Quantitative Photoactivated Localization Microscopy of Membrane Receptor Oligomers

Optical super-resolution microscopy allows the visualization of cellular structures with a spatial resolution of a few tens of nanometers and has revolutionized our understanding in cell biology. However, the spatial resolution achieved with to-date super-resolution microscopy methods in cells is not sufficient to optically resolve proteins within densely packed protein clusters, which themselves represent relevant functional assemblies in cells. Single-molecule localization microscopy (SMLM) offers an opportunity to retrieve this information by analyzing the kinetics of on-off-switching (“blinking”) observed in the fluorescence emission signatures of single fluorophores. We report the theoretical background of kinetics-based molecular quantification of SMLM data, discuss fluorescent probes and methods for protein labeling, and showcase applications in biology.