Point-localization superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. In PALM, serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules is performed. Individual molecules are then localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The aggregate position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities (up to 10,000 molecules/micron squared). While PALM is a powerful approach for investigating protein organization, tools for quantitative, spatial analysis of PALM datasets are largely missing. We developed a pair-correlation analysis method with PALM (PC-PALM) that enables complex patterns of protein organization across the plasma membrane to be analyzed. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrated distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and showed dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. Our results revealed that PC-PALM is an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies. We developed a new way of localizing fluorescent molecules for superresolution imaging that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. Single fluorophores are detected by acquiring a stream of fluorescence images. Fluorophore bleach or blink-off events are then recorded by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We found that BaLM works with all commonly used synthetic fluorescent dyes and genetically expressed fluorescent proteins. We further showed that BaLM can be used in multicolor superresolution experiments, deciphering the molecular distribution of up to four different proteins in a sample. These characteristics indicated that BaLM is a practical and versatile approach for obtaining superresolution images that can either stand alone or be used in conjunction with other superresolution approaches.
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