Correction of Fluorescence Spectra

Abstract

Several methods that can be applied to remove wavelength-dependent instrumental effects from fluorescence emission and excitation spectra have been investigated. Removal of such artifacts is necessary for the comparison of spectra that have been obtained on different instruments. Without correction, spectral line positions and relative intensities will be instrument-determined to a great extent. Furthermore, the application of adequate correction procedures provides excitation spectra which can be directly compared to absorption spectra; comparison of corrected excitation spectra and absorption spectra can be used to interpret the efficiency and pathways of radiative processes. Finally, corrected reflection spectra can be obtained, which can be directly transformed into absorption spectra and are useful for remote sensing applications. The methods that have been studied for the correction of emission spectra are the application of a standard lamp with calibrated spectral output and the use of fluorescence standards. The standards are a series of luminescent phosphors in polymer films and a solution of quinine sulfate dihydrate in perchloric acid, all provided with certified spectral emission values. For correction of excitation spectra, a quantum counter was applied. Several quantum counters were investigated. The best results were obtained for application of a mixture of the dyes basic blue and HITC, which provided good correction for the wavelength range 250 to 820 nm. No good quantum counters have been reported thus far for this (large) wavelength range. Correction for wavelength dependence of the excitation optics was realized by measurement of the excitation light intensity at the sample position and at the reference position with a Si photodiode. Correction factors for the excitation spectra were checked with a number of reference materials. Attention has also been paid to polarization-dependent effects that may occur in fluorescence spectra. The application of correction procedures was demonstrated for phytoplankton fluorescence spectra.