Tecnai spirit biotwin tem

HFF monolayers were grown in T25 culture flasks and were infected with 10⁷ N. caninum Spain-7 (NcSp-7) tachyzoites during 3 h at 37 °C and 5% CO₂. Subsequently, cultures were exposed to continuous treatment with 0.5 μM of each of DCQ, RMB054 or RMB060, and 1 µM RMB055 for 6 h, 9 h, and 48 h. Control cultures were treated with DMSO only. The specimens were fixed and processed for TEM as described earlier [14 (link),16 (link),17 (link)]. Samples were observed on a Philips CM 12 or a FEI Tecnai Spirit BioTwin TEM, both operating at 80 kV.

CHO cells were incubated with Au50 or Au50 + TP peptide in serum-free DMEM medium at 37 °C for 2 h. Then cells were processed at the Characterization Facility of University of Minnesota and imaged by FEI Tecnai Spirit Bio-Twin TEM (FEI, Hillsboro, OR, USA) as previously described [18 (link)].

The spike protein samples were directly analyzed using a Tecnai Spirit Biotwin TEM (FEI). Copper grids with 300 mesh and a thin layer of continuous carbon floated on top (EM Sciences) were glow-discharged at 15 mA for 60 s. The spike protein samples were diluted to 80 nM and applied 3 µL to the grids for 30 s. Uranyl formate (80 µL 1%, wt/vol) was then used to stain the sample on the grid for 1 min. The specimen was finally gently blotted from the side with filter paper and air-dried before imaging. Images were recorded on a Gatan 4 K × 4 K CCD camera with a defocus of −2 μm at a nominal magnification of 68,000× or 98,000×.

Frozen-hydrated specimens were transferred to a Gatan 626 cryo holder (Gatan, Inc.) and maintained under liquid nitrogen until transferred into the TEM. Specimens were imaged using a FEI Tecnai Spirit BioTwin TEM (FEI, Co, Hillsboro, OR) equipped with a LaB₆ filament and operated at an acceleration voltage of 120 kV under low-dose conditions (∼5 electrons/ Ų). Images of transcriptionally-active DLPs were recorded on a FEI Eagle 2k HS CCD camera with a pixel size of 30 μm using a defocus range from -1.0 μm to -3.0 μm at a nominal magnification of 60,000× for a final sampling of 5 Å/pixel at the specimen level.

Two types of acquisition systems were used to image the whole fly brain series, both of which used FEI Tecnai Spirit BioTWIN TEMs. The first, the TEMCA2 system (Figure S2A), was equipped with a custom single-axis Fast Stage (Figure S2B; patent pending) (Price and Bock, 2016a , Price and Bock, 2016b ), vacuum extension, scintillator (5 μm Mylar on a support ring 9 ⁵/₈ inches in diameter, coated with 10 mg fine-grained P43/cm²; Grant Scientific), and four Fairchild SciMOS 2051 Model F2 5.5 megapixel cameras (2560 × 2160 pixel sensor size) configured in a 2 × 2 array (Figure S3E). The second type of system was an Automated Transport and Positioning System (ATPS) (Figures S2E, S2F, and S3I; Videos S2 and S3) (Price and Bock, 2016a , Price and Bock, 2016b ), which was equipped with a custom scintillator (6 mg fine-grain P43/cm2; Grant Scientific), and a single Fairchild SciMOS camera. In both systems, 4:1 minifying C-lenses (AMT Imaging) were mounted on the SciMOS cameras. These systems were previously described in abstract form (Robinson et al., 2016 ). Schematics and model files for the Fast Stage and ATPS are available to non-profit research organizations upon request.