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Calculation of photon-count number distributions via master equations
Kaupo Palo1, Ulo Mets, Vello Loorits
1Evotec Technologies, Harku, Estonia.
Biophysical Journal
|January 3, 2006
Summary
This study introduces a new theory for analyzing fluorescence intensity fluctuations by accounting for molecular emission intensity changes during measurement. This improved photon-count analysis offers significantly better fit quality in experiments.
Area of Science:
- Biophysics
- Spectroscopy
- Fluorescence Microscopy
Background:
- Photon-count number histogram analysis is a key technique for studying fluorescence intensity fluctuations.
- Previous theories often assumed constant molecular emission intensity, an assumption potentially violated in experiments.
- Fluorescence correlation spectroscopy (FCS) is a related technique, but photon-count analysis offers advancements.
Purpose of the Study:
- To develop and validate a new theory for photon-count number distributions that accounts for intensity fluctuations during the counting interval.
- To assess the impact of intensity fluctuations on the accuracy of photon-count analysis.
- To improve the fit quality of experimental fluorescence data compared to existing methods.
Main Methods:
- Developed a new theory for photon-count number distributions incorporating intensity fluctuations.
- Utilized numerical solutions of Master equations (ME) to model diffusion, singlet-triplet transitions, and photon emission.
- Included corrections for detector afterpulsing and dead-time effects.
- Tested the theory by fitting photon-count histograms across various counting time interval lengths.
Main Results:
- The new Master equation (ME) theory significantly improves fit quality compared to earlier fluorescence intensity multiple distribution analysis (FIMDA) methods.
- The ME-theory accurately models photon-count distributions even with intensity fluctuations during the counting interval.
- Demonstrated that previous theories might achieve good fits by indirectly accounting for dynamic processes through spatial brightness distribution adjustments.
Conclusions:
- The developed ME-theory provides a more accurate description of photon-count number distributions by considering intensity fluctuations.
- This advancement enhances the analysis of fluorescence intensity fluctuations, offering deeper insights into molecular dynamics.
- The study highlights the limitations of assuming constant emission intensity and offers a more robust analytical framework.
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