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Polara g2 electron microscope

Manufactured by Thermo Fisher Scientific
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The Polara G2 is an electron microscope designed for high-resolution imaging and analysis of samples at the nanoscale level. It provides advanced imaging capabilities, including low-voltage transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM), enabling detailed visualization and characterization of a wide range of materials and samples.

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Lab products found in correlation

Gatan k2 summit, by Thermo Fisher Scientific (4 mentions) K2 summit, by Ametek (3 mentions) Jem 3200fsc, by JEOL (2 mentions) Holey grids, by Quantifoil (2 mentions) Glow discharged copper grids, by Quantifoil (2 mentions) K2 summit, by Thermo Fisher Scientific (1 mentions)

18 protocols using polara g2 electron microscope

1

Single-particle cryo-EM of purified protein

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Vitrified specimens of the purified protein were prepared by plunge-freezing on Quantifoil holey grids (Quantifoil Micro Tools GmbH) covered with a freshly prepared thin continuous carbon film. For the WT construct, images of frozen-hydrated sample particles were acquired on a Technai G2 Polara electron microscope (FEI) operated at 300 kV using a K2 Summit direct electron detector camera (Gatan). For the mutant construct we used a 300 kV JEM3200FSC (JEOL) with an in-column energy filter (20 eV), which increases particle contrast. Movie stacks were collected in dose fractionation super-resolution counting mode at a nominal magnification of 415,000x (WT) and 425,100x (mutant) on each microscope, corresponding to calibrated physical pixel sizes of 1.26 Å and 1.23 Å, and super-resolution pixel sizes of 0.63 Å and 0.615 Å. The dose rate on the cameras was set to 10 electrons pixel−1s −1. The total exposure time was 10 s, leading to a total accumulated dose of 63 electrons Å−2 on the specimen. Each image stack was fractionated into 50 sub-frames, each with an accumulation time of 0.2 s per frame. Images were acquired at the defocus range of −1 to −3.5 μm (WT) and −0.7 to −3 μm (mutant) (Table 1).
Dosey T.L., Wang Z., Fan G., Zhang Z., Serysheva I.I., Chiu W, & Wensel T.G. (2018). Structures of TRPV2 in distinct conformations provide insight into role of the pore turret. Nature structural & molecular biology, 26(1), 40-49.
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2

Cryo-EM Structure Determination of InsP3R1

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Detergent-solubilized InsP3R1 was purified from rat cerebellum, and its structure–function integrity has been confirmed as described in our earlier study2 (link). The absence of any channel-specific modulatory proteins has been confirmed using immunoprecipitation/mass spectrometry (data not shown)34 (link). The vitrification of the purified protein was performed as previously described2 (link). Images of frozen-hydrated InsP3R1 particles were acquired on a Technai G2 Polara electron microscope (FEI) operated at 300 kV using a K2 Summit direct electron detector camera (Gatan). Images were collected in dose fractionation super-resolution counting mode at a nominal magnification of ×23,000, corresponding to a calibrated physical pixel size of 1.62 Å and super-resolution pixel size of 0.81 Å. The dose rate on the camera was set to ~10 electrons pixel−1 s−1. The total exposure time was 6 s, leading to a total accumulated dose of 22 electrons Å−2 on the specimen. Each image stack was fractionated into 30 subframes, each with an accumulation time of 0.2 s per frame. Images were acquired at the defocus range of −0.6 to −3.5 μm.
Fan G., Baker M.L., Wang Z., Baker M.R., Sinyagovskiy P.A., Chiu W., Ludtke S.J, & Serysheva I.I. (2015). Gating machinery of InsP3R channels revealed by electron cryomicroscopy. Nature, 527(7578), 336-341.
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3

Single-particle cryo-EM of purified protein

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Vitrified specimens of the purified protein were prepared by plunge-freezing on Quantifoil holey grids (Quantifoil Micro Tools GmbH) covered with a freshly prepared thin continuous carbon film. For the WT construct, images of frozen-hydrated sample particles were acquired on a Technai G2 Polara electron microscope (FEI) operated at 300 kV using a K2 Summit direct electron detector camera (Gatan). For the mutant construct we used a 300 kV JEM3200FSC (JEOL) with an in-column energy filter (20 eV), which increases particle contrast. Movie stacks were collected in dose fractionation super-resolution counting mode at a nominal magnification of 415,000x (WT) and 425,100x (mutant) on each microscope, corresponding to calibrated physical pixel sizes of 1.26 Å and 1.23 Å, and super-resolution pixel sizes of 0.63 Å and 0.615 Å. The dose rate on the cameras was set to 10 electrons pixel−1s −1. The total exposure time was 10 s, leading to a total accumulated dose of 63 electrons Å−2 on the specimen. Each image stack was fractionated into 50 sub-frames, each with an accumulation time of 0.2 s per frame. Images were acquired at the defocus range of −1 to −3.5 μm (WT) and −0.7 to −3 μm (mutant) (Table 1).
Dosey T.L., Wang Z., Fan G., Zhang Z., Serysheva I.I., Chiu W, & Wensel T.G. (2018). Structures of TRPV2 in distinct conformations provide insight into role of the pore turret. Nature structural & molecular biology, 26(1), 40-49.
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4

Cryo-EM Vitrification and Imaging Protocol

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Released pDox (5 µl) was deposited onto freshly glow-discharged holey carbon grids for 1 minute. The grids were blotted with What man filter paper and rapidly vitrified in liquid ethane using a gravity-driven plunger apparatus. The resulting frozen-hydrated specimens were imaged at −170 °C using a Polara G2 electron microscope (FEI Company) equipped with a Field Emission Gun and a Direct Detection Camera (Gatan K2 Summit). The microscope was operated at an accelerating voltage of 300 kV with a magnification of 9,400×, resulting the effective pixel size of 4.45 Å. For each area of interest, low dose cryo-EM images at ~5 defocus were recorded on the Direct Detection Camera. The cumulative doses of ~30 e-/A2 were distributed over 30 frames which were subsequently aligned with each other to generate the final high-resolution cryo-EM image.
Xu R., Zhang G., Mai J., Deng X., Segura-Ibarra V., Wu S., Shen J., Liu H., Hu Z., Chen L., Huang Y., Koay E., Huang Y., Liu J., Ensor J.E., Blanco E., Liu X., Ferrari M, & Shen H. (2016). An injectable nanoparticle generator enhances delivery of cancer therapeutics. Nature biotechnology, 34(4), 414-418.
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5

Cryo-EM Imaging of Borrelia burgdorferi

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Tilt series acquisitions were conducted as previously described (Lin et al., 2015 (link), Zhao et al., 2014 (link), Zhao et al., 2013 (link)). Cryo-samples were imaged at −170°C using a 300 keV Polara G2 electron microscope (FEI Company) equipped with a field emission gun. Tilt series of B. burgdorferi cells were collected by SerialEM software, under low dose mode (Mastronarde, 2005 (link)) and were recorded by Direct Detection Camera (Gatan K2 Summit). EM images were acquired at X 9,400 magnification (pixel size of ~4.5 Å), and at ~7 μm defocus. Single axis tilt series were recorded from −60° to +60° with 2°angular increments. The cumulative dose was ~60 e−/Å2 distributed over 61 images. Dose fractionation mode was used during tilt series acquisition with each tilt image being fractionated into 8 frames. The stack of 8 frames were aligned and drift-corrected using Motioncorr (Li et al., 2013 (link)).Tilt series were automatically aligned and reconstructed using a combination of IMOD (Kremer et al., 1996 (link)) and TOMO3D packages (Agulleiro & Fernandez, 2011 (link)).
Moon K.H., Zhao X., Xu H., Liu J, & Motaleb M.A. (2018). A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi. Molecular microbiology, 110(4), 634-647.
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6

Cryogenic Electron Microscopy Tomography

Cited 2 times
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Frozen-hydrated specimens were imaged and data were processed using our previously established protocols (12 (link), 28 (link), 41 (link)). Briefly, specimens were subjected to imaging at −170°C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a direct detection device (Gatan K2 Summit). The microscope was operated at 300 kV at a magnification of ×15,000, resulting in an effective pixel size of 2.5 Å at the specimen level (17 (link)). The tomographic package SerialEM (60 (link)) was used to collect low-dose, single-axis tilt series in the dose fractionation mode with a defocus at ∼6 μm and a cumulative dose of ∼60 e−/Å2 distributed over 35 stacks. Each stack contains ∼8 images. Each tilt series was collected at angles from −51° to 51° with 3° fixed increments. We used Tomoauto (58 (link)) to expedite data processing, which included drift correction of dose-fractionated data using Motioncorr (61 (link)) and assembly of corrected sums into tilt series, automatic fiducial seed model generation, alignment and contrast transfer function correction of tilt series by IMOD (62 (link)), and reconstruction of tilt series into tomograms by Tomo3D (63 (link)). Each tomographic reconstruction was 3,710 by 3,838 by 2,400 pixels and ∼130 Gb in size.
Khara P., Song L., Christie P.J, & Hu B. (2021). In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center. mBio, 12(5), e02465-21.
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7

Cryo-EM Imaging of Hsp104 Hexamer

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The peak fraction corresponding to the Hsp104DWB hexamer was incubated at 22°C for 10 min with 5 mM ATP in buffer consisting of 50 mM MOPS pH 7.5, 2 mM DTT, and 10 mM MgCl2, and kept on ice for another 30 min. 3.0 μl of sample (0.3 mg/ml) was applied to glow discharged copper grids (Quantifoil, Groβlöbichau, Germany), blotted, and frozen in liquid ethane. Frozen hydrated grids were imaged using a 300-kV Polara G2 electron microscope (FEI, Hillsboro, OR) equipped with a field emission gun. Images were recorded on an electron counting K2 Summit direct detection camera (Gatan Inc., Pleasanton, CA) operated in super-resolution mode at 23,000× nominal magnification corresponding to 1.68 Å/pixel. Dose fractionated imaging was performed by semi-automated collection methods using SerialEM (Mastronarde, 2005 (link)). 7.6 s exposures were collected at 200 msec/frame, with a total electron dose of 38 e− per micrograph in 38 frames.
Lee S., Roh S.H., Lee J., Sung N., Liu J, & Tsai F.T. (2019). Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation. Cell reports, 26(1), 29-36.e3.
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8

Cryo-Electron Tomography of Frozen Specimens

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The frozen-hydrated specimens were transferred to a 300-kV Polara G2 electron microscope (FEI) equipped with a Direct Electron Detector (DDD) (Gatan K2 Summit) or with a charge-coupled-device (CCD) camera (TVIPS; GMBH, Germany). Images were recorded at 15,400 × magnification with pixel size of 2.5 Å (for images recorded by K2) or at 31,000 × magnification with pixel size of 5.7 Å (for images recorded by CCD). SerialEM (Mastronarde, 2005 (link)) was used to collect tilt series at −6 to −8 µm defocus, with a cumulative dose of ~100 e-/Å (Terashima et al., 2008 (link)) distributed over 61 images and covering angles from −60°to 60°, with a tilt step of 2°. Images recorded by K2 camera were first drift-corrected using the motioncorr program (Li et al., 2013 (link)). Then all tilt series were aligned using fiducial markers by IMOD (Kremer et al., 1996 (link)), tilt images were contrast transfer function corrected using ‘ctfphaseflip’ function in IMOD, and tomograms were reconstructed by weighted back-projection using TOMO3D (Agulleiro and Fernandez, 2015 (link)).
Chang Y., Moon K.H., Zhao X., Norris S.J., Motaleb M.A, & Liu J. (2019). Structural insights into flagellar stator–rotor interactions. eLife, 8, e48979.
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9

Cryo-Electron Tomography Pipeline

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The frozen-hydrated specimens were imaged at −170 °C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a direct detection device (Gatan K2 Summit). The microscope was operated at 300 kV with a magnification of ×15,500, resulting in an effective pixel size of 2.5 Å at the specimen level. We used SerialEM 26 (link) to collect low-dose, single-axis tilt series with dose fractionation mode at about 5 μm defocus and a cumulative dose of ~50 e−/Å2 distributed over 35 stacks. Each stack contains ~8 images. Over 2,000 tilt series were collected from −51° to 51° with increment of 3°. We used Tomoauto 25 (link) to facilitate data processing which includes drift correction of dose-fractionated data using Motioncorr 27 (link) and assembly of corrected sums into tilt series, automatic fiducial seed model generation, alignment and contrast transfer function correction of tilt series by IMOD 28 (link), and reconstruction of tilt series into tomograms by Tomo3D 29 (link). Each tomographic reconstruction is 3,710 × 3,838 × 2,400 pixels and ~130Gb in size. In total, 2,062 tomographic reconstructions from 7 different strains were generated (Supplementary Information Table 2).
Chetrit D., Hu B., Christie P.J., Roy C.R, & Liu J. (2018). A Unique Cytoplasmic ATPase Complex Defines the Legionella pneumophila Type IV Secretion Channel. Nature microbiology, 3(6), 678-686.
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10

Cryo-EM Tomography of Microbial Specimens

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Frozen-hydrated specimens were imaged at −170 °C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a direct detection device (Gatan K2 Summit). The microscope was operated at 300 kV with a magnification of ×9,400, resulting in an effective pixel size of 4.5 Å at the specimen level. We used SerialEM (38 (link)) to collect low-dose, single-axis tilt series with dose fractionation mode at about 8 µm defocus. Tilt series were collected from −51° to 51° with an increment of 3° and a cumulative dose of ∼70 e−/Å2 distributed over 35 stacks. Each stack contains approximately eight images. We used Tomoauto (39 ) to facilitate data processing, which includes drift correction of dose-fractionated data using Motioncorr (40 (link)) and assembly of corrected sums into tilt series, automatic fiducial seed model generation, alignment, and contrast transfer function correction of tilt series by IMOD (41 (link)) and weighted back projection (WBP) reconstruction of tilt series into tomograms using Tomo3D (42 (link)). Each tomographic reconstruction is 3,710 × 3,838 × 1,600 pixels and ∼1.30 Gb in size. In total, 4,227 tomographic reconstructions from six different strains were generated.
Song L., Perpich J.D., Wu C., Doan T., Nowakowska Z., Potempa J., Christie P.J., Cascales E., Lamont R.J, & Hu B. (2022). A unique bacterial secretion machinery with multiple secretion centers. Proceedings of the National Academy of Sciences of the United States of America, 119(18), e2119907119.
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