Fluorescence Confocal Microscopy of Aspergillus Biofilms

Fluorescence confocal microscopy was performed on an Andor W1 spinning disk confocal with a Nikon Eclipse Ti inverted microscope equipped with a CFI Plan Fluor 20XC MI objective (Nikon). A. fumigatus and A. niger biofilms were cultured for imaging at 10⁵ spores per ml in MatTek dishes (MatTek, P35G-1.0-14-C) in 2 ml of liquid glucose minimal medium for 24 h at 37°C with 5% CO₂ in the dark. For visualization at 405 nm, biofilms were stained with 25 μg/ml calcofluor white (Fluorescent Brightener 28; Sigma, no. F3543) 15 min prior to imaging. The CFI Plan Fluor 20XC MI objective was used with water to image the bottom ∼300 μm of the biofilm with Z-slices collected every 1.2 to 1.5 μm. 3D rendering and image processing were performed in a Nikon Elements Viewer (Nikon). Quantification of biofilm architecture and generation of the heat map figures were carried out as previously described using the BiofilmQ framework written in MatLab (71 (link)). The framework is freely available for download (www.drescherlab.org/data). All biofilm images and single columns in heat maps are representative of a minimum of three independent biological replicates.