Metamorph

The phase-contrast and epifluorescence time-lapse movies on live cells were acquired with an automated Olympus X71 microscope, equipped with 4x, 10x, and 20x objectives while maintaining 37°C, 5% CO₂ partial pressure and 95% relative humidity (Life Science incubator).
The acquisitions were performed with a CCD camera (Retiga 4000R, QImaging) controlled by Metamorph (Universal Imaging). The typical frequency of image acquisitions was 15 minutes.
For confocal time-lapse imaging, we used an inverted laser scanning confocal microscope with spectral detection (LSM700—Zeiss) equipped with a CO₂ incubator to observe fluorescent live cells (SiR-Actin labeling) with a 25x oil objective. The typical duration between acquisitions was 30 minutes.
For super-resolution confocal imaging on fixed cells (Figs. 2 and 4; Figs. S1, 9), we used an inverted Laser Scanning Confocal Microscope with Spectral Detection and Multi-photon Laser (LSM880NLO/MaiTai Laser—Zeiss/Spectra Physics), 63x oil immersion objective combined with super-resolution technique Airyscan.
Tracking experiments were performed by using mixtures with 10% Crispr-labeled Actin mCherry cells. Observations were performed in epifluorescence. Thanks to the relatively large depth of field, cells of both layers could be imaged simultaneously and then tracked manually.

For microscopy of mycelia undergoing sexual development, S. macrospora strains were grown on glass slides with a thin layer of corn meal extract or corn meal medium solidified with 0.8% agar as described [117 (link), 118 (link)]. Fluorescence and light microscopic investigations were carried out with an AxioImager microscope (Zeiss, Jena, Germany). Fluorescence was studied using Chroma (Bellows Falls, VT, USA) filter set 41017 (HQ470/40, HQ525/50, Q495lp) for detection of EGFP, set 49008 (EG560/40x, ET630/75 m, T585lp) for the detection of tdTomato, and set 31000v2 (D350/50, D460/50, 400dclp) for the detection of DAPI and mKalama1. Images were captured with a Photometrix Cool SnapHQ camera (Roper Scientific) and MetaMorph (Universal Imaging). Recorded images were edited with MetaMorph and Adobe Photoshop CS4.