Label-Free Four-Dimensional Visualization of Anaerobically Growing Electroactive Biofilms

Light sheet fluorescence microscopy (LSFM) allows nondestructive, label-free and in vivo imaging of large specimen, even at nontransparent surfaces. We show that LSFM can be applied for label-free analyses of prokaryotes on the example of electroactive biofilms. Biofilm growth is linked to the production of current serving as measure of metabolic activity in vivo by monitoring with high spatial and temporal resolution. After 35 h of exponential growth, a homogeneous biofilm with a thickness of 9 μm was formed. This was followed by a stratification of the biofilm including the formation of 3D structures over the next 100 h. Light reflection was sufficient to visualize the biofilm structure and development over time and the terminal morphology was confirmed using fluorescence staining. This proof of concept on using LSFM for investigation of biofilms opens the door for its application in the entire field of microbial ecology. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.