Data acquisition was done using the SerialEM software28 (link),65 (link) at 300 kV on a Titan Krios (Thermo Fischer Scientific, Eindhoven, NL) equipped with a Quantum post column energy filter (Gatan, Pleasanton, CA, USA) and a K2 Summit direct detector camera (Gatan, Pleasanton, CA, USA). The tilt series was acquired with a pixel size of 0.342 nm, a target defocus of −5 µm, and using dose fractionation mode at 12 frames per second. Individual exposure times between 0.6 and 2.5 sec were automatically adjusted by the software to compensate for thickness variation during tilting. The total dose over the full series was kept below 100 e−/Å2. Data processing and tomogram reconstruction used MotionCor266 (link) and IMOD67 (link) software.
K2 summit direct detector camera
Manufactured by Ametek
Sourced in United States
The K2 Summit direct detector camera is a high-performance imaging device designed for scientific applications. It features a direct detection sensor that captures electron microscopy data with enhanced resolution and sensitivity compared to conventional scintillator-based cameras.
Automatically generated - may contain errors
Lab products found in correlation
Quantum post column energy filter, by Ametek (2 mentions)
Titan krios, by Thermo Fisher Scientific (2 mentions)
Titan krios microscope, by Thermo Fisher Scientific (2 mentions)
Vitrobot, by Thermo Fisher Scientific (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Tecnai arctica microscope, by Thermo Fisher Scientific (1 mentions)
5 protocols using k2 summit direct detector camera
1
Cryo-electron Tomography Data Acquisition
2
Cryo-EM of AtNIT4 Filaments
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Purified AtNIT4 filaments (2.5 μl at ~0.15 mg ml−1) were applied to glow-discharged 1.2/1.3 gold grids (Quantifoil, Micro Tools), incubated for ~30 s, blotted from both sides for ~3.5 s and vitrified using a Vitrobot (FEI) with humidifier turned off (~10% humidity). The grids were imaged on a Titan Krios microscope (FEI) with a K2 Summit direct detector camera (Gatan) using EPU (FEI) at 300 kV. A total of 1266 movies, consisting of 45 frames per movie with a dose rate of 6.5 e− pixel−1 s−1 over 7 s, were collected over a defocus range of −0.75 to −2.0 µm with a physical pixel size of 0.85 Å pixel−1.
3
Cryo-EM Tilt Series Imaging of Cells
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EM grids containing lamellas were transferred into a 300-kV Titan Krios microscope (FEI Thermo Fisher), equipped with a postcolumn energy filter (Gatan) and a K2 Summit direct detector camera (Gatan). Using SerialEM software (76 (link)), tilt series were acquired with 2° steps between −60° and +60° (in two halves, separated at −0° or −20°). Individual tilts were recorded in movie mode at 12 frames per second, at an object pixel size of 3.42 Å and a defocus of −4 to −5.5 µm. The total accumulated dose for the tilt series was kept below ∼100 e−/Å2. Each tomogram was acquired from a separate cell and thus is both a biological and technical replicate. Several different cell cultures and >10 imaging sessions were used to produce the dataset.
4
Cryo-EM Data Collection with Gatan K2
5
Cryo-TEM Imaging of FIB Lamellae
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Cryo-TEM imaging was performed on a Titan Krios (FEI) operated at 300 kV. The microscope was equipped with a field-emission gun, a Quantum post-column energy filter (Gatan, Pleasanton, CA, USA) and a heated phase plate holder (FEI) (Danev et al., 2014) . Data was recorded on a K2 Summit direct detector camera (Gatan) operated in dose fractionation mode. Tilt-series images were collected using SerialEM (Mastronarde, 2005) . Individual frames acquired for each tilt were aligned according to the procedures developed by Li et al. (Li et al., 2013) .
The tilt range for FIB lamella tomography takes into account the pre-tilt of the lamella (8°-15°) and, depending on the direction that the grid was mounted, is typically (±)50° to (±)70°. Tilt-series were acquired with a tilt increment of 2°, an object pixel size of 0.421 nm or 0.342 nm, and a total dose ~60-100 e -/Å 2 .
Alignment and operation of the VPP were carried out essentially as described by Fukuda et al. (Fukuda et al., 2015) . The target defocus was set between 0 and -0.5 m. The tomographic tilt-series presented in this report were collected using standard automated acquisition procedures within SerialEM. Special care was taken to properly align the lamella's pre-tilt axis parallel to the tomographic tilt axis to avoid tracking and focusing errors (described in Section 2.5).
The tilt range for FIB lamella tomography takes into account the pre-tilt of the lamella (8°-15°) and, depending on the direction that the grid was mounted, is typically (±)50° to (±)70°. Tilt-series were acquired with a tilt increment of 2°, an object pixel size of 0.421 nm or 0.342 nm, and a total dose ~60-100 e -/Å 2 .
Alignment and operation of the VPP were carried out essentially as described by Fukuda et al. (Fukuda et al., 2015) . The target defocus was set between 0 and -0.5 m. The tomographic tilt-series presented in this report were collected using standard automated acquisition procedures within SerialEM. Special care was taken to properly align the lamella's pre-tilt axis parallel to the tomographic tilt axis to avoid tracking and focusing errors (described in Section 2.5).
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