A 3 µL aliquot of a suspension of jNPs in PBS at an approximate concentration of 1012 particles/mL was placed on a freshly glow-discharged 75 Quantifoil (Jena, Germany) 400 mesh copper grid overlaid with a 3 nm thick solid carbon layer. The sample grid was held by self-holding forceps vertically attached to a nitrogen gas-driven plunger in a humidified plexiglass freezing station. The grid was blotted with oven-dried filter paper and immediately plunged into supercooled ethane 76 (link). Vitrified samples were stored under liquid nitrogen in a cryogenic storage dewar. At the time of imaging, vitrified samples were transferred under liquid nitrogen into a Gatan Single Tilt Cryoholder using a cryotransfer station (Gatan, Pleasanton, CA) and inserted into a CM12 Transmission Electron Microscope (Philips Electron Optics, Eindhoven, The Netherlands). Images were collected at 120KV, 75,000 X, and a defocus of 1 u with a Tietz 1K x 1K pixel CCD camera (Gauting, Germany) using EMMENU4 Program. A low dose kit minimizes irradiation damage to the sample.
Single tilt cryoholder
Manufactured by Ametek
The Single Tilt Cryoholder is a specialized lab equipment designed for use in electron microscopy applications. It provides a stable platform for holding and tilting samples at cryogenic temperatures, enabling the observation and analysis of delicate or temperature-sensitive specimens.
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2 protocols using single tilt cryoholder
1
Cryo-TEM Imaging of Janus Nanoparticles
2
Cryo-EM Imaging of MC-Salt Mixtures
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Selected MC-salt mixtures (with a 5% wt/vol MC) were subjected to cryo-electron microscopy (cryo-EM). Each mixture at ~5°C was diluted 20-fold with 10 mM HEPES buffer (pH 7.0) containing the same concentration of salt as in the mixture. An aliquot (3 μL) obtained from the diluted mixture was deposited onto a glow-discharged holey carbon film supported on a 200-mesh copper grid (Ted Pella, Redding, CA) held in place by a Mark-IV Vitrobot unit (FEI, Hillsboro, OR) for automated cryo-preparation. The grid was then blotted for 2.5 s and vitrified by plunging into liquid ethane. Next the grid was transferred under liquid nitrogen on to a single-tilt cryo-holder (Gatan, Pleasanton, CA) and observed under low dose using a FEI Tecnai 12 BioTwin electron microscope operating at 120 kV. Images were obtained using a Gatan Orius SC 1000 CCD camera (Gatan Inc., Pleasanton, CA).
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