Super-Resolution Imaging through Single-Molecule Localization

Abstract

Recent advances in super-resolution fluorescence microscopy have revolutionized biological imaging by overcoming the fundamental diffraction barrier, recognized by a Nobel Prize in 2014. It has become an essential tool in biological research. Super-resolution fluorescence microscopy can be generalized into three different approaches: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED), and structured illumination microscopy (SIM). This chapter focuses on single-molecule localization-based super-resolution imaging. It is one of the simplest forms of super-resolution microscopy due to adapting a configuration of wide-field fluorescence microscopy in combination with single-molecule localization through image processing. It covers the main imaging principle, the instrumentation, and various methods to achieve two-dimensional, three-dimensional, and multi-color super-resolution imaging and main data processing steps with a focus on different types of single-molecule localization algorithms. Lastly, we briefly discuss some examples to apply single-molecule localization-based super-resolution microscopy in various areas of biomedical research.