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.