ABSTRACT
This chapter discusses set of steady-state experiments that should prepare the reader for a task on how to properly measure and interpret emission spectra. To avoid problems associated with photo selection by the excitation light and consequential emission polarization, the emission spectra should be measured under Magic Angle conditions. In contrast, fluorescence is measured on a dark background without the possibility of canceling experimental factors. The photomultiplier detector in the spectrofluorometer sees the fluorescence spot created by the excitation light. When the concentration of the dye is low, the excitation light will not be significantly attenuated while passing the cuvette and the fluorescence spot will be homogeneous. Comparison of measurable absorption and fluorescence signal may give a good indication of the sensitivity difference between absorption and fluorescence. Fluorescence measurements often compare signals from different samples, and it is important to realize how various factors may influence the result of the experiment.
