A promising approach to molecular counting problem in superresolution microscopy.

Molecular Counting Problem in Superresolution Microscopy Cellular processes are often controlled by aggregates of protein complexes. Recent studies have witnessed that the stoichiometry, or the relative composition of protein complexes, can be dynamical (1–3). Quantifying the stoichiometry of these complexes, not only at the mean level but also its full distribution, is therefore a grand challenge of molecular biology. This is exactly the issue that Rollins et al. (4) address using state-ofthe-art superresolution imaging data (5–8). Conventional protein-counting methods include quantitative fluorescence microscopy (9) and fluorescence photobleaching (1, 2). These methods all rely on diffraction-limited methods. The maximum spatial resolution is typically a few hundred nanometers for counting proteins. In contrast, superresolution microscopy can overcome the diffraction limit, not only in vitro but also in living cells. One of the superresolution methods is termed photoactivated localization microscopy (PALM) (5). PALM is now broadly available to cell biologists. It works by genetically encoding onto the protein subunit a photoswitchable fluorescent protein (FP). The FPs can then be activated one at a time by exposing the cell to low-intensity light. In the cell, the protein subunits containing FPs have assembled into their respective protein complexes. This process can be captured and quantified by the intensities of FPs; the FPs eventually photobleach one at a time. Therefore, the signals originated from multiple FPs of one protein complex within a diffraction-limited spot can be separated in time, but they cannot be separated in space. The spatial location of each stochastically activated FP can then be identified by searching the center of the spatial Gaussian distribution where the photons originate. Although each fluorescent intensity spike should in principle represent the FP associated with activation of one protein subunit and subsequent corresponding photobleaching, the real situation is more complex. FPs stochastically blink. In other words, they may stochastically turn on and off reversibly before photobleaching. Therefore, registering the number of fluorescent peaks can overcount the number of FPs. To resolve this issue, one may first characterize the photophysics of the FPs in vitro. On the basis of this information, one can then correct for how many times one expects an FP to blink on average. Although this procedure