Open AccessInner Filter Effect Correction for Fluorescence Measurements in Microplates Using Variable Vertical Axis Focus
Author(s) -
Tin Weitner,
Tomislav Friganović,
Davor Šakić
Publication year - 2022
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.2c01031
Subject(s) - chemistry , fluorescence , absorbance , analytical chemistry (journal) , dilution , potassium dichromate , fluorophore , calibration , sample (material) , optics , filter (signal processing) , fluorescence spectrometry , chromatography , physics , inorganic chemistry , computer science , computer vision , thermodynamics , quantum mechanics
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 ≈ 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.