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.