Fluorescence cumulants analysis with non-ideal observation profiles

Area of Science:

• Analytical Chemistry
• Biophysics
• Spectroscopy

Background:

• Accurate brightness profile approximation is a key challenge in fluorescence fluctuation spectroscopy (FFS).
• Fluorescence intensity distribution analysis (FIDA) uses polynomial approximation, while photon counting histogram (PCH) analysis employs additional fitting parameters.
• Differences in brightness profile approximations and normalizations between FIDA and PCH can lead to varying estimates of molecular brightness and number.

Purpose of the Study:

• To apply FIDA and PCH approaches to the theory of time-integrated fluorescence cumulants analysis.
• To demonstrate that developed corrections enhance the accuracy of FFS analysis.
• To derive equations relating molecular brightness and number estimates between PCH and FIDA.

Main Methods:

• Application of polynomial approximation (FIDA) and additional fitting parameters (PCH) to time-integrated fluorescence cumulants analysis.
• Development and implementation of corrections for brightness profile approximations in FFS.
• Validation using simulated and experimental FFS data.

Main Results:

• Developed corrections significantly improve the results of FFS analysis.
• The study provides a theoretical framework for understanding discrepancies between FIDA and PCH.
• New equations are derived to reconcile brightness and molecular number estimates from both methods.

Conclusions:

• The developed theoretical framework and corrections enhance the reliability of FFS data analysis.
• Understanding the impact of different brightness profile approximations is crucial for accurate molecular quantification.
• The derived equations facilitate consistent interpretation of FFS data obtained using PCH and FIDA.