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. 2017 May 17;16(6):900–909. doi: 10.1177/1533034617708960

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© The Author(s) 2017

This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Figure 2. — Image of a TEM stack and layer following freezing. TEM stacks were frozen in the TEM assembly apparatus. Following freezing, TEM stacks were disassembled into individual layers for assessment of iceball diameter as well as cell destruction using fluorescent microscopy. A, Top view of a TEM stack following freezing prior to stack disassembly and (B) image of an individual TEM layer in culture following disassembly. During analysis, identification of the outer edge of the first (orange ring) and second freeze (yellow ring) iceball edge (transition from frozen to nonfrozen tissue which equates to ∼−2°C, nominally) is visible within the gel, thereby allowing for direct measurement of iceball diameter following each freeze event within the protocol.