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 tagged with GFP. ②With the development of biofilm

the ratio of died bacteria was increased gradually 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 significantly

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.