Fluorescence anisotropy in the frequency domain by an optical microscope

Area of Science:

• Biophysics
• Spectroscopy
• Microscopy

Background:

• Fluorescence anisotropy decay spectroscopy is a valuable technique for determining the size and shape of biological molecules.
• Optical microscopy offers reduced excitation volume and applicability to spatially inhomogeneous samples.

Purpose of the Study:

• To couple fluorescence anisotropy decay spectroscopy with optical microscopy.
• To test the dynamic response of the microscope-based system.
• To enable the study of spatially inhomogeneous biological samples.

Main Methods:

• Phase modulated fluorescence anisotropy decay measurements.
• Coupling a phase modulation fluorometer to an optical microscope.
• Comparing results from cuvette measurements and microscope-stage micro-drop measurements.
• Implementing a correction factor for objective depolarization in data analysis.

Main Results:

• The microscope-based setup demonstrated comparable dynamic response to a standard fluorometer.
• Data analysis incorporating an objective depolarization correction factor yielded consistent results between setups.
• The technique was successfully applied to analyze long DNA tracts and short DNA fragments with structural anomalies.

Conclusions:

• Microscope-coupled fluorescence anisotropy decay spectroscopy is a viable method for analyzing biological molecule dynamics.
• The validated technique allows for the investigation of size and shape in inhomogeneous samples.
• This approach provides insights into the structural anomalies of DNA fragments.