Improved FRAP Measurements on Biofilms

Abstract

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. 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.

Passively immobilized cyanobacteria Nostoc species BB 92.2 in a moving bed photobioreactor (MBPBR): Design, cultivation, and characterization

“…It should be briefly noted that mixing time only provides information about macromixing in the reactor, not about mixing within the carrier and diffusion through the biofilm. The former is probably only accessible via CFD simulations, the latter requires complicated measurement setups, for example, with a confocal laser scanning microscope (Hauth et al, 2020) or pH microprobes (Hou et al, 2017). As the biofilm matrix acts both as a nutrient reservoir and as a pH buffer (Flemming et al, 2016), it can be assumed that the values between biofilm and medium will differ anyway.…”

Fluorescence photobleaching and recovery of fluorescein sodium in carbomer film

“…Fluorescence recovery aer photobleaching (FRAP) is an essential technique for studying molecular mobility and diffusion phenomena across various scientic domains. FRAP is widely used to measure slow diffusion 3,4 in various contexts, including high-viscosity solutions, [5][6][7] colloidal systems, 8,9 thin lms, [10][11][12][13] live cells, [14][15][16][17] and others. 18,19 The effectiveness of FRAP in measuring molecular diffusion is inuenced not only by the molecules themselves but also by their environmental medium.…”

The structure and matrix dynamics of bacterial biofilms as revealed by antimicrobial peptides' diffusion

“…The diffusion of solutes in biofilms has been studied for many years, first macroscopically, by measuring the permeability of the solute traversing across biofilms between two separate chambers, 3 and second, by microscopic approaches using optical microscopies, which settled progressively. 4,5 Among the sophisticated techniques used over the last 25 years are fluorescence recovery after photobleaching (FRAP), [4][5][6][7][8] fluorescence correlation spectroscopy (FCS), 7,9,10 including under two-photon excitation, 11,12 time-lapse confocal scanning laser microscopy (CSLM), 10,13 fluorescence lifetime imaging (FLIM), 7 total internal reflection fluorescence (TIRF), 14 multiple particle tracking (MPT), 15 and atomic force microscopy (AFM). 16 Small antibiotic molecules and large macromolecular assemblies such as phages have been studied using these techniques.…”