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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
Time-domain whole-field fluorescence lifetime imaging with optical sectioning
1Femtosecond Optics Group, Department of Physics, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BW, UK.
Journal of Microscopy
|September 14, 2001
Summary
This study presents a whole-field time-domain fluorescence lifetime imaging (FLIM) microscope capable of optical sectioning. The developed FLIM instrument achieves high temporal resolution for advanced chemical and biological tissue imaging.
Area of Science:
- Biomedical Optics
- Fluorescence Spectroscopy
- Microscopy Instrumentation
Background:
- Fluorescence lifetime imaging (FLIM) provides rich information about fluorophore microenvironments.
- Existing FLIM techniques often face limitations in speed, resolution, or portability.
Purpose of the Study:
- To develop and characterize a whole-field time-domain FLIM microscope with optical sectioning capabilities.
- To demonstrate the instrument's utility in chemical and biological imaging applications.
Main Methods:
- Utilized a mode-locked Ti:Sapphire laser for excitation and a gated microchannel plate image intensifier with an intensified CCD camera for detection.
- Acquired time-gated images and applied multi-exponential decay fitting to generate 2-D FLIM maps.
- Incorporated structured illumination for achieving 3D FLIM with optical sectioning.
Main Results:
- Achieved a temporal discrimination of better than 10 picoseconds.
- Successfully performed chemically specific imaging and quantitative analysis of fluorophore mixtures and environments.
- Demonstrated effective imaging and contrasting of biological tissues based on autofluorescence.
Conclusions:
- The developed whole-field time-domain FLIM microscope offers high performance and versatility for various imaging tasks.
- The potential for compact, low-cost, all-solid-state laser configurations suggests real-world application feasibility.
- 3D FLIM with optical sectioning was successfully realized, enhancing imaging depth and specificity.
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