Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

The aim of the following experiment is to generate a carbon platinum specimen replica of biological or other materials for examination by transmission electron microscopy. To achieve this specimens that are prepared as double and single replicas are subjected to ultra rapid freezing rates to limit ice crystal formation. Then the frozen specimen is fractured by remote manipulation at liquid nitrogen called temperatures under high vacuum, which splits the membrane through the lipid bilayer, producing an extracellular face and a face that is proximal to the cytoplasmic aspect.

As an optional additional step. Underlying features can be revealed when water is etched from the surface of the fractured specimen by positioning the cooling microtome arm over the specimen stage to create a temperature differential, which causes water to sublime from the surface. Next, 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 transmission electron microscopy specimen grid. Ultimately freeze fracture freeze etch is used to show three dimensional structural detail of cell membranes, molecular arrays, and nanomaterials upon examination by transmission electron microscopy. The main advantage of this technique over existing methods like examination of ultra thin sections is that objects of interest can be viewed in three dimensions at electron microscopic resolution.

This method can help answer key questions in the cell and molecular biology and nanotechnology field, such as the dimensional and structural characteristics of cell membranes and the organization of molecular and nanomaterial arrays. I first had the idea for this method when I recognized that similar studies had been performed on eukaryotic protests with important discoveries, but that there had been limited attention given to relevant themes in human health. Begin this procedure with preparation of biological specimens for freeze fracture freeze edge, as described in the text protocol.

Select gold or copper specimen stubs of appropriate size and shape for the specimen being processed using fine grade forceps and or small gauge syringe needles. Position small fragments of the specimen on the top of a metal specimen stub. 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 for single replicas.

Use small gauge syringe needles to create a mound of tissue on the stub for double replicas. Invert another stub, position it exactly over the stub and place it lightly onto the specimen surface. Creating a sandwich.

Do not press the specimen out from between the two stubs. Next, fill two insulated duo vessels with liquid nitrogen. 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.

The second vessel is a partition storage holding vessel for briefly maintaining frozen specimens under liquid nitrogen. Use propane only in a chemical hood certified for such use. Take care to avoid extraneous ignition sources to use garments protective against lower temperatures and to maintain adequate ventilation as propane is explosive.

May cause freezing injury on contact and is an asphyxiant. 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 as quickly as possible. Pick up the specimen mount with fine grade forceps and plunge it into the liquified gas in the first vessel for several seconds.

Then quickly transfer to the second holding vessel of liquid nitrogen. Once the specimens are frozen, store under liquid nitrogen until ready to transfer to the freeze fracture plant. 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.

When the chamber has achieved high vacuum and the stage has been called to liquid nitrogen temperature, turn off the pump, bent the chamber and position the mounted specimen stubs onto the specimen table as quickly as possible. Then turn on the pump reestablish high vacuum and use the electronic stage and arm temperature controls to adjust the specimen table temperature to negative 100 degrees Celsius. Also direct liquid nitrogen to the microtome arm to bring it to liquid nitrogen temperature.

Upon achieving high vacuum, a stage temperature of negative 100 degrees Celsius and the microtome arm at liquid nitrogen temperature remotely open the double replica specimen holder, thereby fracturing the specimens on the specimen mounts As an optional additional step. Specimens can be etched to reveal underlying features following fracture To achieve etching use a razor blade maintained in a clamp on the microtome arm to shave the surface of the specimens. Position the cool microtome arm over the fractured surfaces for one to several minutes to sublimate the water in order to reveal true cell surfaces, extracellular matrix, and or molecular assemblies.

Next, activate the platinum carbon electron or resistance gun and allow it to evaporate a thin layer of platinum carbon over the fractured surface from an angle of 30 degrees to 45 degrees. This typically requires approximately 15 to 20 seconds, activate the carbon electron or resistance gun in the same manner, and evaporate a thin layer of carbon to the fractured specimen surface from directly overhead at 90 degrees to give support to the replica. This also typically requires approximately 15 to 20 seconds upon completion of replication, shadowing, and carbon stabilization.

Turn off the vacuum pump and the chamber and remove the specimen mount from the instrument 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. 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. This bath digests the actual biological specimen material from the replica.

When the digestion is complete, transfer the replica back to a clean water surface and retrieve onto a standard copper electron microscopy grid. Store replica supporting grids in commercially available grid boxes where they will remain stable for years. With gentle handling or ultra structural examination of freeze fracture, etch replicas, view replicas in a transmission electron microscope at an accelerating voltage, typically from 50 to 80 kilovolts.

Record the relevant images on standard TEM film or with a high resolution digital camera residing in the cell membrane at the base of each eukaryotic. Cilium and flagellum is an array of membrane associated particles organized into strands ENC circling the shaft of the cilium and known as the ciliary necklace. Specialized membrane particle arrays appear on the luminal border of epithelial cells undergoing Clio Genesis.

These particle arrays represent nascent ciliary necklaces, which reside at the basis of emergent and mature Celia. In various epithelial freeze fracture studies reveal a sophisticated organization of anastomosing strand and groove structures circling the apical aspect of the basolateral borders. Fracture through epithelial cell membranes reveals plasma fracture faces of tight junctions that appear as strands while the extracellular fracture faces exhibit complementary grooves.

Gap junctions appear in freeze fracture preparations of a variety of tissues and are represented by dense arrays of particles on plasma fracture faces and complimentary pits on extracellular fracture. Faces shown here as an example of rapid freezing with deep etching and rotary shadowing. The epithelial cell has been fractured through the cytoplasm and a plasma fracture face is evident behind the arrowheads can be seen.

A true cell surface revealed by subsequent etching Once mastered. This technique can be done in a cycle lasting approximately three hours and yielding one to three specimens if it is performed properly. While attempting this procedure, it's important to remember that the individual steps are very interdependent and that a mistake in one step can render the outcome inadequate for examination and evaluation after its development.

This technique paved the way for researchers in the field of cell and molecular biology to explore how genetic mutations confer structural anomalies in cells, in ways that provoke pathological conditions and disease.