Tecnai g2 polara transmission electron microscope

Reconstructions of structures in the wall of hazel pollen grains were obtained in the electron tomographic experiments described in (Kovacik et al., 2009 (link)). Briefly, hazel pollen grains were chemically fixed, stained by Alcian blue, epon-embedded and postfixed with osmium tetroxide and lanthanum nitrate. Electron tomography of ∼150 nm sections coated with a ∼3 nm thick fine-grained Pt/C layer deposited perpendicular to the sections by electron-beam evaporation was carried out at low temperature (∼−170 °C) with a Tecnai G2 Polara transmission electron microscope (FEI, Hillsboro, OR, USA) equipped with a Gatan energy filter (GIF 2002, Gatan, Pleasanton, USA) and a 2048 × 2048 Gatan CCD camera, operated at 300 kV. High-dose single-axis tilted series were acquired with 1° angular step in the tilting range |±65|° with ∼−6 μm nominal defocus at the magnification of 22,900×. Three-dimensional reconstructions were computed by bilinear interpolation in Fourier space and low-pass filtered with cutoff of 5 nm.

Samples of 4 μL of purified TraI/22-mer ssDNA complex at a concentration of 1mg/mL, with or without 1 mM AMP-PNP/Mg²⁺, were deposited onto glow-discharged lacey carbon grids or Quantifoil R1.2/1.3 gold grids, and plunge-frozen using a Vitrobot, with blot force set to 1, blot time of 4 s, 100% humidity and at room temperature. Grids were imaged in two different conditions: in house using a FEI Tecnai G2 Polara transmission electron microscope and at the eBIC facility at the Diamond synchrotron using a Titan Krios. Both microscopes were operated at 300 kV, and in both cases data was collected on a K2 Summit direct electron detector operated in counting mode, and placed at the end of a Quantum energy filter operated with a slit width of 20 eV. Data on the Polara was acquired manually using SerialEM. It had a final pixel size of 1.86 Å, and was collected using a dose of ∼7 e^-/(pix^∗sec) (equivalent to ∼2 e^-/Å^2∗sec at the specimen level) and a total exposure of 19 s (∼40 e-/Ų) divided into 76 frames. Data on the Krios was automatically acquired with EPU software (FEI). It had a final pixel size of 1.05 Å, and was collected using a dose of 6.3 e^-/(pix^∗sec) (equivalent to 5.7 e^-/Å^2∗sec at the specimen level) and a total exposure of 8 s (∼45 e-/Ų) divided into 20 frames. In both cases defocus ranged between 2 and 3.5 μm.

Tomography was performed on a Tecnai G2 Polara transmission electron microscope (FEI, Eindhoven, Netherlands) equipped with a field emission gun operated at 300 kV, a GIF 2002 post-column energy filter (Gatan, Pleasanton, CA), and either a 2048 × 2048 Gatan slow scan CCD camera or a 3838 × 3710 Gatan K2 Summit direct detection camera (only Figures 7 and 11A–E). Tilt-series acquisition was controlled by SerialEM software (Mastronarde, 2005 (link)) under low-dose conditions (∼100 e/Ų cumulative dose). CCD images were recorded at 2° tilt increments, with −8 μm to −12 μm defocus, at 31,500×, 42,000×, and 52,500× magnifications (pixel sizes of 9.6 Å, 7.1 Å, and 5.7 Å). 52,500× was the highest possible magnification that still prevented excessive electron damage to the sample. K2 images were recorded at 2° tilt increments, with −5 μm defocus, at 11,800× and 14,500× magnifications (pixel sizes of 4.2 Å and 3.4 Å). The dataset for this study consisted of 43 tomograms from 27 FIB lamella preparations.