Three-dimensional morphology of bacterial community developed on the index-matched materials

Herein, we demonstrate that the use of index-matching materials (IMMs) allows direct visualization of microbial cells maintained at a solid–liquid interface through confocal reflection microscopy (CRM). The refractive index mismatch induces a background reflection at the solid–liquid interface that dwarfs the reflection signals from the cells and results in low-contrast images. We found that the IMMs sufficiently suppressed the background reflection at the solid–liquid interface, facilitating the imaging of microbes at the solid surface using CRM. The use of IMMs allowed quantitative analysis of the morphology of the mesh-like structure of Pseudomonas aeruginosa biofilms formed under denitrifying conditions, which led us to propose a novel structural model of the highly porous biofilm structure. These results indicate that the use of CRM coupled with an IMM offers a unique and promising tool for probing the dynamics of biofilm formation, along with visualization of environmental organisms and newly isolated bacteria, for which transformation methods are difficult to establish.