPs), an important phototransformable fluorescent proteins (PTFPs)’s subclass of common use in PALM microscopy, an estimate of the photobleaching quantum yield of mEosFP was obtained (W = 361025) using BSA surface-immobilization [16], and it was suggested in the case of mEos2 and pcDronpa2 [17] that bulk measurements of photobleaching yields may not be valid in singlemolecule microscopy conditions
Estimates of photoconversion efficiencies, another important parameter accounting for the total fraction of fluorescent molecules able to undergo a transition to the active state, were obtained in cellulo for various PCFPs, but again no quantum yield was provided [21]
Summary
Single-molecule localization fluorescence microscopy [1,2,3,4] has become a central tool to investigate cells with unprecedented spatial resolution. A few previous studies have measured photobleaching yields of non-phototransformable fluorescent proteins at the single-molecule level, typically in vitro to ensure a sufficiently sparse molecular density [11,12,13,14,15]. High autofluorescence or background noise due to out-of-focus light is a serious concern when cellular samples are investigated using a widefield geometry Such noise may prevent the detection of many fluorescent spots, which results in a significant number of single-molecule traces being chopped into shorter traces. This in turn translates into increased apparent bleaching and blinking yields. The reduced SNR results in a slight overestimation of the photoblinking regime, whereas at the longest frametime (60 ms) most fluorescence time traces are contained within a single frame, completely obviating the ability to recover correct yields
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