Fig. 2

Typical temperature courses prior to and during high pressure freezing (HPF) of frozen samples. Data as generated during cryofixation by the HPF-device (red line, upper x-axes) and data based on direct measurements of the sample temperature using the temperature measurement adapter (TMA, black lines, lower x-axes), are compared. Solid black line: The frozen samples were transferred to the HPF-device within a precooled transfer block (0 to 15 s) to prevent thawing. When the loading device with the frozen sample was pulled out of the transfer block (1) short intermediate warming of the sample happened. Subsequently, the loading device was quickly positioned in the precooled loading area of the HPF-device and sample temperature declined again (2). During inserting the loading-device into the HPF-device (3) and locking it, sample temperature again slightly increased but remained well below 0 °C as before, because HPF was quickly released within only a few seconds (4). Dashed black line: Samples were treated as before, with the difference that the precooling of the interior of the HPF-device by the precooling device (PD) has been omitted. In this way the sample temperature rapidly began to rise immediately after inserting the loading device into the HPF-device (5) and the warming rate even increased after locking the specimen pod (6). Arrows indicate the further temperature courses (not shown) that ended at − 196 °C