Fluorescent emission and fluorophore concentration are only linearly related below particular concentrations of fluorophore. Theoretically, analysis of fluorescent polarization might allow identification of situations in which local concentrations of fluorophore are above the range of linear response. Using a confocal scanning laser microscope, we demonstrate that progressive depolarization of fluorescent emission from fluorescein and fluorescein analogues occurs over the concentration range where linearity is lost. Critically, depolarization of emission is first seen at concentrations of fluorophore slightly below those at which linearity is lost. Thus, polarization analysis can be used to determine whether the local concentration of fluorophore is such that quantitative analysis can be carried out.
