Fig 1: Modified high-pressure cooler and semi-dry liquid N2 bath. A modified Leica EM ICE is used to high-pressure cool the crystals in capillaries (a). From left to right: the liquid N2 inlet, a microscope, the sample dewar that is pulled out of the machine, a loading groove with the loading station within the table and a screen to monitor the process. Once the samples have been vitrified and stored in the sample dewar they need to be transferred to the semi-dry liquid N2 bath (b). The bath is filled with liquid N2 up to the cover plate to keep the tripod with the vitrified samples (left), the sample-transfer station (middle) and the puck with cryovials cold. Large forceps, a rod and a manipulation tool are stored in the upper right corner. The extensions for elongation of the sample unit to form the sample holder are stored at the top middle. To separate a sample unit from the cartridge it is slid into the sample-transfer station. This is performed with the forceps, with the cartridge facing downwards. Using the rod, the cartridge can be held in place (small bore). With the manipulation tool, an extension is taken (magnetic) and placed onto the sample unit. The sample holder is now complete and is transferred to the cryovial in the puck. The cartridge is removed from the transfer station with the manipulation tool and a new transfer cycle can be started.
Fig 2: The cryo-SEM workflowThis figure shows examples of the equipment that is used for each step. Equipment may vary depending on the manufacturer and model. Equipment shown is (Step 1) slush-freezing: Quorum Technologies Prep-dek®; plunge-freezing: ThermoFisher Vitrobot; and high-pressure freezing (HPF): Leica Microsystems EM ICE. (Steps 3–5) left: Quorum Technologies PP3010 Cryo Preparation Chamber; right: Leica Microsystems EM ACE600 with EM VCT500. (Step 6) JEOL JSM-IT810. Images reproduced with permission from JEOL U.K., Leica Microsystems GmbH, Quorum Technologies, and ThermoFisherScientific.
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