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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
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Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution
Optics Letters
|November 1, 2014
Summary
This study introduces a new fluorescence imaging technique using a high-speed CMOS camera and image intensifier for time-correlated single-photon counting. The method enables fast, sensitive live-cell imaging with low excitation power.
Area of Science:
- Biophysics
- Microscopy
- Photonics
Background:
- Time-correlated single-photon counting (TCSPC) is crucial for fluorescence lifetime imaging microscopy (FLIM).
- Traditional TCSPC systems often face limitations in speed and sensitivity, hindering live-cell applications.
- Achieving high frame rates while maintaining single-photon sensitivity is a key challenge in developing advanced FLIM systems.
Purpose of the Study:
- To develop and demonstrate a wide-field TCSPC imaging system with an ultrafast frame rate and single-photon sensitivity.
- To enable time-resolved imaging of fluorescence decays in biological samples, including live cells.
- To showcase the system's compatibility with low excitation power and short acquisition times.
Main Methods:
- Integration of a 1 MHz frame rate complementary metal-oxide semiconductor (CMOS) camera with an image intensifier for wide-field TCSPC.
- Application of the system on a fluorescence microscope to image ruthenium compound Ru(dpp) decays.
- Validation of live-cell imaging capabilities using europium-containing beads in HeLa cells, with lifetime verification via a standard two-photon excitation scanning FLIM system.
Main Results:
- The developed system successfully achieved ultrafast frame rates (1 MHz) combined with single-photon sensitivity.
- Fluorescence decays of ruthenium compound Ru(dpp) with lifetimes of 1-5 microseconds were imaged.
- Live-cell imaging of europium-containing beads (570 microseconds lifetime) in HeLa cells was demonstrated, confirming the system's applicability.
- Submicrowatt excitation power was sufficient for imaging across the entire field of view.
Conclusions:
- The novel TCSPC imaging system offers a powerful tool for time-resolved live-cell imaging.
- The combination of high speed, sensitivity, and low excitation power overcomes previous limitations in FLIM.
- This approach facilitates efficient and detailed studies of dynamic biological processes at the cellular level.
