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Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
Published on: October 31, 2015
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Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System
Elizabeth M Smith1, Arnaud Gautier2, Elias M Puchner1
1School of Physics and Astronomy , University of Minnesota , Minneapolis , Minnesota 55455 , United States.
ACS Chemical Biology
|May 16, 2019
Summary
The Fluorescence-Activating and absorption-Shifting Tag (FAST) system enables super-resolution imaging and single-molecule tracking. This advanced technique achieves 28 nm resolution, resolving cellular structures and tracking molecules in living cells.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Super-resolution microscopy requires precise labeling for single-molecule localization.
- Existing methods face challenges with dense protein expression and dynamic cellular environments.
Purpose of the Study:
- To develop and validate the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution imaging.
- To enable high-resolution single-molecule tracking in both live and fixed cells.
Main Methods:
- Utilized the FAST system with its fast-off rate fluorogen binding.
- Controlled fluorogen concentration and photobleaching for spatially separated labeling.
- Applied single-molecule localization microscopy (SMLM) techniques.
Main Results:
- Achieved a resolution of 28 nm.
- Successfully resolved Ensconsin-labeled microtubules.
- Tracked single molecules within mitochondria.
- Demonstrated single-molecule measurements in living and fixed cells.
Conclusions:
- The FAST system is highly suitable for SMLM applications.
- Its small size and spectral diversity offer orthogonal labeling advantages.
- FAST can be integrated with existing super-resolution techniques for versatile imaging.
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