Super-STORM: Molecular Modeling to Achieve Single-molecule Localization with STORM Microscopy

Abstract

Summary This protocol introduces the SuperSTORM technique, combining stochastic optical reconstruction microscopy (STORM) and molecular modeling. SuperSTORM is optimized for acquiring and processing STORM images of neutrophil integrins but can be used for any cell-surface molecule with known structure and antibody-binding site(s). SuperSTORM identifies molecular cut-offs for eliminating multiple blinks of STORM imaging, determines colocalization, identifies clusters, and reveals molecular orientations and distributions. This protocol extends STORM imaging to cells in microfluidic systems. Improved resolution is achieved by using biomolecule-inherent parameters. For complete information on the generation and use of this protocol, please refer to the paper by Fan et al. (2019) .

Visualization of integrin molecules by fluorescence imaging and techniques

“…Using Fab fragments of mAb24 and KIM127, the distribution of E − H + , E + H − , and E + H + β2 integrins on neutrophil footprint during arrest was visualized by STORM ( Fan et al ., 2019 ). Combined with molecular modeling, the SuperSTORM technique was developed ( Fan et al ., 2020 ), and the orientation of E − H + , E + H − , and E + H + β2 integrins were indicated. This work enabled visualizing integrin molecules at the single molecular level and was the first to show the orientation of different conformation integrins.…”