freeze-etch and freeze-fracture methods for

electron microscopy
Freeze-fracture electron microscopy
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.

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 emerging
form 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 in
our understanding of the structural organization of membranes and organelles that were
impossible to achieve by conventional thin-section electron microscopy (EM). Currently, a
revival 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 tendency
of 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's
ability to reveal the distribution and organization of integral membrane proteins as
intramembrane particles in the membrane plane.

Essential methodology of freeze fracture

There 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 an
optional 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
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 to
glycerol (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, a
limitation 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 platinum–carbon
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 sodium
hypochlorite 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 ice—hence
the 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 absence
of 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 major
features 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 glycerol
cannot 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 reveal
some 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 is
to such applications that the term “etching” should be confined. To be effective in this mode, the
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