Automated AFM for Large-Area Biofilm Imaging—A Breakthrough in Biofilm Characterization

Biofilms, complex microbial communities encased in an extracellular matrix, are found in a variety of environments, from natural ecosystems to industrial and clinical settings. Diverse chemical and nutritional gradients drive variations in cell structure and function across the biofilm architecture. The complex architecture of the biofilm imparts unique resilience to the biofilm community and enhances antibiotic resistance, making them difficult to control in healthcare environments. Traditional methods for biofilm analysis often fail to capture their full complexity due to limited spatial resolution and preparation artifacts. To address this, we developed an automated platform for large-area Atomic Force Microscopy (AFM) imaging, which enables high-resolution mapping of biofilm morphology over millimeter-scale areas. Our system, integrated with advanced image stitching algorithms and machine learning-based analysis, provides a comprehensive view of biofilm structures, capturing details such as flagella, pili, and cellular arrangements while preserving spatial relationships. This innovation significantly enhances the study of biofilms, allowing us to quantify key parameters like cell count, confluency, and gap size across extensive regions.
Our platform was applied to biofilms formed by Pantoea sp. YR343, revealing a honeycomb morphology on hydrophobic surfaces and highlighting the critical role of flagella in biofilm stability. The integration of gradient-structured surfaces allowed us to explore how varying surface properties influence biofilm development, offering new insights into biofilm-surface interactions. By automating the AFM process, we overcame the limitations of traditional AFM’s small imaging area, enabling the examination of biofilms at scales relevant to their natural environments. This advancement opens new possibilities for biofilm research, providing a powerful tool for understanding biofilm resilience and informing future strategies for controlling biofilm formation in various settings.