Inner Filter Effect Correction for Fluorescence Detection in AUC by Difference Invariant

When employing the fluorescence detection system for analytical ultracentrifuge (AU-FDS) with confocal optical structure, the inner filter effect (IFE) introduces a nonlinear relationship between fluorescence intensity and concentration at high concentrations. This phenomenon limits the potential applications of AU-FDS. To address the challenge, we propose a correction method specifically designed for the confocal optical structure, which substitutes uncorrected data with a concentration-proportional, difference-invariant <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1-f$ </tex-math></inline-formula>. By performing real-time calculation of system parameters, this method achieves self-calibration, ensuring that the difference-invariant <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1-f$ </tex-math></inline-formula> is not affected by systematic noise and variations in environmental factors, including fluorescent material adhering to the upper window, temperature fluctuation, and excitation light power shift. Experiments conducted on the rotation equipment demonstrate that, by applying our method, the linear correlation coefficient of fluorescein solutions within the concentration range of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$0.86\sim 109.9~\mu $ </tex-math></inline-formula>M is improved from 0.9166 to 0.9965.