The effect of excitation beam absorption on measured values of fluorescence has been studied wlth a computer-centered spectrofiuorimeter capable of measuring fluorescence and absorbance slmuitaneously. This effect appears to be independent of the nature of the absorbing species and the excitation and emisslon wavelengths. A model is proposed and tested which corrects fluorescence, observed at 90°, for the attenuation of the excitation beam caused by the absorbance of the fluorophore and any chromophores present in the cell. The resuitlng absorptlon-corrected fluorescence is linear with the concentration of the fiuorophore in solutions wlth total absorbances as high as 2.0.
3(b), 3(c), and 3(d), one could induce a 90' (i.e. , 180' for two-photon) phase shift between every other pair of coherent pulses into the sample cell.By subtracting the resulting fluorescence from pairs of coherent pulses with and without the phase shift, one could eliminate the diffraction background and thus automatically double the interference signal. A detailed study of this scheme will appear elsewhere. 'To conclude, we mention a possible variant of the previous method. Instead of two pulses, one could use a sequence of N equally spaced pulses obtained, for example, by sending the initial pulse into a confocal resonator. ' The optical analog of such a system would be a grating with N lines. One should expect in that case that the frequency spectrum experienced by the atom would be a series of peaks (N times narrower in width), separated by the frequency interval 1/T, «.
A computer centered spectrophotometer-spectrofluorimeter combination instrument has been fabricated that will allow simultaneous absorption and fluorescence measurements.The dedicated computer applies corrections to the data collected for many of the instrumental and photophysical variables of fluorescence measurements.
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