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Table 1 Workflow for high pressure freezing of already frozen biological samples

From: A new technical approach for preparing frozen biological samples for electron microscopy

Time

Action

Effects/notes

− 15 min

Set air temperature inside the Plexiglas® chamber to 0 °C

Insert a loading device and release HPF in advance

Slow precooling of the loading area

Precooling of the internal components of the HPF-device

− 5 min

Insert and lock the precooling device (PD)

Set air temperature inside the Plexiglas® chamber to − 5 °C. The following disclosures relate to the experiment on R. glacialis and to the usage of the Leica Empact HPF-device (Leica Microsystems, Vienna, Austria)

Increased precooling of the loading area and of the internal components of the HPF-device

− 30 s

Fill LN2 directly into the pan (up to the rim)

Prevents the HPF-device from activating the release delay due to low LN2-level in the pan for 2–3 min

− 10 s

Switch the fan inside the Plexiglas® chamber permanently on and remove the lid

Develops a cold (− 5 to − 10 °C) atmosphere around and above the loading area. Important for transferring the loading device from the transfer block to the loading area of the HPF-device

0 s

Unlock and remove the PD

Pull out the loading device from the transfer block and quickly insert it into the HPF-device

Promptly press ‘Lock’ and start HPF

This step must be executed within max. 8 s

Otherwise the sample will thaw before HPF is completed

  1. The following disclosures relate to the experiment on R. glacialis and to the usage of the Leica Empact HPF-device (Leica Microsystems, Vienna, Austria)