Related Experiment Video
Updated: May 13, 2025
14:43
Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Published on: August 27, 2014
11.5K
A General Strategy for Enhanced Single-Molecule Imaging Through Intramolecular Energy Transfer
Song Chen1, Fan Ding1, Chang Liu1
1Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Fudan University, Shanghai, 200438, China.
Angewandte Chemie (International Ed. in English)
|April 16, 2025
Summary
Researchers developed a novel intramolecular energy transfer (IMET) strategy using xanthene-based fluorophores to significantly enhance photostability and photon budget for single-molecule imaging applications.
Area of Science:
- Photochemistry and Spectroscopy
- Biophysics
- Materials Science
Background:
- Single-molecule imaging requires fluorophores with high photostability and photon budget.
- Existing fluorophores often bleach rapidly under high excitation power, limiting imaging duration.
- Optimizing energy transfer is crucial for improving fluorophore performance.
Purpose of the Study:
- To develop and evaluate an intramolecular energy transfer (IMET) strategy for enhancing fluorophore photostability.
- To create novel xanthene-based IMET cassettes for advanced single-molecule imaging.
- To investigate the efficiency and timescale of energy transfer in optimized fluorophore systems.
Main Methods:
- Covalent linkage of donor (rhodamine) and acceptor (Si-rhodamine) fluorophores into xanthene-based IMET cassettes.
- Characterization of energy transfer efficiency using spectroscopic methods.
- Assessment of photostability and photobleaching pathways under high excitation power.
- Time-resolved transient absorption spectroscopy to determine energy transfer kinetics.
- Application of IMET cassettes in single-molecule tracking on supported lipid bilayers and live cells.
Main Results:
- Optimized IMET cassettes achieved 94.8% energy transfer efficiency.
- 94.9% of molecules showed donor photobleaching before acceptor photobleaching, minimizing direct excitation-induced acceptor bleaching.
- A 670% enhancement in photostability was observed due to efficient energy channeling.
- Intramolecular energy transfer occurred on a picosecond timescale, faster than fluorescence relaxation and photobleaching.
- Superior performance in live-cell tracking of epidermal growth factor receptor (EGFR) dynamics.
Conclusions:
- The developed IMET strategy significantly enhances fluorophore photostability and photon budget for single-molecule imaging.
- Optimized rhodamine-Si-rhodamine IMET cassettes provide superior performance compared to conventional fluorophores.
- This approach offers a powerful tool for advancing various single-molecule imaging and tracking applications, including live-cell studies.
