Agitation modules: Flexible means to accelerate automated freeze substitution Journal Article


Author(s): Reipert, Siegfried; Goldammer, Helmuth; Richardson, Christine; Goldberg, Martin W; Hawkins, Timothy J; Hollergschwandtner, Elena; Kaufmann, Walter A; Antreich, Sebastian; Stierhof, York-Dieter
Article Title: Agitation modules: Flexible means to accelerate automated freeze substitution
Affiliation IST Austria
Abstract: For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate 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.
Keywords: transmission electron microscopy; cryofixation; high-pressure freezing; rapid freeze substitution
Journal Title: Journal of Histochemistry and Cytochemistry
ISSN: 0022-1554
Publisher: Histochemical Society  
Date Published: 2018-07-03
Start Page: epub ahead of print
DOI: 10.1369/0022155418786698
Open access: no