We apply confocal fluorescence microscopy for real time studies of reversible conformational changes of poly(methacryloyloxyethyl phosphate) (PMEP) brushes chemically grafted onto gold substrates. Oregon green 488 fluorophores chemically attached onto the PMEP polymers were used as reporters for probing the conformational changes. Use of a specially designed liquid flow microchamber allowed dynamic imaging of the brushes under varying environmental conditions. The fluorescence intensities exhibited fully reversible brightness changes on alternation of the solution in the chamber between water and KCl. This reversible quenching behavior is consistent with a conformational change between an extended and a collapsed brush configuration. The fluorescence quenching behavior of the brushes was found to be dependent on ion concentration as well as polymer grafting density and was caused by nonradiative energy transfer to the polymer scaffold and the gold substrate.