The influence of the biofilm matrix on molecular diffusion is commonly hypothesized to be responsible for emergent characteristics of biofilms such as nutrient trapping, signal accumulation and antibiotic tolerance. Hence quantifying the molecular diffusion coefficient is important to determine whether there is an influence of biofilm microenvironment on the mobility of molecules. Here, we use single plane illumination microscopy fluorescence correlation spectroscopy (SPIM-FCS) to obtain 3D diffusion coefficient maps with micrometre spatial and millisecond temporal resolution of entire Pseudomonas aeruginosa microcolonies. We probed how molecular properties such as size and charge as well as biofilm properties such as microcolony size and depth influence diffusion of fluorescently labelled dextrans inside biofilms. The 2 MDa dextran showed uneven penetration and a reduction in diffusion coefficient suggesting that the biofilm acts as a molecular sieve. Its diffusion coefficient was negatively correlated with the size of the microcolony. Positively charged dextran molecules and positively charged antibiotic tobramycin preferentially partitioned into the biofilm and remained mobile inside the microcolony, albeit with a reduced diffusion coefficient. Lastly, we measured changes of diffusion upon induction of dispersal and detected an increase in diffusion coefficient inside the biofilm before any loss of biomass. Thus, the change in diffusion is a proxy to detect early stages of dispersal. Our work shows that 3D diffusion maps are very sensitive to physiological changes in biofilms, viz. dispersal. However, this study also shows that diffusion, as mediated by the biofilm matrix, does not account for the high level of antibiotic tolerance associated with biofilms.

通常假设生物膜基质对分子扩散的影响是造成生物膜新兴特征的原因，例如营养物捕获，信号积累和抗生素耐受性。因此，量化分子扩散系数对于确定生物膜微环境是否对分子迁移具有影响很重要。在这里，我们使用单平面照明显微镜荧光相关光谱法（SPIM-FCS）获得具有整个铜绿假单胞菌的微米级空间和毫秒时间分辨率的3D扩散系数图小殖民地。我们探讨了分子特性（例如大小和电荷）以及生物膜特性（例如小菌落的大小和深度）如何影响荧光标记的葡聚糖在生物膜内部的扩散。2 MDa葡聚糖显示出不均匀的渗透和扩散系数的降低，表明该生物膜起着分子筛的作用。其扩散系数与小菌落的大小负相关。带正电的右旋糖酐分子和带正电的抗生素妥布霉素优先分配进入生物膜，并在微菌落内部保持活动性，尽管扩散系数降低。最后，我们测量了诱导分散后扩散的变化，并检测到生物膜内部扩散系数的增加，然后才损失生物质。因此，扩散的变化是检测早期扩散的代理。我们的工作表明3D扩散图对生物膜中的生理变化非常敏感，即。分散。但是，这项研究还表明，由生物膜基质介导的扩散不能解释与生物膜相关的高水平的抗生素耐受性。