Fig. 9: Relative z-position of single bacteria within the embedding matrix from mono- and dual-species biofilms after expansion.

Cells were labelled with Alexa Fluor 555 NHS ester (red) and matrix was visualized by Concanavalin A-488 (green). Top. 2D reconstruction image from YZ + XZ projections from single bacterium (n = 10). For the reconstruction, images were centred using the brightest red fluorescence pixel. Dimensions of cropped images are 20 μm × 4.8 μm (vertical and horizontal directions). Middle. Fluorescence z-distribution of both the bacterial and matrix channels. An overlap above 60% is considered to describe a system with bacteria embedded into the biofilm matrix (S. aureus, BS4 and BS6) whereas below 60%, a carpet configuration is considered. Z arrow bar is 20 μm. For a better visualization, curves are normalized. Bottom. Boxplots of ΔHeight, defined as the difference between the maximum value for red fluorescence z-distribution and the maximum value for green fluorescence z-distribution. Average ± SD values are: L. reuteri: 2.9 ± 1.2 μm (n = 17); E. faecalis: 2.0 ± 0.4 μm (n = 17); S. marcescens: 3.2 ± 0.5 μm (n = 18); (BS1) L. reuteri + E. faecalis: 2.5 ± 0.7 μm (n = 18); (BS2) L. reuteri + S. marcescens: 1.7 ± 0.3 μm (n = 5); (BS3) L. reuteri + S. aureus: 3.3 ± 2.3 μm (n = 13) and (BS5) E. faecalis + S. aureus: 3.2 ± 0.6 μm (n = 16). Boxplot elements are black square-mean; centre line-median; box limits-upper and lower quartiles; whiskers- extreme values; diamonds-experimental data; curved line: normal distribution.