10..Freeze fracture, etching of techniques

freeze fracture

1. Freeze Fracture Technique

2. •Freeze fracture replication technique is used to study the ultrastructure of frozen
cells.
•Small pieces of tissue to be freeze fractured are placed on a small metal disk and
rapidly frozen by first impregnating with glycerol and then freezing in liquid freaon
(-130 ºC) or in liquid nitrogen (-196 ºC).
•The disk is then mounted on a cooled stage within a vacuum chamber, and the
frozen tissue block is struck by a knife edge (also maintained at about 100 ºC
using liquid nitrogen ).
•The resulting fracture plane spreads out from the point of contact, splitting the
tissue into two pieces, not unlike the way that an axe blade splits a piece of wood
in two.
•A fracture plane spreads through a cell containing a variety of organelles of
different composition. These structures tend to cause deviations in the fracture
plane, either upward or downward, giving the fracture face elevations,
depressions, and ridges that reflect the contours of the protoplasm traversed.
•The surfaces exposed by the fracture contain information about the contents of
the cell.
•To make this information visible, the fractured surface is used as a template on
which a heavy-metal layer is deposited.
•The heavy metal is deposited onto the newly exposed surface of the frozen tissue
in the same chamber where the tissue was fractured.
•The metal is deposited at an angle to provide shadows that accentuate local
topography.

3. •A carbon layer is then deposited on top of the metal layer to cement the patches
of metal into a solid surface.
•Once this cast has been made, the tissue that provided the template can be
thawed, removed, and discarded.
•The metal–carbon replica is placed on the specimen grid and viewed in the
electron microscope.
•Variations in thickness of the metal in different parts of the replica cause
variations in the numbers of penetrating electrons to reach the viewing screen,
producing the necessary contrast in the image.
•The fractured planes take the path of least resistance through the frozen block,
which often carries them through the center of cellular membranes.

Significance
•This technique is particularly well suited for examining the distribution of integral
membrane proteins as they span the lipid bilayer. Such studies carried out by
Daniel Branton and others played an important role in the formulation of the fluid
mosaic structure of cellular membranes in the early 1970s.

•Allows an investigation of the micro-heterogenity of the membrane.

•Localized differences in parts of the membrane stand out in these replicas and
can be identified.

4. Freeze Etching technique
The Freeze-etching technique. In steps(a) and (b), a frozen eucaryotic cell is fractured with
a cold knife. Etching by sublimation is depicted in (c). Shadowing with platinum plus carbon
and replica formation are shown in (d) and (e).

6. •It is a variation of freeze fracture technique which allows visualization of the external
surface of cell membrane in addition to their interior faces.
•In freeze etching the tissue is frozen and fractured while it is still in place within the
cold chamber.
•The tissue is then exposed to a vacuum at an elevated temperature for one to a few
minutes, during which a layer of ice can evaporate (sublime) from the exposed
surface.
•Once some of the ice has been removed, the surface of the structure can be coated
with heavy metal and carbon to create a metallic replica that reveals both the
external surface and internal structure of cellular membranes.
•After the specimen has been removed chemically, this replica is studied in TEM and
provides a detailed three dimensional view of intracellular structure.
•An advantage of freeze-etching is that it minimises the danger of artifacts because
the cells are frozen quickly rather than being subjected to chemical fixation,
dehydration and plastic embedding.

7. •The technique delivers very high resolution and can be used to reveal the
structure and the distribution of macromolecular complexes, such as those of
the cytoskeleton.
•The TEM discloses the shape of organelles within microorganisms if specimens
are prepared by freeze-etching procedure.

8. Negative Staining
•The given micrograph shows bacteriophage visualized by negative staining.
•Negative staining is useful for the visualization of intact biological structures
such as viruses, ribosomes, multisubunit enzymes, cytoskeletal elements, and
protein complexes.
•The shapes of individual proteins and nucleic acids can also be resolved as
long as they are made to have sufficient contrast from their surroundings.

9. •In this method, the biological specimen is deposited on a supporting film and a
heavy metal stain is allowed to dry around its surface.
•The unstained sample (small particles such as viruses or macromolecules) is
surrounded by an electron dense material, such as phosphotungstic acid.
•Phosphotungistic acid permeates the open superficial interstices of sample.
•This produces an image in which The sample appears light (i.e. electron
transparent) that are high lightened by the surrounding dark background.
•Thus, in this technique the background is stained, leaving the actual specimen
untouched.

10. Positive Staining
•In positive staining, tissue specimens are cut into
thin sections and stained with heavy metal salts
(osmium tetroxide, uranyl acetate, lead citrate)
that reacts with lipids, proteins and nucleic acids.
•These heavy metal ions bind to a variety of cell
structures which consequently appear dark in
final image.
•Alternating positive staining procedures can also
be used to identify specific macromolecules
within the cells. For e.g. antibodies labeled with
electron dense heavy metals (gold particles) are
frequently used to determine the subcellular
location of specific proteins under the electron
microscope.