Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy

Area of Science:

• Fluorescence Spectroscopy
• Biophysical Chemistry
• Data Analysis Techniques

Background:

• Traditional fluorescence fluctuation analysis methods like FCA and PCH have limitations with arbitrary sampling times.
• Longer sampling times can enhance signal-to-noise ratios in fluorescence data, but existing theories do not fully support this regime.

Purpose of the Study:

• Introduce a novel analysis technique, time-integrated fluorescence cumulant analysis (TIFCA), for fluorescence fluctuation data.
• Develop a theoretical framework for cumulant functions applicable to arbitrary sampling times.
• Provide tools for rigorous error analysis in TIFCA.

Main Methods:

• Derived a theoretical model for cumulant functions valid for any sampling time.
• Calculated factorial cumulants of photon counts by data rebinning.
• Developed and tested expressions for cumulant variance for error analysis.

Main Results:

• TIFCA is the first exact theory to account for sampling time effects in fluorescence fluctuation experiments.
• Rebinning data can reduce the relative error of higher-order cumulants, enhancing signal-to-noise.
• Successfully applied TIFCA to simple dye systems, demonstrating its ability to resolve species.

Conclusions:

• TIFCA offers a robust method for analyzing fluorescence fluctuation data, especially with long sampling times.
• The technique accurately determines species brightness, occupation number, and diffusion time.
• TIFCA provides a significant advancement in fluorescence data analysis, improving accuracy and species resolution.