Extended photon counting histogram and fluorescence intensity distribution analysis approaches for optically biased...

Area of Science:

• Physical Chemistry
• Spectroscopy
• Biophysics

Background:

• Laser-induced gradient potentials can bias particle diffusion dynamics.
• This bias affects photon counting statistics in fluorescence fluctuation spectroscopy (FFS).
• Existing analysis methods may not accurately retrieve unbiased parameters under these conditions.

Purpose of the Study:

• To extend the theories of photon counting histogram (PCH) and fluorescence intensity distribution analysis (FIDA).
• To accurately fit biased experimental data and retrieve unbiased parameters (N, ε, α).
• To demonstrate the equivalence of the extended PCH and FIDA approaches.

Main Methods:

• Theoretical extension of PCH and FIDA approaches.
• Monte Carlo simulations for single- and double-component systems.
• Numerical and analytical proofs of method equivalence.

Main Results:

• Successfully extended PCH and FIDA theories to account for laser-induced bias.
• Validated the extended theories using simulations.
• Proved the analytical and numerical equivalence of the extended PCH and FIDA methods.

Conclusions:

• Extended PCH and FIDA provide accurate, unbiased parameter retrieval from biased FFS data.
• The extended methods are robust for both single- and multi-component systems.
• Discussed practical implementations and potential applications for these advanced analytical tools.