Rapid freeze substitution for cryopreparation of frozen cells and tissues for (electron) microscopy

What is it about?

We developed and manufactured modules for sample agitation in the cryochambers of the most widely used automated freeze substitution systems, AFS(1) and AFS2 (Leica Microsystems), and demonstrated that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

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Why is it important?

Conventional chemical fixation and processing at room temperature lead to artifacts due to shrinkage, extraction, aggregation and redistribution of biological material. High-pressure freezing followed by freeze substitution avoids such artifacts. Since freeze substitution is notoriously slow, it is important to accelerate the substitution process by agitation. We tested magnetic force-driven agitation modules that offer the possibility of straightforward refitting of automated freeze substitution systems (AFS) that are currently in use because it does not interfere with the electronics and construction of the AFS itself. Given the widespread distribution of AFS in EM labs world-wide, we conclude, that the acceleration of FS with agitation modules, offers a cost-effective way to generate cryopreparations in much shorter times, at high quality.

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