The quantification of fluorescent emission from biological samples using analysis of polarization

The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth-dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission.