Eagle ccd camera

Cells were fixed with 2.5% glutaraldehyde in phosphate buffer and kept in the fixative during 1 h at room temperature. They were washed and postfixed with 1% osmium tetroxide in the same buffer containing 0.8% potassium ferricyanide at 4°C. The samples were dehydrated in acetone, infiltrated with Epon resin during 2 days, embedded in the same resin and polymerised at 60°C during 48 h. Ultrathin sections were obtained using a Leica Ultracut UC6 ultramicrotome (Leica Microsystems, Vienna) and mounted on Formvar-coated copper grids. They were stained with 2% uranyl acetate in water and lead citrate. Sections were observed in a Tecnai Spirit electron microscope equipped with an Eagle CCD camera (FEI, Eindhoven, The Netherlands).

Electron microscopy was used to examine the DnaT-phiX174 ssDNA complex with a published method that was applied for the analysis of the (39 (link)) PriB-phiX174 ssDNA complex (21 (link)). For preparing the DnaT-phiX174 ssDNA nucleoprotein filaments, the purified DnaT protein (8.0 μL; 10 mg/ml) was incubated with phiX174 ssDNA (1.6 μL; 50 μg/ml) and added buffer A to a final volume of 50 μl for 30 min at 20°C. The reaction products were diluted 4-fold with buffer A for Electron microscopy (EM) study. The final concentration of DnaT applied in the EM study was around 20 μM (400 μg/ml) and the substrate, circular phiX-174 ssDNA (5386 nt), was 0.24 nM (0.4 μg/ml) which was quite low. Other samples were prepared similar to this. For each sample, ∼3 μl protein-ssDNA complex was applied to a glow-discharged carbon-coated 400-mesh Cu EM specimen grid. Then, the sample was stained by 0.7% (w/w) uranyl formate. We imaged the protein-ssDNA particles under a low-dose condition using an FEI Tecnai F20 microscope (200 kV accelerating voltage, ∼0.6–0.8 μm underfocus) with an FEI Eagle CCD camera at 62 000× or 150 000× magnification. The final pixel sizes were 3.54 or 1.46 Å after 2-fold pixel averaging.

All samples were measured with an FEI Tecnai spirit TEM (FEI, Hillsboro, Oregon, USA) at 120 kV. Images were recorded with a Veleta CCD camera 2048 × 2048 (Olympus-SIS, Münster, Germany) or Eagle CCD camera 4096 × 4096 (FEI, Hillsboro, Oregon, USA) and processed using ImageJ software as described in the supporting information (Section 1 of Additional file 1: Supplementary information).
Single AuNPs were characterized using conventional TEM: Briefly, single AuNPs suspension (10 μL) was deposited onto a 400 mesh carbon-coated copper grid and let dried at room temperature. Images were recorded with the Veleta camera.
Aggregated AuNPs were characterized using cryo-TEM: Aggregated AuNPs suspension (5 μL) was deposited on a carbon-coated copper grid and liquid excess was carefully removed with filter paper. The grid was then plunged into a liquid ethane bath cooled by liquid nitrogen. The resulting vitrified sample was then stored in liquid nitrogen prior to analysis. Images were recorded using the Eagle camera.

HeLa cells were grown in wells, infected with C. trachomatis LGV serovar L2 strain 434 at an MOI of 0.1 and carefully trypsinized at the indicated time points. The cells were then washed with PBS once and fixed with 0.1 M cacodylate and 2.5% glutaraldehyde at room temperature for at least 30 min. PATAg staining was performed as described elsewhere (Thiéry, 1967 ). Briefly, thin sections were incubated in 1% periodic acid for 25 min and then washed several times in water, followed by an incubation step in 0.2% thiocarbohydrazide in 20% acetic acid for 45 min. Several washing steps in a graded acetic acid series to water were carried out and the thin sections were stained with 1% silver proteinate for 30 min. Samples were observed within a week after preparation. Images were obtained using a Tecnai T12 transmission electron microscope (FEI) at 100 kV and captured using an Eagle CCD camera (FEI).

Ty3-expressing yeast cells were prepared for ET as described by Kukulski et al. (64 (link)). The yeast cell paste was high-pressure-frozen (Empact 2; Leica), processed by freeze substitution, and embedded in Lowicryl resin using an AFS2 (Leica). The samples were sectioned and mounted onto EM grids. Tomographic data were collected using an F30 Tecnai microscope (FEI) equipped with an Eagle CCD camera (FEI) with a pixel size at the specimen level of 11.8 Å. Dual-axis tilt series were collected with a 1° increment in a ±60° range. Tomograms were reconstructed in IMOD (65 (link)).

5 μL of sample (Fig. 2a–d: VIPP1 Prep #4 alone and mixed with 200 μM 95:5 PC:PG or 95:5 PC:PI4P liposomes; Fig. 2e: VIPP1 Preps #1 and #2) was applied to glow discharged, 200 mesh copper grids (G2200C, Plano GmbH) that had been coated with homemade carbon film. The sample was incubated for 2 min, blotted, washed three times with water, and then stained with 2% uranyl acetate for 30 s. Images were recorded using a FEI Tecnai F20 FEG microscope operated at 200 kV with an FEI Eagle CCD camera, a magnification of 50,000 × (2.21 Å per pixel) and a defocus range of −2 to −5 μm. Measurements of negative stain images were performed with IMOD^{63 (link)} and ImageJ^{64 (link)} software packages.