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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
Supercontinuum stimulated emission depletion fluorescence lifetime imaging
Michael D Lesoine1, Sayantan Bose, Jacob W Petrich
1U.S. Department of Energy, Ames Laboratory , Ames, Iowa, United States; Department of Chemistry, Iowa State University, Ames, Iowa, United States.
The Journal of Physical Chemistry. B
|June 15, 2012
Summary
Supercontinuum stimulated emission depletion (STED) fluorescence lifetime imaging achieved 36-40 nm resolution, surpassing the diffraction limit. This advanced technique accurately measured cellular actin structures, enabling new biological insights.
Area of Science:
- Biophotonics and advanced microscopy techniques.
- Cellular imaging and structural biology.
Background:
- Confocal microscopy is limited by the diffraction limit of light, hindering the visualization of sub-diffraction cellular structures.
- Stimulated emission depletion (STED) microscopy offers enhanced spatial resolution beyond the diffraction limit.
- Fluorescence lifetime imaging provides quantitative information about the molecular environment.
Purpose of the Study:
- To demonstrate supercontinuum (SC) STED fluorescence lifetime imaging using time-correlated single-photon counting (TCSPC).
- To achieve and validate sub-diffraction spatial resolution for cellular imaging.
- To apply the developed STED system for analyzing fine cellular structures like F-actin.
Main Methods:
- Utilized a supercontinuum light source for STED microscopy.
- Employed time-correlated single-photon counting (TCSPC) for fluorescence lifetime detection.
- Measured spatial resolution using 40-nm fluorescent beads and analyzed F-actin structures in cultured cells.
Main Results:
- Achieved a spatial resolution of 36 ± 9 nm (X) and 40 ± 10 nm (Y) with the SC-STED system.
- Demonstrated that underfilling the objective and time gating were crucial for reaching STED resolution.
- Confirmed consistent fluorescence lifetime measurements (2.0 ± 0.1 ns) between confocal and STED imaging for beads.
- Successfully imaged Alexa Fluor 594-phalloidin labeled F-actin projections with dimensions below the diffraction limit.
- Measured fluorescence lifetimes for actin-rich projections ranging from 2.2 to 2.9 ns.
Conclusions:
- Supercontinuum STED fluorescence lifetime imaging provides superior spatial resolution for biological samples.
- The developed instrument enables the study of sub-diffraction cellular structures with quantitative lifetime information.
- This technique holds potential for advancing research in cell biology and understanding dynamic cellular processes.
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