Single‐Molecule Localization Microscopy ( SMLM )

The resolution of an optical microscope is restricted to approximately 200 nm, owing to the diffraction of light. Many advanced super-resolution techniques have been developed to surpass the diffraction limitation, which has resulted in significant improvement in resolution. Among them, single-molecule localization microscopy (SMLM) stands out as a powerful technique owing to the relatively high achievable resolution and the simple optical setup. This chapter focuses on SMLM techniques and provides an overview of the diverse SMLM approaches, primarily in terms of the different single-molecule detection schemes, including stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy, and point accumulation for imaging in nanoscale topography (PAINT), and single-molecule localization algorithms. Unlike stimulated emission depletion microscopy, SMLM is based on wide-field imaging. The basic setup for the STORM technique is very similar to that of conventional fluorescence microscopy, with only a few modifications.