Analysis of fluorescence anisotropy decays by a least square method

Area of Science:

• Biophysics
• Physical Chemistry

Background:

• Fluorescence anisotropy decay analysis is crucial for studying molecular dynamics.
• Existing methods may not fully account for instrument response functions, potentially introducing errors.

Purpose of the Study:

• To develop and validate a novel method for fluorescence anisotropy decay analysis.
• To rigorously incorporate the apparatus response function into the analysis.
• To improve accuracy and reduce errors in determining fluorescence decay and correlation times.

Main Methods:

• A non-linear least squares fitting procedure was employed.
• The transient anisotropy r(ex)(t) was fitted to the ratio of convolutions of the apparatus response function g(t) with sums of exponential functions.
• The method was tested using simulated data from air and nitrogen lamp response functions.

Main Results:

• The new method accurately accounts for the apparatus response function.
• It is applicable to various decay shapes and time values.
• Statistical standard errors for anisotropy decay parameters were smaller compared to the moment method.
• Systematic errors in decay time had a reduced impact on correlation time.

Conclusions:

• The developed method provides accurate and convenient fluorescence anisotropy decay analysis.
• It offers improved precision in determining molecular dynamics parameters.
• This approach is robust and adaptable to different experimental conditions.