Prior to plunge-freezing, 200 mesh Cu grids with a lacey carbon membrane (EMS Cat# LC200-CU-100) were glow discharged in a Pelco easiGlow glow discharger (Ted Pella) using an atmosphere plasma generated at 15 mA for 15 s with a pressure of 0.24 mbar. This treatment created a negative charge on the carbon membrane, allowing liquid samples to spread evenly over the grid. Four microliters of the sample was pipetted onto the grid and blotted for 5 s with a blot offset of +0.5 mm, followed by immediate plunging into liquid ethane within a FEI Vitrobot Mark III plunge freezing instrument (Thermo Fisher Scientific). Grids were then transferred to liquid nitrogen for storage. The plunge-frozen grids were kept vitreous at –180 °C in a Gatan Cryo Transfer Holder model 626.6 (Gatan) while viewing in a JEOL JEM1230 LaB6 emission TEM (JEOL USA) at 100 keV. Image data were collected by a Gatan Orius SC1000 CCD camera Model 831 (Gatan).
Orius sc1000 ccd camera model 831
Manufactured by Ametek
Sourced in United States
The Orius SC1000 CCD camera Model 831 is a high-performance scientific camera designed for laboratory applications. It features a large-format CCD sensor with high quantum efficiency and low readout noise, enabling the capture of high-quality images and data. The camera offers a range of customizable settings, including exposure time, gain, and binning, to optimize performance for specific applications.
Automatically generated - may contain errors
Lab products found in correlation
Pelco easiglow glow discharger, by Ted Pella (2 mentions)
Cryo transfer holder model 626, by Ametek (2 mentions)
Jem1230 lab6 emission tem, by JEOL (2 mentions)
Lc200 cu 100, by Ted Pella (1 mentions)
Fei vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
Mark 3, by Thermo Fisher Scientific (1 mentions)
Jem1230 lab6 emission tem, by Ametek (1 mentions)
Jem 1230 lab6, by JEOL (1 mentions)
Uc7 ultramicrotome, by Leica (1 mentions)
Lc200 cu, by Ted Pella (1 mentions)
4 protocols using orius sc1000 ccd camera model 831
1
Cryo-EM Sample Preparation Technique
2
Cryo-EM Specimen Preparation Protocol
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Lacey Carbon 200 mesh Cu grids (EMS Cat# LC200-CU) were glow discharged in a Pelco easiGlow glow discharger for 30 seconds at 30 mA. 4 μL of sample solution was carefully pipetted onto the grids and plunge frozen in liquid ethane in a FEI Vitrobot Mark III with a blotting time of 5 seconds and blot offset of 0.5 mm. Grids were stored in liquid nitrogen and loaded into a Gatan 626.6 Cryo Transfer Holder cooled down to -170°C prior to observation in a JEOL JEM-1230 LaB6 emission TEM running at 100 kV. Images were collected with a Gatan Orius SC1000 CCD Camera, Model 831.
3
Ultrastructural Analysis of Biological Samples
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Samples were pelleted at 21,000 x G in an Eppendorf 5424 microcentrifuge for 10 minutes. Pelleted samples were fixed with 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1M PBS, post-fixed with 1% (w/v) OsO4 and 1% (w/v) UA, dehydrated in a graded series of ethanol, infiltrated with EMBed 812 epoxy resin, and embedded in beam capsules. The embedded samples were polymerized at 60°C for 48 hours prior to ultra-thin sectioning utilizing a Leica UC7 ultramicrotome. Sections were collected on 150 mesh Cu grids with a formvar/carbon membrane and stained with 3% (w/v) UA in 50% (v/v) methanol and Reynold’s lead citrate to further enhance contrast. The samples were observed in a JEOL JEM-1230 LaB6 emission TEM at 100 kV. Images were collected with a Gatan Orius SC1000 CCD Camera, Model 831.
4
Cryo-TEM Specimen Preparation Protocol
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For cryo-TEM measurement, 200 mesh Cu grids with a lacey carbon membrane (EMS Cat. # LC200-CU) were placed in a Pelco easiGlow glow discharger (Ted Pella Inc., Redding, CA, USA) and an atmosphere plasma was introduced on the surface of the grids for 30 seconds with a current of 15 mA at a pressure of 0.24 mbar. This treatment creates a negative charge on the carbon membrane, allowing for aqueous liquid samples to spread evenly over of the grid. 4 µL of sample was pipetted onto the grid and blotted for 5 seconds with a blot offset of +0.5 mm, followed by immediate plunging into liquid ethane within a FEI Vitrobot Mark III plunge freezing instrument (Thermo Fisher Scientific, Waltham, MA, USA). Grids were then transferred to liquid nitrogen for storage. The plunge-frozen grids were kept vitreous at -172 °C in a Gatan Cryo Transfer Holder model 626.6 (Gatan Inc., Pleasanton, CA, USA) while viewing in a JEOL JEM1230 LaB6 emission TEM (JEOL USA, Inc., Peabody, MA,) at 120 keV. Image data was collected by a Gatan Orius SC1000 CCD camera Model 831 (Gatan Inc., Pleasanton, CA, USA).
Image analysis was done using Image J.
Image analysis was done using Image J.
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