Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System.
| Autor: | Smith EM; School of Physics and Astronomy , University of Minnesota , Minneapolis , Minnesota 55455 , United States., Gautier A; PASTEUR, Département de Chimie , École Normale Supérieure, PSL University, Sorbonne Université, CNRS , 75005 Paris , France., Puchner EM; School of Physics and Astronomy , University of Minnesota , Minneapolis , Minnesota 55455 , United States. |
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| Jazyk: | angličtina |
| Zdroj: | ACS chemical biology [ACS Chem Biol] 2019 Jun 21; Vol. 14 (6), pp. 1115-1120. Date of Electronic Publication: 2019 May 23. |
| DOI: | 10.1021/acschembio.9b00149 |
| Abstrakt: | 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. |
| Databáze: | MEDLINE |
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