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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
Precise fluorophore lifetime mapping in live-cell, multi-photon excitation microscopy
Ching-Wei Chang1, Mary-Ann Mycek
1Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109-2099, USA.
Optics Express
|July 1, 2010
Summary
We improved precision in live-cell fluorescence lifetime imaging microscopy (FLIM) using temporal optimization and spatial denoising. This method enhances cellular optical molecular imaging and quantitative sensing by minimizing light damage.
Area of Science:
- Cellular optical molecular imaging
- Bioimage informatics
- Quantitative sensing
Background:
- Fluorophore excited state lifetime offers insights into cellular micro-environments.
- Precise lifetime determination is crucial for quantitative sensing but challenging in live-cell imaging.
- Existing methods face limitations due to experimental conditions favoring live cells.
Purpose of the Study:
- To enhance lifetime precision in live-cell fluorescence lifetime imaging microscopy (FLIM).
- To introduce temporal optimization and spatial denoising methods for two-photon TCSPC FLIM.
- To minimize adverse effects of excitation light on live cells during imaging.
Main Methods:
- Application of temporal optimization techniques.
- Implementation of spatial denoising algorithms.
- Utilizing two-photon time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM).
Main Results:
- Achieved a greater than five-fold improvement in lifetime precision.
- Demonstrated successful application of new methods to live-cell images.
- Minimized detrimental effects of excitation light on cellular samples.
Conclusions:
- Temporal optimization and spatial denoising significantly improve FLIM precision in live cells.
- This approach benefits high-content analysis and bioimage informatics.
- The developed methods offer a robust solution for quantitative sensing in live-cell imaging.
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