Inner Filter Effects: Conducting Fluorescence Experiments With Highly Absorbing Samples

Luca, Ceresa

Publication

Inner Filter Effects: Conducting Fluorescence Experiments With Highly Absorbing Samples

Luca, Ceresa

Date

2020

Abstract

Fluorescence technology has enabled many practical applications in chemistry, biology, and medicine. However, the fluorescence intensity dependence on fluorophore absorption/concentration can develop non-linearity because of the Primary Inner-Filter Effect (IFE). The primary IFE is a consequence of the progressive excitation light attenuation due to sample absorption. For low sample concentrations/absorptions, this effect is minimal, yet still detectable (even for absorbances < 0.05). The Inner-Filter Effect has been recognized for many decades, yet there is no method that adequately corrects the loss in linearity. Hence, I am proposing an original strategy based on the calculation of a spectrofluorometer’s “geometrical sensitivity factor”, which can be found for any instrument. Using a thin layer sample (= 1 mm) that is mounted on a movable holder, we can determine the sensitivity factor that will allow us to correct the emission/excitation spectra and restore the linear dependence.