Ems 002 rapid immersion freezer

Cells cultured for 48 h in 2D culture plates, or for 14 d in 3DPS, were gently washed once in cell medium and fixated for 1 h at room temperature in 2.5% glutaraldehyde (Product ref G7651-10ML, Sigma-Aldrich Sweden AB, Stockholm, Sweden) diluted in cell medium. Scaffolds were washed once in TBS (50 mM Trizma-HCl, Sigma-Aldrich, Stockholm, Sweden; 150 mM NaCl, Merck, Stockholm, Sweden; pH 7.5) with 0.1 mM CaCl₂ (Merck, Stockholm, Sweden), fixated for 1 h at room temperature in 1% osmium tetroxide (Sigma-Aldrich, Stockholm, Sweden) in TBS (50 mM Trizma-HCl (Sigma-Aldrich, Stockholm, Sweden); 150 mM NaCl, (Merck, Stockholm, Sweden); pH 7.5) with 0.1 mM CaCl₂ (Merck, Stockholm, Sweden), rinsed in deionized water, plunge-frozen in liquid propane (EMS-002 Rapid Immersion Freezer, Electron Microscopy Sciences) and freeze-dried overnight (VirTis Sentry 2.0 Benchtop Freeze Dryer, SP Scientific, Warminster, PA, USA). Samples were mounted on aluminum stubs by adhesive carbon tabs (Agar Scientific) and coated with a 10 nm Au/Pd conducting thin film by sputter-coater (PECS Mod 682, Gatan Inc., Pleasanton, CA, USA). Samples were imaged by Zeiss SUPRA 40VP scanning electron microscope operated in secondary electron mode at 3.0–4.1 kV acceleration voltage, 9–17 mm working distance and 100–250,000× magnification range.

P. patens was sub-cultured and grown five days in a lighted 23 °C incubator on cellophane disks overlaying solid basal medium with ammonium tartrate40 . An actively growing protonemal colony was mounted in 1 μl rehydrated yeast paste between two copper planchets, frozen within a few seconds in liquid-nitrogen-cooled resolidifying propane using an automated plunger (EMS 002 Rapid Immersion Freezer; Electron Microscopy Sciences, Hatfield PA), and stored in liquid nitrogen.