Cm200 electron microscope

Recombinant proteins were visualized using a TEM (CM-200 Electron Microscope, Philips, CA, USA) operating at an acceleration voltage of 80 kV. For the preparation of TEM samples, one drop of each capsid particle suspension was placed onto a 200-mesh copper grid precoated with carbon. After 2 min of deposition, distilled water was removed by air-drying. Negative staining was applied using a droplet of 2% (w/v) aqueous uranyl acetate solution. The particle size of the recombinant proteins was measured using DLS (NanoBrook 90Plus, Brookhaven, NY, USA).

TEM bright field images were acquired using a Philips CM200 electron microscope operating at 200 kV equipped with a field emission gun filament. A Gatan US 1000 CCD camera was used and 2048 × 2048 pixel images with 256 grey levels were recorded. The suspension was dropped onto a 200 mesh carbon-coated copper grid and air dried for several hours before analysis. All the samples were visualized without the negative staining procedure.

Grids (Cu, 200 mesh) were negatively stained by uranyl acetate (1%). After being dried (24 h; room temperature), the grids were placed on CDplex samples (in 20 mm HEPES at pH 7.4). Grids were then dried and imaged using a Philips CM200 electron microscope. Measurements were performed in duplicate at the Centre of Biological Research Services (CIBMS-CSIC) in Madrid (Spain).

1 × 10⁷ conidia of the relevant strains were grown statically at 37°C in MM for 24 h. Mycelia were harvested and immediately fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 2.5% (v/v) of glutaraldehyde and 2% (w/v) of paraformaldehyde for 24 h at 4°C. Samples were encapsulated in agar (2% w/v) and subjected to fixation (1% OsO₄), contrasting (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with Spurr resin (Electron Microscopy Sciences) of 16 and 3 h at room temperature. Additional infiltration was provided under vacuum at room temperature before embedment in BEEM capsules (Electron Microscopy Sciences) and polymerization at 60°C for 72 h. Semithin (0.5-μm) survey sections were stained with toluidine blue to identify the areas of best cell density. Ultrathin sections (60 nm) were prepared and contrasted again with uranyl acetate (1%) and lead citrate (2%). TEM images were obtained using a Philips CM-200 electron microscope at an acceleration voltage of 120 kV using a MegaView3 camera. Cell wall thickness of 100 sections of different germlings were measured and images analyzed with the ImageJ software. Statistical differences were evaluated by using one-way ANOVA with Dunnett's multiple comparison test.