Quantum efficiency independence of the time integrated emission from a fluorescent molecule.

The sensitivity of chemical or biological analyses using fluorescent tagged reagents is limited by concentration quenching of the fluorescence at high tag concentrations and by photochemical bleaching if high illumination intensities are used. This paper shows by theoretical analysis and experimental verification that concentration quenching lengthens bleaching lifetimes, and the integrated fluorescent emission obtained on complete bleaching is invariant with fluorescent quantum efficiency, absorption cross section, and the illumination's intensity and duration. This can be used to produce extremely sensitive fluorescent tagging procedes. The procedure also improves photometric reproducibility, allows a fluorescent stain to act as a probe of several properties in its microenvironment, and allows sample background differentiation even when the free stain background is more fluorescent than the bound one.