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 |