Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Summary

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Abstract

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Introduction

The concept and practical application of freeze-fracture processing of biological specimens was introduced by Steere¹ over half a century ago. The early apparatus appropriated disparate components into a working self-contained unit¹. The original apparatus was modified and refined into commercially available instruments in order to accommodate the critical need for remote manipulation, maintenance of high vacuum, and the evaporation of carbon and metals to produce a replica suitable for examination by transmission electron microscopy (Figure 1 and Figure 2).

The typical instrument cons....

Protocol

1. Preparation of Biological Specimens for Freeze-fracture/Freeze-etch

1. Use a conventional EM fixative formulation such as 2% glutaraldehyde + 2% paraformaldehyde in 0.1 M phosphate buffer for 1 hr. to perform primary fixation of biological tissues. NOTE: While it may be desirable to freeze some types of specimens without prior fixation, universal blood borne pathogen precautions mandate appropriate fixation where the specimen consists of human tissue.
2. Following primary fixation, rinse the specimen in the same buffer supplemented with 0.2 M sucrose for no more than 1 hr.
3. Transfer the specimen to a cryoprotectant solution consisting of 25....

Results

The key premise of freeze-fracture image interpretation is that fracture planes pass through the lipid bilayer of membranes conferring two fracture faces, called by convention the PF-face (plasma fracture-face) and EF-face (extracellular fracture-face) (Figure 3). The PF-face is the half of the membrane lipid bilayer adjacent to the cytoplasm of the cell and the EF-face is the half of the membrane lipid bilayer adjacent to the extracellular milieu. The freeze-fracture technique is particularly useful for.......

Discussion

In the years following its introduction and commercial availability, freeze-fracture/etch procedures were widely utilized for investigations of biological membrane structure. Indeed, the best perspectives of some of the structural specializations of membranes have been obtained in freeze-fracture/etch preparations. These studies not only contributed to understanding of the structural organization of membranes but also provided insights into how structure and function are related.

The advent of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Balzers Freeze-fracture/freeze-etch plant	Balzers	BAF400T
Standard buffers	various suppliers
Standard aldehyde fixatives	various suppliers
Sodium dichromate	various suppliers
Sulfuric acid	various suppliers
Disposable supplies for Platinum/Carbon Evaporation	Technotrade International
Liquid nitrogen	various suppliers
Propane	various suppliers
Disposable supplies for electron microscopy	Electron Microscopy Sciences
Transmission electron microscope	Carl Zeiss Inc.