EFFECT OF CONCENTRATION QUENCHING ON FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING MEASUREMENTS

Standard analysis of fluorescence recovery after photobleaching (FRAP) data is valid only if the quantum yield of unphotobleached fluorophor es is independent of concentration, yet close molecular packing in two -dimensional systems may lead to significant fluorescence concentratio n quenching. Using total internal reflection fluorescence, we quantifi ed the surface concentration dependence of the relative quantum yield of fluorescein isothiocyanate-labeled proteins adsorbed to polymeric s urfaces before performing measurements of fluorescence recovery after pattern photobleaching. Adsorbed layers of FITC-labeled ribonuclease A displayed significant concentration quenching, and thus the standard FRAP analysis method was unacceptable. We present an extended FRAP ana lysis procedure that accounts for the changing quantum yield of diffus ing fluorophores in systems that are influenced by concentration quenc hing. The extended analysis shows that if concentration quenching cond itions prevail, there may be significant error in the transport parame ters obtained from FRAP measurements by using the standard procedures.