Use of epifluorescence widefield deconvolution microscopy for imaging and three-dimensional rendering of Pseudomonas aeruginosa biofilms and extracellular matrix materials

Microbial biofilms form on surfaces in most aqueous environments and play key roles in many biological processes, ranging from nutrient cycling in the environment to persistence of infectious diseases. Microscopic imaging is a core technique used to help understand microbial biofilms, including their structure and development, location of gene expression, extracellular matrix production, and community composition. Due to the speed of imaging, epifluorescence widefield microscopy is well suited for high throughput image analysis and screening of biofilm samples. However, epifluorescence microscopy is not ideal for obtaining high resolution images of three-dimensional biofilm structures, due to the high background signal from out-of-focus light, which compromises the lateral and axial resolution of the biofilms. Here, we demonstrate the application of epifluorescence widefield deconvolution microscopy (WF-DCM) for imaging Pseudomonas aeruginosa PAO1 biofilms. Using fluorescent stains for P. aeruginosa PAO1 extracellular matrix components, we demonstrate that WF-DCM allows quantification of biofilm components, including cell numbers and biovolumes of matrix materials. We show that environmental conditions, such as calcium addition, affects P. aeruginosa PAO1 biofilm structure, and determine the effects of extracellular polysaccharide production on early P. aeruginosa biofilm development. The results demonstrate that WF-DCM provides an imaging strategy based on epifluorescence microscopy for obtaining clear in-focus images of three-dimensional biofilms. The technique may be used to quantify biofilm parameters, including the effects of environmental conditions on biofilm structure, and it may be used as a mutant screening strategy that is based on biofilm imaging.