ABSTRACTWe expand the standard FRAP model introduced by Axelrod et al. in 1976. Our goal is to capture some common artifacts observed in the fluorescence measurements obtained with a confocal laser scanning microscope (CLSM) in biofilms: 1) linear drift, 2) exponential decrease (due to bleaching during the measurements), 3) stochastic Gaussian noise, and 4) uncertainty in the exact time point of the onset of fluorescence recovery. In order to fit the resulting stochastic model to data from FRAP measurements and to estimate all unknown model parameters, we apply a suitably adapted Metropolis-Hastings algorithm. In this way, a more accurate estimation of the diffusion coefficient of the fluorophore is achieved. The method was tested on data obtained from FRAP measurements on a cultivated biofilm.STATEMENT OF SIGNIFICANCEDiffusion and mass transport in biofilms is presumed to play an important role in the resistance against antimicrobial agents and the secretion thereof. FRAP measurements give insight into these transport processes. In this article, the authors expand the standard FRAP model by a blackbox part which addresses some artifacts commonly observed in the measurements. This is done in order to improve the estimations of diffusion coefficients considerably, aiming at a more comprehensive description of diffusion processes inside the biofilm. We expect that the methods are transferable to FRAP measurements on materials other than biofilms.