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Optical Engineering

On-Line Computer Capability In Fluorescence Spectroscopy
Author(s): Werner Schmidt
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Paper Abstract

Fluorescence excitation spectra of chlorophyll a and flavin, corrected either by employing the common quantum counter rhodamine B or by comparison with the corresponding absorption spectra, are discussed.absorption spectra are not necessarily identical, as commonly assumed. functions, "kernels," is used for smoothing. Cross-correlations with two other, more complex kernels demonstrate their equivalence with the first and fourth derivative. The latter procedures, in turn, have been known for some time to improve wavelength resolution. For the determination of fluorescence quantum efficiencies, spectra are converted from wavelength to wave number on the x-axis, and from energy to quanta absorption on the y-axis. Based on thermodynamic arguments, the fluorescence emission spectrum of chlorophyll a is calculated from its absorption spectrum. Measured and calculated emission spectra are practically identical. Similarly, the integrated absorption spectrum of chlorophyll a was used to calculate the intrinsic fluorescence lifetime and, taking the quantum efficiency of fluorescence into account, the ap-parent lifetime. Continuous fluorescence polarization spectra have been obtained for various commonly used fluorophores. Based on a well-known formula originally deduced by Perrin, "angle spectra" are calculated from these polarization spectra, which immediately exhibit the angles between the electronic transition dipoles. In addition, for chlorophyll a and flavin, "anisotropy spectra" have been determined. Taking the dependency of fluorescence polarization on temperature or on the concentration of the fluorophore into account, both its rotational relaxation time and the value of Rc are determined. For all thirteen fluorophores investigated, the corrected and normalized fluorescence excitation and emission spectra are given, including the apparent 0-0 transition energies as deduced from the wavelength of their intersection.

Paper Details

Date Published: 1 October 1983
PDF: 7 pages
Opt. Eng. 22(5) doi: 10.1117/12.7973199
Published in: Optical Engineering Volume 22, Issue 5
Show Author Affiliations
Werner Schmidt, Fakultat fur Biologie (Federal Republic of Germany)


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