Tomograms of the GTP-bound DRP1 rings labelled with Ni-NTA-Nanogold were collected with the FEI Tecnai G2 F20 cryo-STEM at a nominal magnification of 25,000X corresponding to a pixel size of 0.44 nm (with negative-stained samples) using tilt range between −60° to 60°. The images were aligned, cropped, and binned using the IMOD [61 (link)]. The 3D visualization and interpretation was carried out with Chimera [27 (link), 60 (link)].
Tecnai g2 f20 cryo stem
Manufactured by Thermo Fisher Scientific
The Tecnai G2 F20 cryo-STEM is a transmission electron microscope designed for cryogenic (low-temperature) imaging and analysis of biological samples. It combines the capabilities of a scanning transmission electron microscope (STEM) with the ability to maintain samples at cryogenic temperatures. The core function of this instrument is to provide high-resolution, low-dose imaging and analysis of delicate, hydrated biological specimens in their native state.
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4 protocols using tecnai g2 f20 cryo stem
1
Cryo-STEM Imaging of DRP1 Rings
2
Cryo-STEM Imaging of DRP1 Helices
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Tomograms of the GMP-PNP-bound DRP1 helices attached with Ni-NTA-Nanogold were acquired with the FEI Tecnai G2 F20 cryo-STEM at 200 kV. Images were recorded at a nominal magnification of 25,000X corresponding to a pixel size of 0.48 nm (for the NanoVan-stained samples). The tomograms for DRP1 helices unbound to the Nanogold were collected with the FEI Titan Krios cryo-STEM at 300 kV using a tilt range between −60° to 60° at 18,300 fold magnification corresponding to a pixel size of 0.820 nm. The images were aligned, cropped, and binned using IMOD [61 (link)–63 (link)]. One representative tomogram (acquired in cryo condition), binned twice to 1.64 nm/pixel, was manually traced in 3dmod, Version 4.5 [60 (link)]. Chimera was used to compare the DRP1 model helices to the extracted helix data traced in 3dmod. Additionally, the pitches and diameters of the helices were manually measured on the digital images.
3
Cryo-Electron Tomography of Eggshell and Synthetic Calcite
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FIB-cut sections approximately 80 nm thick from eggshell and synthetic calcite crystals grown with 5.9 μM OPN were collected on a copper grid. A series of single–axis tilt images was collected with a Tecnai G2 F20 cryo-S/TEM (FEI) operated at an accelerating voltage of 200 kV equipped with a Gatan UltraScan 4000 4k × 4k digital CCD camera system (model 895). Images were captured at a magnification of 62,000 over a tilt range of −40° to +60° for the eggshell samples and −50° to +50° for the synthetic calcite crystals (2° increments in both low tilts and high tilts on the 80-nm-thick sections). The resulting images had pixel sizes of 0.19 nm. The images from the tilt series were aligned, filtered, and reconstructed into a tomogram using the IMOD software package (54 (link)). The movies for the raw tilt series and reconstruction were carried out using IMOD, whereas the movies with 3D volume with solid and surface rendering were generated using UCSF Chimera (version 1.10.1).
4
Cryo-Electron Tomography of Otoconia
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The 80-nm-thick FIB-cut section of an otoconium collected on a TEM copper grid was used to collect a series of single-axis tilt images at an accelerating voltage of 200 kV using a Tecnai G2 F20 cryo-S/TEM (FEI) equipped with a Gatan Ultrascan 4000 4k × 4k digital CCD camera system (model 895). Images were taken at a magnification of 62,000X over a tilt range from -40° to +50° for the samples (2° increments in both low tilts and high tilts). The resulting images had pixel sizes of 0.19 nm. For electron tomography in a scanning transmission electron microscope (STEM) (data shown in Supplementary Material), images were recorded using a ThermoFisher 300 kV Titan 3 Themis X-FEG S/TEM at a magnification of 62,000 over a tilt range from -50° to +70° (2° increments in low tilts and 1° high tilt step on the 80-nm-thick sections). The images from the tilt series were aligned, filtered and reconstructed into a tomogram using the IMOD software package (Kremer et al., 1996) . The movies for the raw tilt series and reconstruction were done in IMOD, whereas the movies with 3D volume with solid and surface rendering were generated using UCSF Chimera (version 1.10.1) software.
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