Realistic representation of Bacillus subtilis biofilms architecture using combined microscopy (CLSM, ESEM and FESEM)

•We used a combinatorial microscopic approach to decipher Bacillus subtilis biofilm.•B. subtilis forms protruding structures of several hundred of microns on surface.•Cells were organized in aligned bundles as showed by ESEM and CLSM.•Cells were connected at their pole by dense N-acetyl glucosamine aggregates.•Fibrillar structures in matrix were revealed by SEM and CLSM with thioflavine dying.

In this contribution, we used a set of microscopic techniques including confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM) and field emission scanning electron microscopy (FESEM) to analyze the three-dimensional spatial arrangement of cells and their surrounding matrix in Bacillus subtilis biofilm. The combination of the different techniques enabled a deeper and realistic deciphering of biofilm architecture by providing the opportunity to overcome the limits of each single technique.