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 consists of a high vacuum chamber with specimen table and microtome arm having regulable liquid nitrogen throughputs (Figure 1). The chamber also houses two electron guns, one for stabilizing carbon evaporation positioned at a 90º angle to the specimen stage and the other for platinum/carbon shadowing at an adjustable angle, typically 15º - 45º (Figure 2). Power to the unit is applied to operate the vacuum pump and electronic panels regulate temperature adjustment and electron gun control.

Originally conceived as a means to achieve improved imaging of viruses, freeze-fracture gained even more popularity as a technique for the examination and analysis of cell membranes and their specializations^2,3. Indeed, this procedure has been integral to elucidating structure/function relationships in cells and tissues and many of these studies stand as classic contributions to cell and molecular biology^4-9. The major goal and rationale for the development of the freeze-fracture technique was to limit artifacts observable at electron microscopic resolution deriving from chemical fixation and processing used in conventional biological electron microscopy. Here the goal is to limit chemical fixation and to freeze the specimen with sufficient speed and frequently in the presence of a cryoprotectant in order to limit ice crystal formation and other freezing artifacts. More recently, this technique has found a resurgence of interest from molecular biologists and materials science investigators for examination of nanoparticles and nanomaterials.

Freeze-fracture and freeze-etch images exhibit a three-dimensional character and sometimes are mistaken for scanning electron micrographs. However, freeze-fracture preparations are examined by transmission electron microscopy and their major contribution to high resolution morphologic studies is their unique representation of structure/function elements of cell membranes. Freeze-fracture processing is initiated by freezing cells and tissues with sufficient speed to limit ice crystallization and/or with the use of cryoprotectant agents such as glycerol. The specimens are then fractured under vacuum and a replica is generated by evaporation of carbon and platinum over the fractured surface. The original specimen is digested from the replica which is retrieved onto a standard EM specimen grid. Another common misinterpretation of freeze-fracture images is that they depict cell surfaces. However the basic premise of freeze-fracture is that biological membranes are split through the lipid bilayer by the fracture process (Figure 3). This process in biological membranes yields two fracture faces, one which reveals the organization of the half of the membrane adjacent to the cytoplasm, the PF-face, and one which reveals the half of the bimolecular leaflet of the membrane that is adjacent to the extracellular milieu, the EF-face. True cell surfaces are not represented in freeze-fracture images but only appear when the subsequent added step of freeze-etching following the fracture procedure is used. In order to effectively etch previously fractured specimens to reveal surface detail, specimens must be frozen at a rapid rate and without unetchablecryoprotectant. Etching of water from the surface of the fractured specimen revealing underlying features is accomplished by positioning the cooling microtome arm over the specimen stage creating a temperature differential between the stage holding the specimen and the cooling microtome arm which causes water to sublime from the surface. When water is sublimed from the surface of the fractured specimen during the freeze-etching maneuver, then aspects of actual cell surfaces, extracellular matrix, cytoskeletal structures, and molecular assemblies may be revealed at high resolution. Thus freeze-fracture and freeze-etch are not interchangeable terms but rather reflect a step-wise process the latter of which may not be necessary or desirable depending on the needs of the particular study.

Following the freeze-fracture/freeze-etch procedures, the fractured surfaces are subjected to directed evaporative coats of carbon and platinum in order to provide support and imaging contrast to the replica. The platinum/carbon imaging evaporation may be unidirectional or rotary and is accomplished by either resistance or electron guns. Unidirectional shadowing from a known angle, typically 30º - 45º, is useful in performing certain morphometric calculations. Specimens that have been subjected to deep-etching typically are rotary shadowed and the resultant images of these specimens are photographically reversed for evaluation.

The historical as well as a present goal of the freeze-fracture/freeze-etch technique is to limit chemical fixation and processing specimen artifacts that are associated with more conventional transmission electron microscopy procedures. However, this technique provides a substantive advantage in its ability to confer three-dimensional detail and thus facilitate acquisition of morphometric data in biological, material science, and nanotechnology specimens. Freeze-fracture and freeze-etch procedures are complex and multi-faceted and some aspects of its application are customized. This presentation offers a survey view of the major features of the process and the reader is referred to comprehensive published protocols^10,11 in order to address the details and customize the process for specific research needs.

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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% glycerol in 0.1 M phosphate buffer for no more than 1 hr.

2. Freezing and Storage of Specimens In Advance of Freeze-fracture

1. Select gold or copper specimen stubs of appropriate size and shape for the specimen being processed (Figure 4).
2. Using fine grade forceps and/or small gauge syringe needles position small fragments of the specimen on the top of a metal specimen stub.
   1. Reduce the liquid content of the specimen to a sticky, slightly glue like consistency by drawing off liquid from the edge of the stub with filter paper.
   2. For single replicas, use small gauge syringe needles to create a mound of tissue on the stub. For double replicas, invert another stub and position it exactly over the stub and place lightly onto the specimen surface creating a sandwich (Figure 5A). Do not press the specimen out from between the two stubs.
3. Fill two insulated dewar vessels with liquid nitrogen. NOTE: The first vessel accommodates a metal post containing a small well which is used to liquefy a small volume of propane gas from a commercially available cylinder (Figure 5B). The second vessel is a partitioned storage/holding vessel for briefly maintaining frozen specimens under liquid nitrogen.
   1. Using the cylinder nozzle positioned in the propane well, open the cylinder valve and allow gas to flow into the well of the first vessel where it will liquefy. NOTE: CAUTION!! PROPANE IS EXPLOSIVE, MAY CAUSE FREEZING INJURY ON CONTACT, AND IS AN ASPHYXIANT. Use propane only in a chemical hood certified for such use taking care also to avoid extraneous ignition sources, using garments protective against low temperatures, and maintaining adequate ventilation.
   2. As quickly as possible, pick up the specimen mount with fine grade forceps and plunge it into the liquefied gas in the first vessel for several seconds then quickly transfer to the second holding vessel of liquid nitrogen (Figure 5B).
   3. Once the specimens are frozen, store under liquid nitrogen until ready to transfer to the freeze-fracture plant. Transfer the stubs to a larger storage liquid nitrogen Dewar container for extended storage if desired, however care should be taken not to allow the stubs to thaw.

3. Operation of the Freeze-fracture Instrument and Specimen Processing

1. Loading specimens on the brass holder and into the chamber
   1. Load the gold specimen stubs into a booklet-type double replica specimen holder or clamp device under liquid nitrogen and maintain there until ready to position in the specimen chamber (Figure 6).
   2. When the chamber has achieved high vacuum (approximately 2 x 10^-6 mbar) and the stage has been cooled to liquid nitrogen temperature, turn off the pump, vent the chamber, and position the mounted specimen stubs onto the specimen table as quickly as possible.
   3. Turn on the pump, reestablish high vacuum, and use electronic stage and arm temperature controls to adjust the specimen table temperature to -100 °C and direct liquid nitrogen to the microtome arm and bring it to liquid nitrogen temperature.
2. The fracture process
   1. Upon achieving high vacuum, a stage temperature of -100 ºC and microtome arm at liquid nitrogen temperature, remotely open the double replica specimen holder thereby fracturing the specimens on the specimen mounts (freeze-fracture_movie1.mov).
   2. In the case of specimens intended for etching following fracture, use a razor blade maintained in a clamp on the microtome arm to shave (i.e., fracture) the surface of the specimens in lieu of step 3.2.1 (freeze-etch_movie2.mov).
   3. If the added freeze-etching step is to be performed, position the cooled microtome arm over the fractured surfaces for one to several minutes. NOTE: This maneuver will sublime (i.e., etch) water from the fractured surface. The effect of this maneuver is to reveal true cell surfaces, extracellular matrix, and/or molecular assemblies by sublimation of water, in addition to fracture faces passing through the lipid bilayer of membranes.
3. Metal shadowing and replica support evaporation using electron guns
   1. Activate the platinum/carbon electron or resistance gun and allow it to evaporate a thin layer (approximately 2 nm) of platinum/carbon over the fractured surface from an angle of 30º - 45º. This typically requires approximately 15 - 20 sec.
   2. Activate the carbon electron or resistance gun as in step 3.3.1 and evaporate a thin layer of carbon to the fractured specimen surface from directly overhead (90º) to give support to the replica. This typically requires approximately 15 - 20 sec.
4. Retrieval of replicas
   1. Upon completion of replication shadowing and carbon stabilization, turn off the vacuum pump, vent the chamber, and remove the specimen mount from the instrument.
   2. Using a pair of fine grade forceps remove each gold specimen stub from the double replica booklet/clamp and allow to thaw for several seconds before gently lowering the stub onto a water surface in a spot plate (Figure 7). NOTE: The carbon/platinum replica containing adherent specimen material will float onto the water surface.
   3. Maneuver the replicas using either a fine wire loop or a conventional copper electron microscope grid held by fine grade forceps and transfer to another spot plate containing 5% sodium dichromate in 50% sulfuric acid for one to several hours. NOTE: This bath digests the actual biological specimen material from the replica. Full strength household bleach may be substituted for the dichromate/sulfuric acid solution.
   4. When the digestion is complete, transfer the replica back to a clean water surface and retrieve onto a standard copper electron microscopy grid. (NOTE: Replicas may fragment into smaller pieces during digestion but even very small replicas may contain substantial information and should be retrieved and examined.) Store replica supporting grids in commercially available grid boxes where they will remain stable for years with gentle handling.

4. Ultrastructural Examination of Freeze-fracture/etch Replicas

1. View replicas in a transmission electron microscope at an accelerating voltage typically from 50 - 80 kV.
2. Record relevant images on standard TEM film or with a high resolution digital camera.

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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...

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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...

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Materials

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.