A new technique combining scanning electrochemicalmicroscopy (SECM) and single-molecule fluorescencespectroscopy was developed to accomplish locally andtemporally defined pH adjustments in buffer solutions andon surfaces monitored by fluorescence alteration of pH-sensitive fluorophores in real time. Local pH gradientswere created by electrochemical generation of H
+ or OH
-during redox reactions at ultramicro- or nanoelectrodeswith radii from 5
m to 35 nm. Ratiometric fluorescencemeasurements were performed with a confocal lasermicroscope using two detectors for different spectralregions. Time-resolved pH measurements were carriedout with freely diffusing SNARF-1-dextran. For pH measurements on surfaces, total internal reflection fluorescence microscopy was used in combination with a CCDcamera. The fluorophore SNAFL-succinimidyl ester wasbound to amino-terminated octadecylsilane-coated coverslips. Local pH determinations could be accomplishedwith an accuracy of 0.2 unit. The measured pH profilesshowed a strong dependence on the tip diameter, thebuffer/mediator concentration ratio, and the tip-surfacedistance. As an application for bionanotechnology usingSECM-induced pH changes on the molecular level, theproton-driven ATP synthesis by single membrane-boundF
0F
1-ATP synthases was investigated. ATP synthesisresulted in stepwise subunit rotation within the enzymethat was monitored by single-molecule fluorescence resonance energy transfer.