By conjugating proteins with a common pH-sensitive fluorescent label, fluorescein isothiocyanate (FITC), and controlling the ionic strength, we provide a means to decrease the characteristic length scale of the total internal reflection fluorescence (TIRF) technique by two orders of magnitude. The usual characteristic length scale for TIRF is an optical length, specifically the evanescent wave penetration depth (on the order of 100 nm). In our experiments the penetration depth is replaced by the Debye screening length as the characteristic length scale. This is readily controlled to match the dimensions of an adsorbed protein layer (on the order of 1 nm). We achieve this length scale reduction by coupling the well-known pH-sensitivity of fluorescence emission by FITC-labeled proteins with the variation of electrostatic potential near a negatively charged surface. Using this fine-resolution TIRF capability in combination with scanning angle reflectometry, we find that lateral repulsions induce a dramatic reconfiguration of adsorbed lysozyme layers on negatively charged silica surfaces. This occurs as the surface concentration approaches the jamming limit for random sequential adsorption. The reconfiguration evidently optimizes electrostatic interactions in the adsorbed layer and decreases the effective excluded area per lysozyme. The decrease in effective excluded area allows adsorption to continue beyond the jamming limit to ultimately attain a hexagonal close packed monolayer of horizontally oriented lysozyme molecules. The adsorption kinetics switch abruptly from being transport-limited to surface-limited after the reconfiguration.

Standard analysis of fluorescence recovery after photobleaching (FRAP) data is valid only if the quantum yield of unphotobleached fluorophores is independent of concentration, yet close molecular packing in two-dimensional systems may lead to significant fluorescence concentration quenching. Using total internal reflection fluorescence, we quantified the surface concentration dependence of the relative quantum yield of fluorescein isothiocyanate-labeled proteins adsorbed to polymeric surfaces 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 analysis procedure that accounts for the changing quantum yield of diffusing fluorophores in systems that are influenced by concentration quenching. The extended analysis shows that if concentration quenching conditions prevail, there may be significant error in the transport parameters obtained from FRAP measurements by using the standard procedures.

No abstract