Experimental method to correct fluorescence intensities for the inner filter effect

Abstract

An empirical procedure for the correction of measured fluorescence intensity for the inner filter effect is described. The procedure is more reliable than those described previously as fluorophores with the fluorescence properties of interest are sequestered from the external aqueous phase. The inner filter effect can therefore be assessed in the absence of chemical reaction or Forster energy transfer between the fluorophore and chromophore. A correction curve for the inner filter effect is obtained by measuring the fluorescence intensity of the sequestered fluorophore after adding a chromophore to increase the absorbance of the solution. The procedure was tested with liposomes containing calcein and two commercial polymer bead preparations containing embedded fluorophores. The former have the advantage of a wider choice of fluorophores and the latter of stability and convenience. The inner filter effect was varied using small aliquots of potassium chromate or pyridinium chloride solution. It was shown that the magnitude of the inner filter effect must be experimentally determined for each instrument and whenever the instrumental configuration is altered if an accurate correction for the inner filter effect is to be obtained. The magnitude of the correction depends on the wavelength range and the pathlength but not on slit-width or sample turbidity for the instrument employed for most of the experiments reported here. This empirical procedure makes possible studies of protein fluorescence quenching by agents previously unsuitable because of high absorbances.