Mycobacterium smegmatis and Mtb cultures were fixed as above prior to vitrification. Grids were prepared following standard procedures and observed at liquid nitrogen temperatures in a JEM-2200FS/CR transmission electron microscope (JEOL Europe, Croissy-sur-Seine, France) operated at 200 kV. An in-column omega energy filter helped to record images with improved signal/noise ratio by zero-loss filtering. The energy selecting slit width was set at 9 eV. Digital images were recorded on an UltraScan4000 CCD camera under low-dose conditions at a magnification of 55,058 obtaining a final pixel size of 2.7 Å/pixel.
Jem 2200fs cr transmission
Manufactured by JEOL
Sourced in Japan
The JEM-2200FS/CR is a high-resolution transmission electron microscope (TEM) designed for advanced materials characterization. It features a field emission gun for high-brightness electron illumination and a multi-functional imaging and spectroscopy system. The TEM provides direct, high-resolution imaging of nanoscale structures and compositions.
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5 protocols using jem 2200fs cr transmission
1
Transmission Electron Microscopy of Mycobacterial Cells
2
Ultrastructural Analysis of Biological Samples
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Cells were fixed with 2% glutaraldehyde in 0.1 M cacodylate at room temperature for 2 h, and then incubated overnight in 4% formaldehyde, 1% glutaraldehyde, and 0.1% PBS. After fixation, samples were stained for 90 min in 2% osmium, serially dehydrated in ethanol, and embedded in Spurrs epoxy resin. Thin sections were obtained on an Ultracut UCT (Reichert) and stained with 1% uranyl acetate. Samples were examined in a JEOL JEM1010 operating at 100 kV. For Cryo‐EM, grids were prepared following the standard procedures and observed at liquid nitrogen temperatures in a JEOL JEM‐2200FS/CR transmission electron microscope operated at 200 kV. An in‐column omega energy filter helped to record images with improved signal/noise ratio by zero‐loss filtering. The energy selecting slit width was set at 9 eV. Digital images were recorded on an UltraScan4000 CCD camera under low‐dose conditions at a magnification of 55,058 obtaining a final pixel size of 2.7 Å/pixel.
3
Cryo-EM Imaging of Extracellular Vesicles
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EVs were directly adsorbed onto glow‐discharged holey carbon grids (QUANTIFOIL, Germany) and processed as described.21 Images were obtained in a JEM‐2200FS/CR transmission cryo‐electron microscope (JEOL, Japan).
4
Cryo-EM Imaging of Vitrified Samples
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EV preparations were directly adsorbed onto glow‐discharged holey carbon grids (QUANTIFOIL, Germany). Grids were blotted at 95% humidity and rapidly plunged into liquid ethane with the aid of VITROBOT (Maastricht Instruments BV, Netherlands). Vitrified samples were imaged at liquid nitrogen temperature using a JEM‐2200FS/CR transmission cryo‐electron microscope (JEOL, Japan) equipped with a field emission gun and operated at an acceleration voltage of 200 kV. In cryo‐electron microscope sessions, digital images were taken using a low‐dose technique by means of an ULTRASCAN 4000SP (4096 × 4096) cooled slow‐scan charged‐coupled device camera (GATAN). An in‐column energy filter (Omega Filter) was used to improve the signal‐to‐noise ratio of these images by zero‐loss filtering.
5
Cryo-EM Imaging of Concentrated EVs
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Concentrated EV preparations were immobilised onto glow‐discharging holey carbon 200‐mesh copper grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Grids were vitrified using a Vitrobot (Maastricht Instruments BV, Maastricht, NL). Vitrified samples were imaged at liquid nitrogen temperature using a JEM‐2200FS/CR transmission cryo‐electron microscope (JEOL), as described previously (Yeung et al., 2018 (link)), with ≥6 microscopic fields assessed per sample.
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