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 language | English (US) |
|---|---|
| Pages (from-to) | 569-579 |
| Number of pages | 11 |
| Journal | Journal of Structural Biology |
| Volume | 192 |
| Issue number | 3 |
| DOIs | |
| State | Published - Dec 2015 |
Funding
This work was funded by the NSF Major Research Instrumentation program ( NSF MRI-1229693 ), the NSF Biomaterials program ( DMR-1106208 ), and the Northwestern University Materials Research Center ( DMR-1121262 ). This work made use of NU ANCE -EPIC and OMM, Northwestern University core facilities that are supported by the MRSEC program (NSF DMR-1121262), the International Institute for Nanotechnology (IIN), and the State of Illinois . We thank Dr. Reiner Bleher for discussion and Dr. Thomas O’Halloran for providing the mouse liver tissues.
Keywords
- Cryo-SEM
- Ion milling
- Sample preparation
- Ultrastructure
ASJC Scopus subject areas
- Structural Biology