中文摘要：

目的探讨铜绿假单胞菌细菌生物被膜（biofilm，BF）形成发展过程中空间立体结构变化。方法SYTO9／PI荧光探针标记PAO1菌株，体外建立6h、1d、3d及6d时间组铜绿假单胞菌PAO1菌株BF模型，激光共聚焦显微镜（confocal laser scanning microscopy．CLSM）摄取BF形成发展各阶段不同层面的图片堆，经图像结构分析软件（image structure analyzer，ISA）分析获得PAO1菌株BF相关空间结构参数定量化数据。结果①CLSM结合SYTO9／PI荧光探针标记，实现了对PAO1菌株BF动态形成过程的观察。②随着BF形成时间的延长，各层死菌比例逐渐增加，且死菌多分布于微菌落中心。③ISA软件定量化分析显示，随着BF发展，厚度显著增加，前3d增加程度显著大于后3d；区域孔率显著下降趋势；平均扩散距离呈逐渐增加趋势；结构熵呈显著上升趋势。结论利用ISA程序结合SYTO9／PI荧光探针可以表征PAO1菌株BF的空间结构特征和BF各层细菌的生存状态。

英文摘要：

Objective To investigate the sequential development of spatial structure of biofilm with Image Structure Analyzer （ISA） software so as to found a basis for further research on biological behavior of biofilm. Methods In vitro biofilm model of Pseudomonas Aeruginosa （ P. aeruginosa） PAO1 tagged with SYTO9/PI was established on glass slice, and its biofilm development was monitored at different time points （6 h, 1, 3, and 6 d）. The fluorescence images stack of different layers in biofilm model were obtained by confocal laser scanning microscopy （CLSM） based on fluorophores from PAO1. Quantitative parameters describing biofilm spatial structure were acquired after the image information was calculated by ISA software. Results ①The PAO1 biofilm process was investigated successfully by CLSM after genetically velopment of biofilm, the ratio of died bacteria was increased tagged with GFP. ②With the degradually in each layer and most of them were distributed in the core of microcolony. ③The quantitative data from ISA software showed that the thickness of biofilm was increased significantly during biofilm growth; meanwhile the areal porosity （AP） was decreased signifi- cantly, the average diffusion distance （ADD） indicated an increased tendency; and the textural entropy （TE） increased significantly. Conclusion The ISA software combined with SYTO9/PI tagging provide useful information of bacterial biofilm spatial structure in PAO1 biofilm process. Quantifying bacterial biofilm structure permits correlating biofilm development with biofilm performance.