Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System.

ABSTRACT: We develop and employ the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution (SR) imaging and single-molecule tracking based on single-molecule localizations. The fast off rate of fluorogen binding, combined with its spatially well-separated labeling of the densely expressed FAST fusion proteins, allowed single-molecule measurements to be performed in both living and fixed cells. The well-separated fluorescence localization density was achieved by either reversibly controlling the fluorogen concentration or by irreversibly photobleaching the FAST-fluorogen complex. The experimentally determined resolution of 28 nm allowed us to resolve Ensconsin-labeled microtubules and to track single molecules in mitochondria. Our results demonstrate that FAST is well-

SUBMITTER: Smith EM 

PROVIDER: S-EPMC8608280 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature