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

Abstract

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 (R² > 0.999), up to A_ex ≈ 2 and A_em ≈ 0.5. The correction extended linear fluorescence response over ≈98% of the concentration range with ≈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 geometric parameters with similar results. Both methods are available online at https://ninfe.science.

Figure 1

Geometric parameters of the microplate reader used for the ZINFE, eqs 3–5. The values of the parameters used for the calculations can be found in Table S2, Supporting Information.

Figure 2

Results of the ZINFE correction: left: Q concentration series in UV-transparent microplates (data set 1); right: Q-v concentration series in non-transparent microplates (data set 4); F₁ (blue diamond solid), F₂ (brown box solid), F_Z (green triangle up solid), F_N (purple multiplication), F_A (blue asterisk), and IFS (orange hyphen). Ordinate values were calculated as F_x,norm, and abscissa values were calculated as c_norm. All results can be found in Figure S9, Supporting Information.

Figure 3

Comparison of uncorrected fluorescence (F₁) and IFE-corrected fluorescence (F_A, F_Z, and F_N) in UV-transparent microplates: left: LOD % from Table 1: F₁ (blue box solid), F_A (red box solid), F_Z (green box solid), and F_N (violet box solid); right: b % from Table 1: F₁ (blue box solid), F_A (red box solid) F_Z (green box solid), and F_N (violet box solid). Data are shown only for UV-transparent microplates and the data for non-transparent microplates are shown in Figures S19 and S20, Supporting Information.

Figure 4

Comparison of ZINFE and NINFE corrections (F_Z and F_N) in UV-transparent (T) and non-transparent (NT) microplates. Left: LOD % from Table 1: F_Z (green box solid) and F_N (violet box solid); right: b % from Table 1: F_Z (green box solid) and F_N (violet box solid).