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Breaking the nanoscale labeling and detection limit using Fab-PAINT (Conference Presentation)
Author(s): Ying S. Hu

Paper Abstract

Spatially resolving protein clusters and protein-protein interactions requires high labeling and detection efficiencies at the nanoscale. Increasing super-resolution data suggest that high-density labeling by immunostaining is greatly affected by increased steric hindrance and variable antibody specificity. Photobleaching during extended acquisition further limits the achievable resolution in single molecule localization microscopy. Here, we present an innovative technique utilizing dye-conjugated antibody fragments in point accumulation in imaging nanoscale topology. Termed Fab-PAINT, the super-resolution technique reduces the binding affinity of full-length antibodies and promotes transient binding without losing their specificity. The binding avidity and affinity can be modulated by antibody fragmentation and a cocktail of the imaging buffer containing chaotropic agents. While the level of modulation is highly dependent upon non-covalent interactions of the antigen-antibody binding, Fab-PAINT has been found effective on antibodies recognizing common polypeptide tags, including the human influenza hemagglutinin tag (HA) and myc tag. Upon optimization, we demonstrate much higher molecular densities detected by Fab-PAINT compared to dSTORM and DNA-PAINT. The combined high labeling and detection efficiencies enable Fab-PAINT to uncover unprecedented details of membrane protein clusters critical for early signaling events during T cell activation. Combined with the multiplexing capability, Fab-PAINT turns single molecule localization microscopy into a nanoscale quantitative bioanalytical tool for investigating protein clusters and protein-protein interactions in biological systems.

Paper Details

Date Published: 4 March 2019
Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 1088403 (4 March 2019); doi: 10.1117/12.2507668
Show Author Affiliations
Ying S. Hu, Univ. of Illinois at Chicago (United States)

Published in SPIE Proceedings Vol. 10884:
Single Molecule Spectroscopy and Superresolution Imaging XII
Zygmunt Karol Gryczynski; Ingo Gregor; Felix Koberling, Editor(s)

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