Extended Data Fig. 2: Examples of the data visualization and plot categories that are possible within BiofilmQ.

All graphs shown in this figure were produced directly by BiofilmQ, except panel f. a, Screenshot showing several key elements of the data visualization tab in the BiofilmQ graphical user interface, depicting how to choose the axis of figures to be plotted, and the plot type. b, Left, a kymograph quantifies fluorescent reporter expression as a space-time heatmap for n = 1 biofilm. In this example, the fluorescence of an RpoS-mRuby3 translational fusion is plotted over time and space during V. cholerae C6706 WT biofilm development. Centre, a 1.5D histogram shows the relation between fluorescent reporter intensity and position in the biofilm for a single time-point, mean values are plotted with error bars representing the standard deviation of 100–1500 cubes with similar distance from the surface. Right, the heatmap represents a demograph of n = 19 different biofilms, which reveals spatially-resolved differences between biofilms, for a particular cube-level parameter (here: RpoS-mRuby3 fluorescence as a function of height in V. cholerae biofilms after 18 h of growth). c, Left, several global biofilm parameters can be plotted into the same 2D graph for better comparison. In this example, the biofilm roughness and volume during biofilm development of a V. cholerae N16961 rugose ΔcrvA strain are plotted for n = 1 biofilm. Right, to analyze the behavior of a parameter in a time series, other time-related quantities, such as the number of cubes in the biofilm, can be used as the timescale on the x-axis for the same biofilm as in the left panel; showing mean +/- error bars represent std. dev. of all of the 3000–5000 cubes at a given time point. d, Top left, analogous to flow cytometry, BiofilmQ can perform biofilm image cytometry, comparing two fluorescent reporters or any other cube parameter. In this example, results are shown from a biofilm co-culture of two V. cholerae N16961 WT strains that constitutively produce sfGFP (n = 1 dataset of two biofilms merging during growth); one of these strains additionally produces mRuby2 constitutively. The segmentation was performed on the sfGFP channel. The gating/filtering option enables the separation of two populations; properties of each gated population can then be visualized separately. Top right, in a 2D+colour scatter plot, extracted cube parameters can be visualized; this example shows the biofilm thickness distribution in space at a specific timepoint (22 h) during the development of n = 1 biofilm of V. cholerae C6706 WT. Bottom left, a 3D+colour scatterplot visualizes quantified cube parameters, but provides one additional axis. In this example, the spatial distribution of the local density during V. cholerae C6706 WT biofilm growth is shown at a particular timepoint (12 h, n = 1 biofilm). Bottom right shows another example of a 3D+colour plot, visualizing two tracked V. cholerae N16961 rugose ΔcrvA biofilm colonies with the same constitutive fluorescent protein expression (sfGFP), growing together over time (n = 1 dataset of two biofilms merging during growth). Separation of lineages is performed via cube tracking. e, Histograms of quantified cube parameters: These examples show the fluorescence signal of an RpoS-mRuby3 translational fusion reporter (left) and the distance of each cube to the biofilm center (right), for a V. cholerae C6706 WT n = 1 biofilm grown for 22 h. f, The location and relative abundance of three different V. cholerae N16961 rugose strains (differing by the colour of a constitutively expressed fluorescent protein marker: mTFP1, mKOκ, mKate2) were quantified using BiofilmQ and the result was exported and rendered in 3D with the ParaView software for visualization.