Improved FRAP measurements on biofilms

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>We 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.</jats:p><jats:sec><jats:title>STATEMENT OF SIGNIFICANCE</jats:title><jats:p>Diffusion 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.</jats:p></jats:sec>

Diffusion profiles in L. lactis biofilms under different conditions

“…Biofilms of Lactococcus lactis (DSM 20729) were anaerobically cultivated in a flow‐cell system described by Schlegel [29], with an optimized dye inlet and chamber [28]. Pre‐culture was carried out in a 100 mL bottle at 30°C in 100 mL MRS (de Man, Rogosa, Sharpe) medium [30] at a pH of 7 with 20 g·L −1 glucose inoculated from the same cryo batch for all experiments.…”

“…FRAP data were processed and diffusion constants were calculated according to the improved FRAP model by Hauth et al. [28]. From the diffusional constants the adjusted diffusion could be calculated., with D B being the diffusion constant in the biofilm and D W being the diffusion constant in the medium (= water control).…”

“…Biofilms of Lactococcus lactis (DSM 20729) were anaerobically cultivated in a flow-cell system described by Schlegel [29], with an optimized dye inlet and chamber [28]. Preculture was carried out in a 100 mL bottle at 30 • C in 100 mL…”

“…FRAP data were processed and diffusion constants were calculated according to the improved FRAP model by Hauth et al [28]. From the diffusional constants the adjusted diffusion…”

“…In contrast, a non-invasive method for assessing diffusion in biofilms is fluorescence recovery after photobleaching (FRAP), where a short, but strong laser pulse bleaches a region of interest and the recovery time of fluorescence is measured, from which then diffusional constants can be calculated. Since the 70s, several diffusion models have been developed [26,27], with the latest model employing Markov-Chain Monte Carlo methods to reduce common artefacts found in FRAP measurements [28]. With this model, it is possible to calculate diffusional constants more exactly from FRAP measurements.…”