Complex homogeneous and heterogeneous fluorescence anisotropy decays: enhancing analysis accuracy

Area of Science:

• Biophysics
• Photochemistry
• Computational Biology

Background:

• Biological macromolecules exhibit complex motions affecting fluorescence properties.
• Analyzing time-correlated single-photon counting data is challenging due to multiple decay parameters and instrument response function (IRF) deconvolution sensitivity.

Purpose of the Study:

• To evaluate different numerical discretizations of the IRF for improved analysis of fluorescence anisotropy decays.
• To determine the most effective method for distinguishing heterogeneous from homogeneous rotational relaxation times.

Main Methods:

• Simulations were used to compare various IRF discretization methods, including linear, quadratic, and cubic local approximations.
• The Grinvald-Steinberg discretization was used as a benchmark.
• Information criteria, specifically the Akaike information criterion, were assessed for detecting heterogeneity.

Main Results:

• Quadratic and cubic IRF approximations provided more accurate estimations of short rotational relaxation times and lifetimes compared to the Grinvald-Steinberg method.
• Cubic approximation demonstrated superior ability in discriminating between complex heterogeneous and homogeneous anisotropy decays.
• The Akaike information criterion effectively detected heterogeneity in rotational relaxation times, even when anisotropy decays appeared homogeneous within statistical error.

Conclusions:

• Advanced IRF discretizations, particularly cubic approximations, significantly improve the accuracy of fluorescence decay analysis for biological macromolecules.
• The Akaike information criterion is a robust tool for identifying heterogeneity in molecular dynamics, enhancing the interpretation of fluorescence anisotropy data.