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 (, 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 radiation transfer simulations are performed to better understand these effects. Several general observations, 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 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.

中文翻译：

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辐射传输对细菌薄膜和悬浮液荧光成像的影响


荧光成像和光谱学是分析化学样品（包括细菌生物膜和悬浮液等生物材料）的方便、快速且简单的方法。原则上，这些技术可用于诊断或区分皮肤或眼表感染中的感染性细菌（MK）。然而，将这些技术扩展到强烈吸收和散射光的宏观浑浊介质是很困难的。辐射传输效应模糊了微观散射和吸收特性与宏观可观察量（例如荧光强度、透射和反射）之间的关系。结合水性细菌细胞悬浮液的实验测量和辐射传输模拟，以更好地了解这些效应。详细讨论了一些一般观察结果，即荧光强度在散射主导的介质中最大化。研究发现，即使在中等光密度（OD 1；远低于扩散极限）下，也可以观察到与波长相关的辐射传输效应。需要使用严格的物理模型仔细考虑辐射传输效应，以确定单细胞散射和吸收特性，并在大多数感兴趣的情况下准确解释定量荧光测量。提供了辐射传输效应和分析模型的详细讨论。在表面感染和 MK 的背景下，发现在某些情况下，模型细菌的辐射传输效应可以忽略不计。然而，需要对微生物光学特性进行更准确的测量来证实这一结论并将其推广到其他物种。 总体而言，这项工作表明细菌薄膜和悬浮液的定量荧光成像和光谱是可行的，但需要详细的样品表征并仔细考虑辐射传输效应。