Inner Filter Effect Correction for Fluorescence Measurements in Microplates Using Variable Vertical Axis Focus

The inner filter effect (IFE) hinders fluorescence measurements, limiting linear dependence of fluorescence signals to low sample concentrations. Modern microplate readers allow movement of the optical element in the vertical axis, changing the relative position of the focus and thus the sample geometry. The proposed Z-position IFE correction method requires only two fluorescence measurements at different known vertical axis positions (z-positions) of the optical element for the same sample. Samples of quinine sulfate, both pure and in mixtures with potassium dichromate, showed a linear dependence of corrected fluorescence on fluorophore concentration (R-2 > 0.999), up to A(ex) approximate to 2 and A(em) approximate to 0.5. The correction extended linear fluorescence response over approximate to 98% of the concentration range with approximate to 1% deviation of the calibration slope, effectively eliminating the need for sample dilution or separate absorbance measurements to account for IFE. The companion numerical IFE correction method further eliminates the need for any gematric parameters with similar results. Both methods are available online at https://ninfe.science .

Automated summary

The study proposes a method for correcting the inner filter effect by changing the position of the optical element, allowing for a linear relationship between fluorescence signals and sample concentration. The results show that the corrected fluorescence signal exhibits a linear response within the concentration range, with minimal calibration slope deviation, effectively eliminating the need for sample dilution or separate absorbance measurements.