Cell membranes consist of phospholipids and attached or embedded proteins. Membrane proteins play vital roles in the metabolism and life of the cell. You cannot use ordinary microscopy to visualize or characterize adhesion proteins, transport proteins and protein channels in the cell membrane. Using electron microscopy and a technique called "freeze fracture," which splits frozen cell membranes apart, allows visualization of the membrane structure and the organization of proteins within the sea of phospholipids. Combining other methods with freeze fracturing not only helps us to understand the structure of different cell membranes and membrane proteins, but allows for the visualization and detailed analysis of the function of specific proteins, bacteria and viruses.
Basic Steps in Freeze Fracture
Using liquid nitrogen, biological tissue samples or cells are rapidly frozen to immobilize cell constituents. Cell membranes are composed of two layers of phospholipids, called a bilayer, where the hydrophobic, or water-hating, lipid tails point to the inside of the membrane and the hydrophilic, or water-loving, ends of the lipid molecule point outward and toward the inside of the cell. The frozen sample is cracked or fractured with a microtome, which is a knife-like instrument for cutting thin tissue slices. This causes the cell membrane to split apart precisely between the two layers because the attraction between the hydrophobic lipid tails represents the weakest point. Following fracturing, the sample undergoes a vacuum procedure, called "freeze etching." The surface of the fractured sample is shadowed with carbon and platinum vapor to make a stable replica, which follows the contours of the fracture plane. Acid is used to digest organic material adhering to the replica, leaving a thin platinum shell of the fractured membrane surface. This shell is then analyzed by electron microscopy.
Freeze etching is the vacuum-drying of an unfixed, frozen and freeze-fractured biological sample. The vacuum-drying procedure is similar to freeze drying fruits and vegetables that are packaged and sold at grocery stores. Without freeze etching many details of cellular structure are obscured by ice crystals. The deep- or freeze-etching step improves and extends the original freeze fracture method, allowing the observation of cell membranes during various activities. It allows for the analysis of not only the membrane structure, but also of intracellular components and provides detailed structural information on bacteria, viruses and large cellular protein complexes.
Electron microscopy can reveal and magnify more than a million times the tiniest organisms or structures, such as bacteria, viruses, intracellular components and even proteins. Visualization is created by bombarding an ultra-thin sample with a beam of electrons. The two electron microscopy methods are scanning electron microscopy, or SEM, and transmission electron microscopy, or TEM. Freeze fracture samples are routinely analyzed with TEM. TEM has better resolution than SEM and offers structural information down to 3 nanometers of replicas.
Revealing Cell Membrane Structure
The development and use of freeze fracture electron microscopy showed that cell plasma membranes are made up of lipid bilayers and clarified how proteins are organized within cell membranes. Freeze fracture gives a unique look at the interior of cell membranes, because it splits and separates membrane phospholipids into two opposite and complementary sheets or faces. In the more than 50 years since the introduction of the first freeze fracture machine, making a platinum replica is still the only way to obtain structural information about the cell membrane. The technique shows whether specific proteins float or are anchored in the cell membrane, and whether and how some proteins aggregate. A newer method -- using antibodies that target specific proteins -- is combined with freeze fracture to identify proteins and their function in the cell membrane.
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