Cryo-planing of frozen-hydrated samples using cryo triple ion gun milling (CryoTIGM™)

Irene Y T Chang, Derk Joester*

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Cryo-SEM is a high throughput technique for imaging biological ultrastructure in its most pristine state, i.e. without chemical fixation, embedding, or drying. Freeze fracture is routinely used to prepare internal surfaces for cryo-SEM imaging. However, the propagation of the fracture plane is highly dependent on sample properties, and the resulting surface frequently shows substantial topography, which can complicate image analysis and interpretation. We have developed a broad ion beam milling technique, called cryogenic triple ion gun milling (CryoTIGM™ ['krī-ə-,tīm]), for cryo-planing frozen-hydrated biological specimens. Comparing sample preparation by CryoTIGM™ and freeze fracture in three model systems, Baker's yeast, mouse liver tissue, and whole sea urchin embryos, we find that CryoTIGM™ yields very large (~700,000μm2) and smooth sections that present ultrastructural details at similar or better quality than freeze-fractured samples. A particular strength of CryoTIGM™ is the ability to section samples with hard-soft contrast such as brittle calcite (CaCO3) spicules in the sea urchin embryo.

Original languageEnglish (US)
Pages (from-to)569-579
Number of pages11
JournalJournal of Structural Biology
Volume192
Issue number3
DOIs
StatePublished - Dec 1 2015

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Keywords

  • Cryo-SEM
  • Ion milling
  • Sample preparation
  • Ultrastructure

ASJC Scopus subject areas

  • Structural Biology

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