Axio observer microscope platform

Inguinal snap-frozen tissues were fixed overnight in 4% buffered formalin, dehydrated, and embedded in paraffin blocks. 4-μm-thick sections were cut and stained for hematoxylin and eosin. For Oil-Red-O staining, cells were fixed in 4% wt/vol. paraformaldehyde solution (AppliChem, Darmstadt, Germany) for 30 min on ice, rinsed in dH₂O, and dehydrated in 60% vol./vol. isoproterenol. Cells were stained with a solution of 0.3% wt/vol. Oil-Red-O (O0625; Sigma–Aldrich, Buchs, Switzerland) dissolved in 100% isoproterenol that was freshly diluted to 60% vol./vol. in dH₂O. After 5–10 min, cells were rinsed twice in dH₂O. Tissue slices or cells were imaged using an Axio Observer microscope platform (Zeiss, Jena, Germany). Tile scans were analyzed with ImageJ software to calculate the Oil-Red-O positive signal per well.

A fluorescence spinning disc microscope (Axio Observer microscope-platform equipped with Cell Observer SD, Zeiss, Oberkochen, Germany) was used to visualize BFIs at 0 h, 24 h, and 48 h after bacterial inoculation. For each time point, 16 images were taken at random positions of the microchannels, including both ends of the BFI device (N = 16 technical replicates). Brightfield images were captured using a laser intensity of 7.1 V and an exposure time of 100 ms. Images of bacterial cells (M. rhizoxinica wild type, M. rhizoxinica Δmtal1, M. rhizoxinica Δmtal1 pBBR∅, M. rhizoxinica Δmtal1 pBBR-mtal1, M. rhizoxinica ΔsctT, M. rhizoxinica ΔsctC, and M. rhizoxinica ΔrhiG), stained with SYTO9, were captured at 485/498 nm with an exposure time of 100 ms. As a reference, wild-type R. microsporus ATCC62417, naturally containing endosymbionts, was also analyzed, without additional bacterial cultures being added through the inlet. Each reinfection experiment was performed three times independently (N = 3 biological replicates).