Fig. 1: Quantification of microbial community properties with spatial resolution in BiofilmQ.

a, Examples of different biofilm image categories that can be analysed using BiofilmQ: E. coli macrocolony, V. cholerae meso- and microcolonies, Bacillus subtilis pellicle and floating aggregates, V. cholerae biofilm (yellow) on mouse intestinal villi (blue). Many file formats are supported, based on the Bio-Formats toolbox³⁶. Scale bars, 1 mm (top left), 30 µm (middle and bottom left, and top right), 40 µm (middle right), 100 µm (bottom right). b, The BiofilmQ image processing pipeline for a 3D biofilm image. The raw fluorescence image is filtered and thresholded to obtain a binary representation of the biofilm. These 3D binary data are then dissected into cubes of a user-defined size, and the cubes are then used to quantify the biofilm properties. Alternatively, binary images of the biofilm or single cells can be imported. Here, each cube in the biofilm is coloured according to the local biovolume density, which is one of the cube properties that can be extracted. c,d, Many parameters can be quantified for each cube (c) and for the whole biofilm (d). e, A B. subtilis microcolony (left) on an agar pad consisting of two strains (a strain constitutively expressing superfolder green fluorescent protein (sfGFP) and a strain constitutively expressing mKate2) was analysed using cube-based image cytometry, single-cell image cytometry and flow cytometry. The image cytometry results are qualitatively similar, and the flow cytometry results also show two additional cell populations (non-fluorescent cells and cells with fluorescence in both channels). The data shown are one example out of n = 3 experiments, which all showed the same qualitative results. Scale bar, 10 µm. f, For an E. coli macrocolony (left) on agar consisting of two strains (a strain constitutively expressing sfGFP and a strain constitutively expressing mRuby3), we compared the results from cube-based image cytometry and flow cytometry. The data shown are one example out of n = 3 experiments, which all showed the same qualitative results. Scale bar, 500 µm. a.u., arbitrary units.