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Updated: Nov 4, 2025
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Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
Published on: June 26, 2010
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The Positive Switching Fluorescent Protein Padron2 Enables Live-Cell Reversible Saturable Optical Linear Fluorescence
Timo Konen1, Daniel Stumpf1, Tim Grotjohann1
1Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
ACS Nano
|May 21, 2021
Summary
Researchers developed Padron2, a novel positive switching reversibly switchable fluorescent protein (RSFP). This advancement enables advanced Reversible Saturable Optical Linear transitions (RESOLFT) nanoscopy in live cells without sequential light switching.
Area of Science:
- Biophysics
- Microscopy
- Molecular Biology
Background:
- Reversibly switchable fluorescent proteins (RSFPs) enable optical switching between fluorescent states.
- Reversible Saturable Optical Linear (RESOLFT) nanoscopy achieves super-resolution imaging.
- Existing RESOLFT methods often rely on negative switching RSFPs and sequential illumination.
Purpose of the Study:
- To engineer a positive switching RSFP for improved RESOLFT nanoscopy.
- To develop a RESOLFT imaging scheme that avoids sequential light switching.
- To demonstrate live-cell RESOLFT imaging with the new RSFP.
Main Methods:
- Engineering of the green fluorescent RSFP Padron into a positive switching variant, Padron2.
- Characterization of Padron2's switching properties, including cycle count and contrast ratio.
- Development and application of a non-sequential beam scanning RESOLFT imaging scheme using Padron2.
Main Results:
- Padron2 exhibits 50-fold more switching cycles than its predecessor.
- Padron2 achieves an on/off contrast ratio exceeding 100:1.
- Successful demonstration of live-cell RESOLFT nanoscopy without sequential illumination.
Conclusions:
- Padron2 is a robust positive switching RSFP suitable for advanced RESOLFT nanoscopy.
- The developed non-sequential RESOLFT method simplifies imaging and enhances applicability.
- This work paves the way for more efficient super-resolution live-cell imaging.
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