Open AccessSingle-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System
Author(s) -
Elizabeth M. Smith,
Arnaud Gautier,
Elias M. Puchner
Publication year - 2019
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.9b00149
Subject(s) - photobleaching , fluorescence , fluorescence microscope , microscopy , resolution (logic) , absorption (acoustics) , molecule , biophysics , chemistry , fluorescence recovery after photobleaching , fluorescence lifetime imaging microscopy , super resolution microscopy , microscope , nanotechnology , materials science , optics , computer science , physics , biology , organic chemistry , artificial intelligence
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-suited for single-molecule localization microscopy (SMLM). The small size and the availability of spectrally distinct fluorogens present unique advantages of the FAST system as a potential orthogonal labeling strategy that could be applied in conjunction with existing super-resolution dyes and photoactivatable proteins in versatile imaging applications.