On the impact of radiative transfer in fluorescence imaging of bacterial films and suspensions

Abstract

Fluorescence imaging and spectroscopy are convenient, rapid, and simple methods to analyze chemical samples including biological materials such as bacterial biofilms and suspensions. In principle, these techniques could be used to diagnose or discriminate between infectious bacteria in infections of the skin or ocular surface (microbial keratitis, MK). However, the extension of these techniques to macroscopic turbid media that strongly absorb and scatter light is difficult. Radiative transfer effects obscure the relationship between microscopic scattering and absorption properties and macroscopically observable quantities such as fluorescence intensity, transmission, and reflection. A combination of experimental measurements of aqueous bacteria cell suspensions and Monte Carlo radiation transfer simulations are performed to better understand these effects. Several general observations, e.g., that fluorescence intensity is maximized in scattering-dominated media, are discussed in detail. It was found that wavelength-dependent radiative transfer effects are observable even at moderate optical densities (OD ∼ 1; well below the diffusion limit). Careful consideration of radiative transfer effects using physically rigorous models is needed to determine single-cell scattering and absorption properties and interpret quantitative fluorescence measurements accurately in most cases of interest. A detailed discussion of radiative transfer effects and analytical models is provided. In the context of surface infections and MK, it was found that radiative transfer effects may be negligible for the model bacteria E. coli in some cases; however, more accurate measurements of microbe optical properties are needed to confirm and extend this conclusion to other species. Overall this work demonstrates that quantitative fluorescence imaging and spectroscopy of bacterial films and suspensions is feasible, but requires detailed sample characterization and careful consideration of radiative transfer effects.