Selective dequenching by photobleaching increases fluorescence probe visibility

Self- quenching properties of fluorescent dyes have been developed to improve the sensitivity of fluorescent measurement. Photobleaching of Calcein at a concentration greater than the critical, self- quenching concentration actually increased fluorescence, whereas at lower concentrations photobleaching decreased fluorescence, enhancing signal to noise by almost 4000. The photobleaching-dequenching principle has been demonstrated in giant liposomes encapsulating Calcein at higher quenched concentrations. Upon photobleaching background fluorescence was reduced and the liposome fluorescence increased. Liposomes invisible in the presence of background fluorescence became visible upon photobleaching. Fluorescent lifetime was unaffected by photobleaching, whereas the lifetime decreased significantly upon dilution, allowing distinction between photobleached fluorescence particularly upon dequenching. The principle may be suited to improving fluorescence imaging and resolving fluorescent probes in particle- based assays.