Geometry-Encoded Multireceiver Fluorometry Enables Full-Range Nonlinear Quantification under the Inner Filter Effect

Abstract

The inner filter effect (IFE) transforms the nominally linear fluorescence-concentration relationship into a geometry-dependent and often nonmonotonic response, resulting in reduced sensitivity, concentration ambiguity, and inaccurate underestimation at high optical densities. Here, we introduce a spatially encoded multireceiver fluorometric strategy that does not eliminate or correct the IFE, but instead harnesses the spatial fluorescence attenuation induced by IFE as an additional quantitative encoding dimension. Fluorescence generated along the excitation axis is integrated over independently positioned receiver windows, and concentration is recovered by nonlinear optimization of the joint fluorescent intensity vector. Tryptophan was selected as a biomedically relevant model fluorophore to validate the proposed strategy. Single-window calibration exhibited vanishing-gradient boundary and two-valued concentration inversions, whereas two spatially separated receiver windows restored global identifiability across the full concentration range. Screening of 35 two-receiver geometries identified the minimum-uncertainty configuration, achieving an average 95% error half-width of mean E_95= 0.884 mg/L, and the maximum-sensitivity configuration, reaching a noise-normalized response sensitivity of mean S_N=3.832 a.u./(mg/L). By converting spatial attenuation into a multidimensional concentration coordinate, this approach extends quantitative fluorescence analysis without dilution, a separate absorbance measurement, or piecewise calibration and provides a general metrology framework for fluorescence metrology even under strong IFE conditions.