Uc6 fc6

Additional corneal specimens were fixed with 2.5% glutaraldehyde in Sorense-Gomori’s PBS (pH 7.4) for 2 h at 4 °C. After being washed three times with PBS, specimens were post-fixed using 1% osmium tetroxide (Wako Pure Chemical Industries, Tokyo, Japan) for 2 h at 0 °C, dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections of 70 nm were cut using an ultra-microtome (Leica UC6/FC6, Leica Microsystems, Wetzlar, Germany) equipped with a diamond knife, and mounted on grids. Sections were observed by TEM with accelerating voltage at 200 kV (LEO 922 Omega, Carl Zeiss, Germany).

Pt/CB powder was dispersed in water at a concentration of 10^-4 wt% and deposited on the lacey carbon films on copper mesh TEM grids. The CLs were held between the epoxy resin blocks and sectioned to a thickness of ~200 nm using an ultramicrotome (Leica UC6/FC6, Leica Microsystems GmbH, Wetzlar, Germany) at −80 °C. The ultrathin sections were transferred to lacey carbon films on copper mesh TEM grids. TEM observations were carried out on a JEM-ARM300F (JEOL Ltd., Akishima, Japan) instrument operating at 300 kV and equipped with a CMOS camera (OneView, Gatan Inc., Pleasanton, CA, USA).

The vegetative cells and spores from B. subtilis, S. violaceoruber, A. chroococcum, M. xanthus and A. cylindrica were prepared as described above; where necessary, culture volumes were doubled or tripled to obtain a higher biomass. The samples were resuspended in phosphate-buffered saline (PBS) supplemented with 30 % dextran (D1662 from Sigma-Aldrich, St Louis, MI, USA). They were then cooled under high pressure (2000 bar) to liquid nitrogen temperature (−196 °C) within milliseconds using an EM-PACT2 machine (Leica Microsystems, Wetzlar, Germany). These conditions prevent the formation of ice crystals, which can damage cellular structures [50 (link)]. The frozen samples were then cut into thin slices (50 nm) in a cryo-ultramicrotome UC6 FC6 (Leica Microsystems, Wetzlar, Germany) and placed on a R3.5–1 holey carbon EM grid (Quantifoil, Großlöbichau, Germany). Samples prepared in this way were then loaded into a Tecnai F20 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) on a cryo-holder model 626 (Gatan, Pleasanton, CA, USA) keeping the sample temperature low (−180 °C) and analysed by the low-dose method at 200 kV acceleration voltage.

Transmission electron microscopy (TEM) was performed as described by Zhao et al. with a minor modification [51 (link)]. The first true leaves were cut into small sections (1–2 mm in length) and then fixed in 2.5% glutaraldehyde under 4 °C overnight. The tissue samples were washed 3× in 0.1 M phosphate buffer and fixed in 2% osmic acid (OsO₄) at 4 °C for 2 h. The tissues were washed three times with 0.1 M phosphate buffer and then dehydrated with an ethanol series of 50% v/v, 70% v/v, and 90% v/v for 15 min per ethanol concentration. The tissue samples were infiltrated with mixtures of 90% ethanol and 90% acetone (1:1), 90% acetone, and 100% acetone, acetone and resin (1:1, 1:2), and 100% resin for 12 h. The tissue samples were embedded and polymerized at 60 °C for 48 h. Thin sections (50–70 nm) were prepared with a cryo-ultramicrotome (UC6-FC6, Leica Microsystems, Wetzlar, Germany) and double-stained with uranyl acetate-lead citrate. The tissue samples were examined under a transmission electron microscope (Tecnai G2 Spirit BioTWIN, Thermo Fisher Scientific, Waltham, MA, USA).