Chromophore-driven optical path reduction as an indirect inner filter effect mitigation strategy in fluorescence...

Microplate readers have become indispensable for high-throughput fluorescence measurements across diverse scientific fields, yet the Inner Filter Effect (IFE) frequently compromises their accuracy. IFE arises when the sample absorbs excitation or emission photons, undermining the linear fluorescence-concentration relationship essential for reliable quantification. Here, we present a practical method to mitigate the secondary IFE (sIFE) in microplate assays, enabling accurate fluorescence measurements in samples with high optical densities. In this study, we present a strategy that leverages a strongly absorbing chromophore to enhance primary IFE (pIFE) in highly absorbing fluorophore solutions. By confining fluorescence generation to a shallow region near the sample surface, sIFE is effectively mitigated, even at emission optical densities up to 79 absorbance units per cm. We confirmed linear fluorescence-concentration responses (R² > 0.99) over a broad dynamic range, demonstrating minimal loss in signal fidelity at very high analyte concentrations. This approach eliminates the need for post-measurement mathematical corrections: samples are simply mixed with a fixed amount of absorber, and fluorescence can be measured directly in either transparent or non-transparent microplates. Because the method physically reduces the optical path length for emitted photons, it remains robust across various microplate types and instrument configurations, delivering consistent, accurate results without complex protocols or specialized instrumentation. This work introduces a novel physical solution to mitigating sIFE in microplate fluorescence assays. By strategically adjusting the sample's optical geometry with an added absorber, we dramatically expand the dynamic range for high-density fluorophore measurements, surpassing the limitations of conventional correction formulas. The presented method was developed specifically for microplate readers but could also be promising for front-face fluorescence measurements in conventional fluorometers.