Oneview cmos detector

After fluorescence imaging, grids were either stored in liquid nitrogen or immediately transferred to a Gatan 626 cryogenic sample holder and inserted into a Tecnai T12 transmission electron microscope (FEI) operated at 120 kV and equipped with a OneView CMOS detector (Gatan). All images were recorded in low-dose mode (< 1 electron per Ų) to avoid damage to the sample that could otherwise have been interpreted as due to exposure to laser light. The data presented in Fig. 3b is an exception: electron tomography on the cellular samples was done using a Talos Arctica 200 kV cryo-TEM (Thermo Fisher Scientific) equipped with a K3 direct electron detector and BioContinuum energy filter (Gatan). Tilt series were acquired at × 31 k magnification with a symmetrical dose regime from ± 54° in 3° increments with a total dose of 60 e^-/Ų and a defocus range of 2–6 μm underfocus. Cryo-TEM and cryo-fluorescence images were correlated manually by matching features visible in both images and adjusting the transform of the TEM images such that these features overlapped.

Non-purified crystals of Cry11Aa WT were visualized using a Thermofisher TF20 electron microscope from the IBS electron microscopy platform. For negative staining TEM, samples were diluted 5 times in H₂O and 4 µL of the diluted sample was introduced to the interface of an amorphous carbon film evaporated on a mica sheet. The carbon film was then floated off the mica sheet in ~200 µL 2% sodium silicotungstate (SST) solution. The carbon film with the crystal sample was then recovered onto a Cu 300 mesh TEM grid after 30 s, let dry, and imaged at 200 keV. Images were recorded on a Gatan OneView CMOS detector. Non-purified crystals of Cry11Ba WT were visualized using a FEI Tecnai T12 electron microscope within the UCLA California Nanoscience Institute, EICN facility. For negative staining TEM, samples were prepared by adding 5 µL of pure crystal fractions in 10 µL ultrapure H₂O. In total, 2.5 µL of this sample was added to 300 mesh Cu F/C grids that were positively glow discharged. These samples were then wicked away using Whatman 1 filter paper; washed with 2.5 µL ultrapure H₂O, wicked; and negatively stained with 2.5 µL 2% uranyl acetate, wicked. These were allowed to dry overnight to ensure all moisture had evaporated and imaged at 120 keV. Images were recorded on a Gatan 2 K x 2 K CCD.

Grids were transferred to a Gatan 626 cryo holder (Gatan, Pleasanton, USA) and inserted into a Tecnai T12 transmission electron microscope (FEI Company, USA) operating at 120 kV. Images were recorded on a OneView CMOS detector (Gatan Inc., Pleasanton, USA) using low-dose conditions. EM images used for Fig. 5 were collected on a Tecnai F20 transmission electron microscope (FEI Company, USA), operating at 200 kV. Images were recorded on a US 4000 (Gatan Inc, Pleasanton). A tilt series were acquired using the software package Xplore3D (FEI Company, Eindhoven, NL), using a discontinuous tilt scheme from −54 ° to +60 °, using 3 ° increments, under low dose conditions. The tomogram was reconstructed using IMOD and 6 iterations of the simultaneous iterative reconstruction technique.