Freeze-fracture electron microscopy

Abstract

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum–carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.

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Figure 1: Photograph of the Balzers BAF 400 T machine.
Figure 2: Cutaway diagram of the apparatus for freeze fracture in the vacuum chamber of the Balzers BAF 400T machine.
Figure 3: View of the apparatus in the vacuum chamber with the door opened.
Figure 4: Diagram illustrating the two principal methods for fracturing frozen specimens, knife fracture and tensile fracture.
Figure 5: The principal types of specimen carrier designed for mounting specimens for knife fracture and tensile fracture.
Figure 6: Detachable tables for holding specimen carriers for knife fracture in the Balzers BAF 400.
Figure 7: Detachable cold table for tensile fracture (original type), designed for use with specimen sandwiches mounted between pairs of gold-stub carriers.
Figure 8: Detachable cold table for tensile fracture, with spring-loaded hinged partners, designed for use with specimen sandwiches mounted between pairs of copper hat carriers.
Figure 9: Liquid nitrogen bench dewar with stainless steel basket for holding frozen specimens.
Figure 10: Electron beam evaporation gun (type EK 552) as used in the Balzers BAF 400 machine.
Figure 11: To set up the guns, a filament centering rod is used to position the filament.
Figure 12: Loading gold carrier-mounted frozen specimens onto a standard specimen table.
Figure 13: Loading sandwich-mounted frozen specimens onto a tensile fracture specimen table.
Figure 14: Inserting the loaded specimen table into the vacuum chamber and onto the cold stage.
Figure 15: Replica mounted on an electron microscope grid.
Figure 16: Low-power survey freeze-fracture image of a capillary in heart muscle.
Figure 17: High-magnification freeze-fracture micrograph illustrating plasma membrane structure of adjacent endothelial cells.
Figure 18: Freeze-fracture view of the interior of a Chlorella cell.
Figure 19: Nomenclature for describing the aspects of the plasma membrane revealed by freeze fracture and etching.
Figure 20: Diagram illustrating the three basic fracture paths through frozen spheroidal cells, with corresponding examples from freeze-fractured yeast cells.
Figure 21: Freeze-fracture views of human airway epithelial cells.
Figure 22: Freeze-fracture imaging of the intercalated disk plasma membrane of cardiac muscle cells.
Figure 23: A gap junction, fractured in a similar manner to that in Figure 22, depicted at higher magnification.
Figure 24: Freeze-fracture view of the luminal part of the plasma membrane of rat urinary bladder, seen in E-face view.
Figure 25: E surface of the bladder luminal membrane revealed by deep etching to expose the surface.
Figure 26: Examples of the application of ultrarapid freezing.
Figure 27: Apart from the study of membrane and organelle structure in cells and tissues, freeze fracture has wide application to model systems, for example cell cultures (a), isolated membranes (b) and artificial liposomes (c).

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Correspondence to Nicholas J Severs.

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Severs, N. Freeze-fracture electron microscopy. Nat Protoc 2, 547–576 (2007). https://doi.org/10.1038/nprot.2007.55

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