Related Experiment Videos
Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)
Andrew H A Clayton1, Quentin S Hanley, Donna J Arndt-Jovin
1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
Biophysical Journal
|August 31, 2002
Summary
We developed anisotropy-FLIM (rFLIM), a novel microscopy technique for wide-field measurement of fluorophore anisotropy decay. This method images cellular rotational dynamics and molecular proximity, offering new insights into biological structures.
Area of Science:
- Biophysics
- Microscopy
- Cellular Imaging
Background:
- Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique.
- Measuring fluorophore anisotropy decay provides insights into molecular dynamics.
- Existing FLIM methods have limitations in wide-field anisotropy measurements.
Purpose of the Study:
- To introduce and validate a novel variant of FLIM, termed anisotropy-FLIM (rFLIM).
- To enable wide-field, pixel-by-pixel measurement of fluorophore anisotropy decay.
- To develop a theoretical framework for extracting molecular dynamics parameters.
Main Methods:
- Adaptation of frequency-domain FLIM technology.
- Incorporation of linear polarizers in excitation and emission paths.
- Recording phase delay and intensity ratios for polarized fluorescence components.
- Utilizing a CCD camera for 2D imaging.
Main Results:
- Demonstrated rFLIM's capability to measure anisotropy decay.
- Developed theory to extract hindered rotator model parameters from single-frequency measurements.
- Illustrated rFLIM with fluorescein-glycerol and enhanced green fluorescent protein (EGFP) in bacteria.
- Observed concentration depolarization due to emFRET in EGFP-expressing bacteria.
Conclusions:
- rFLIM is a powerful new tool for cellular imaging.
- The technique is based on rotational dynamics and molecular proximity.
- rFLIM provides insights into complex biological structures and phenomena like emFRET.