Quantitative Single-Molecule Localization Microscopy

Abstract

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.

A dynamic role for transcription factors in restoring transcription through mitosis

“…Recent innovations have enabled an unprecedented increase in temporal and spatial resolution well beyond the diffraction limit, giving rise to various methods collectively known as ‘super-resolution’ microscopy [ 35 ]. One such technique that has been used to study TF dynamics is single-molecule localization microscopy coupled with single particle tracking analysis (SMLM-SPT) [ 36 , 37 ]. In this assay, fluorescently labeled TFs are localized individually and tracked over time, and varying image acquisition rates allow for kinetic measurements of DNA bound and freely diffusing molecules.…”