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A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants
Cristina Flors1, Jun-ichi Hotta, Hiroshi Uji-i
1Department of Chemistry and Institute for Nanoscale Physics and Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium.
Journal of the American Chemical Society
|October 25, 2007
Summary
New fluorescent proteins Dronpa-2 and Dronpa-3 exhibit faster photoswitching and dark recovery than Dronpa. This improved performance enables optimized imaging for advanced microscopy techniques like photoactivation-localization microscopy (PALM).
Area of Science:
- Biophysics
- Molecular Biology
- Microscopy
Background:
- Fluorescent proteins are crucial tools in biological imaging.
- Reversible photoswitchable fluorescent proteins (RSFPs) offer dynamic control over fluorescence.
- Dronpa and its variants are key RSFPs with applications in super-resolution microscopy.
Purpose of the Study:
- To investigate the photophysical properties and photoswitching mechanisms of Dronpa-2 and Dronpa-3.
- To compare the performance of Dronpa-2 and Dronpa-3 with the precursor Dronpa.
- To optimize imaging parameters for photoactivation-localization microscopy (PALM) using these proteins.
Main Methods:
- Ensemble and single-molecule fluorescence spectroscopy.
- Analysis of single-molecule fluorescence traces to determine switching kinetics.
- Development of optimized acquisition parameters for camera-based super-resolution imaging.
Main Results:
- Dronpa-2 and Dronpa-3 demonstrate faster light response and dark recovery compared to Dronpa, both in bulk and at the single-molecule level.
- Key differences in forward and backward switching efficiencies and rate constants were identified between the mutants and Dronpa.
- Higher conformational freedom of the chromophore in the Dronpa mutants was proposed as the underlying reason for improved performance.
Conclusions:
- Dronpa-2 and Dronpa-3 exhibit enhanced photoswitching properties, making them superior to Dronpa for advanced imaging.
- Optimized photophysical parameters facilitate improved performance in camera-based sub-diffraction-limit imaging.
- These RSFPs are valuable for fast photoactivation-localization microscopy (PALM) and enable new approaches like simultaneous two-color stroboscopic illumination.
