Advancing Cellular Ultrastructure Research

Freeze-Fracture and Freeze-Etching

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Freeze-fracture and freeze-etching are useful tools for studying flexible membrane-associated structures such as tight junctions or the enteric glycocalyx. Freeze-fracture and etching are two complementary methods for exposing membrane associated macromolecules, by means of sample vitrification for the preservation of targeted structure and then breaking a frozen specimen to reveal internal structures. Freeze etching is a follow-up step in which surface ice sublimates under vacuum to reveal further details. In these techniques, coating with metal or carbon enables the sample to be imaged in a cryo-SEM directly or in a TEM as a replica film. This pair of techniques is used to investigate cell organelles, membranes, layers and emulsions and is particularly useful for flexible structures that are not resolved by conventional or cryo-electron microscopy.

The ultimate resolution of these techniques is determined by how well the biological surface is replicated.  Amorphous carbon replicas far better replicate biologic details than conventional metal coatings and using phase-contrast electron microscopy, provide unparalleled insight into these elusive cellular components.

Speaker: Evan Krystofiak, PhD.

Evan Krystofiak is a staff scientist in the Cell Imaging Shared Resource (CISR) core facility at Vanderbilt University, performing electron microscopy for over 90 labs using a combination of conventional and cryo-based approaches. Dr. Krystofiak’s main research interests lie in understanding macromolecular ultrastructure in the in-situ cellular context. His research includes development of high-resolution freeze-etch using amorphous replicas, freeze-substitution of difficult to preserve samples, and Fourier space analysis to understand biological paracrystal packing. He has used these techniques to examine the structure of tight junction fibrils and revealed the hydrated structure of enterocyte glycocalyx. 

Key Learnings

  • Outline the basic methodology of Freeze-Fracture and Freeze-Etch techniques
  • Discuss the role of the enterocyte glycocalyx and similar extracellular structures in situ
  • Illustrate how to optimize these replica techniques for maximum biological detail

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