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freeze-etch and freeze-fracture methods for

electron microscopy

Freeze-fracture electron microscopyThe 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 platinumcarbon 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 iscarried 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 microscopictechniques 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 shapedour understanding of the functional morphology of the cell.

History and principles

Freeze-fracture electron microscopy has been firmly established as a major technique in

ultrastructure research for well over 30 years. Although elements of the technique in emergingform can be traced back to the 1950s, it was not until the 1960s that images of such persuasive

beauty were obtained that its potential captured the imagination of the cell biologists of the day.

Initially, interpretative controversies held up the application of freeze fracture but, once these

had been resolved, the technique flourished during the 1970s and 1980s, providing advances inour understanding of the structural organization of membranes and organelles that were

impossible to achieve by conventional thin-section electron microscopy (EM). Currently, arevival of interest has taken place with the development of effective approaches in freeze-fracture cytochemistry, providing new tools to address hitherto unresolved questions in cell

biology.

The critical feature of the freeze-fracture technique on which its success depends is the tendencyof the fracture plane to follow a plane through the central hydrophobic core of frozen

membranes, splitting them into half-membrane leaflets. The resulting en face views of

membranes give spectacular three-dimensional perspectives of cellular organization and details

of membrane structure at macromolecular resolution. Of particular importance is the technique'sability to reveal the distribution and organization of integral membrane proteins as

intramembrane particles in the membrane plane.

Essential methodology of freeze fractureThere are four essential steps in making a freeze-fracture replica: (i) rapid freezing of the

specimen, (ii) fracturing the specimen, (iii) making the replica of the frozen fractured surface by

vacuum-deposition of platinum and carbon and (iv) cleaning the replica to remove all the

biological material. In addition, the freezing step is commonly preceded by pretreatment, and anoptional etching step may be interposed between fracturing and making the replica.

The standard method of rapid freezing is to immerse swiftly a suitably mounted sample into a

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liquid coolant (e.g., subcooled liquid nitrogen). Unfortunately, most biological samples frozen in

this way show ice crystal damage at the ultrastructural level unless they are treated beforehand

with a cryoprotectant. The most commonly used cryoprotectant is glycerol. As exposure toglycerol (or other cryoprotectants) may lead to artifacts in membrane structure, the standard

practice is to carry out chemical fixation with glutaraldehyde first. Resorting to these

pretreatment steps falls short of the ideal of directly freezing cells from the living state, alimitation that has been one of the driving forces in the development of more specialized

ultrarapid freezing techniques.

Fracturing the frozen specimen is usually carried out under vacuum by using a liquid-nitrogen-cooled microtome blade or by breaking the frozen specimen apart in a hinged device. In some

simple versions of the technique, fracturing is carried out with a razor blade under liquid nitrogen

at atmospheric pressure.

Making the replica involves two steps, shadowing and backing. In the standard procedure,oblique, unidirectional shadowing is carried out by evaporating a fine layer of platinumcarbon

onto the specimen; this is followed immediately by a strengthening (backing) layer of electron-

lucent carbon, evaporated from above. The topographical features of the frozen, fractured surface

are thus converted into variations in thickness of the deposited platinum layer of the replica.After the replica has been made, the sample is brought to atmospheric pressure and allowed to

warm to room temperature. The biological material is removed from the replica using sodiumhypochlorite solution, chromic acid or other cleaning agents. After washing in distilled water,

pieces of replica are mounted on grids for examination in the transmission electron microscope.

Freeze etching

The term freeze etching is sometimes used synonymously with freeze fracturing. This usage,which arises for historical reasons, is to be discouraged. Etching is defined as removal of ice

from the surface of the fractured specimen by vacuum sublimation (freeze drying), before

making the replica. In the early days of the technique, it was thought that the planar aspects

viewed in replicas represented true surfaces of membranes, exposed by removal of icehencethe technique was originally designated freeze etching. However, it was subsequently

conclusively demonstrated that the planar membrane views (fracture faces) so characteristic of

the technique are revealed just as effectively when freeze fracturing is carried out in the absenceof etching; moreover, in suitably prepared samples, a narrow margin representing the true

surface of the membrane (alongside the fracture face) is exposed specifically by etching. Thus,

freeze fracture is the preferred term for the technique in its standard form, where the majorfeatures of relief observed in the replica are generated by the splitting of membranes.

The traditional etching step, when executed after fracturing standard glycerol-treated specimens,

does not lower the ice table sufficiently to reveal underlying structural detail because glycerolcannot be removed by vacuum sublimation. Only small ice crystals that have been broken apart

by the fracture plane are removed; etching is halted once the surrounding glycerol eutectic is

reached. Consequently, etching has little effect other than giving the cytoplasmic, nuclear and

extracellular matrix a fine granular texture. In a few specific instances, etching may help revealsome extra detail, but for most glycerol-treated specimens, etching is redundant. When applied to

non-glycerol-treated specimens, however, etching (or freeze-drying) is a valuable method in its

own right for exposing the natural surfaces of membranes or other cellular components, and it isto such applications that the term etching should be confined. To be effective in this mode, the

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etching period is typically longer than that traditionally applied, and the specimens are frozen

directly in distilled water, dilute buffer or buffer mixed with methanol