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

Area of Science:

• Analytical Chemistry
• Spectroscopy

Background:

• The inner filter effect (IFE) significantly limits the linear range of fluorescence measurements.
• Accurate fluorescence quantification is often hindered by IFE, necessitating sample dilution or absorbance corrections.

Purpose of the Study:

• To develop a novel method for correcting the inner filter effect (IFE) in fluorescence measurements.
• To extend the linear relationship between fluorescence signal and fluorophore concentration.

Main Methods:

• A Z-position correction method was proposed, utilizing two fluorescence measurements at different vertical optical element positions.
• The method was validated using quinine sulfate samples, pure and mixed with potassium dichromate.
• A companion numerical IFE correction method was developed, requiring no geometric parameters.

Main Results:

• The Z-position method achieved linear fluorescence response (R² > 0.999) up to high absorbance values (Aex ≈ 2, Aem ≈ 0.5).
• The correction extended linear response over ~98% of the concentration range with ~1% deviation.
• Both methods effectively eliminated the need for sample dilution or separate absorbance measurements.

Conclusions:

• The Z-position IFE correction method offers a simple and effective way to improve fluorescence quantification.
• The developed methods significantly enhance the utility of fluorescence measurements in various applications.
• Accessible online tools are provided for implementing these IFE correction techniques.