Cryo-TEM - Molecules frozen in time

Since most biological cells are about 70% water, they require some method of preservation that will counteract the effects of the electron beam and the high vacuum environment of the electron microscope. In contrast to older methods for preserving structures for electron microscopy, rapid freezing of fully hydrated whole cells, macromolecular complexes or single macromolecules allows the structural details to be captured in their essentially native state to near atomic resolution.

The 1950’s marked the early development of chemical fixation, dehydration and embedding protocols for biological specimens. In order for these bulk specimens to be transparent to the electron beam, it was necessary to slice thin sections of the embedded blocks of tissues using an ultramicrotome. Staining the thin sections with heavy metal stains provided differential contrast in the electron microscope due to elastic scatting of the electron beam as it passed through the section, resulting in sub-cellular image detail at magnifications not previously afforded by light microscopic methods. The advent of negative staining techniques in the 1960’s allowed very tiny specimens, such as viruses and isolated sub-cellular macromolecules, to be fixed and differentially stained by placing a small drop of the sample in an aqueous suspension onto a suitable substrate, such as a carbon reinforced plastic film mounted to a copper EM grid. Specimens prepared by these methods were dry by the time they were placed into the electron microscope for viewing and image recording, thereby overcoming limitations imposed by the high vacuum of the microscope. Heavy metals, which are bound to the cellular architecture during and after fixation and embedding and which provide contrast, are relatively impervious to the radiation damage from a high dose electron beam. Such specimens can be examined at leisure with less concern about damage from exposure to the beam.

The main disadvantage of classical techniques used in specimen preparations for electron microscopy is that chemical fixation does not perfectly capture the native structure of the once hydrated sample. The 1980’s marked a major milestone in the development of techniques that preserve the native structure of the specimen. These new techniques involved changing the embedding medium of the specimen and then viewing and recording images at very low temperature using low electron dose imaging techniques to protect the specimen from excessive damage from the electron beam. This technique was called cryo-electron microscopy.

In the case of 2D biological crystals, it was discovered that high resolution detail could be preserved by the use of very high concentrations of sugar solutions (such as sucrose, tannin and trehalose) to protect the native structure of the specimen. Since the crystals were small and thin enough to be transparent to the electron beam, a small drop of the crystal/sugar suspension could be directly applied to the surface of, for example, a continuous carbon foil mounted to a copper EM grid. The grid could then be gently blotted to remove excess fluid and then transported to the electron microscope using a cryo-transfer holder and cooled to approximately -180 °C for viewing and recording images or diffraction patterns of the native crystalline structure.

Other specimens, such as a solution of biological macromolecules, could be prepared by directly applying a small drop of the solution directly onto the surface of a suitable substrate, such as a holey carbon foil on a copper EM grid. The grid could then be quickly blotted with filter paper to produce a thin fluid layer (~100 nm.) With the aid of a homemade plunging device, the grid was then plunged into a small container of a liquid cryogen (ethane) that was cooled to just above the freezing point (-196 °C) using liquid nitrogen. The ability of the ethane to freeze the specimen almost instantaneously prevented the formation cubic ice crystals. This resulted in the specimen being embedded in a thin layer of glassy (vitreous) ice. As long as the specimen grid was maintained at a temperature below the devitrification point of about -150 °C for all subsequent steps, including recording the data in the electron microscope using low electron dose imaging techniques, its native structure remained intact and high resolution images of the structure could be recorded.

Every technique has its drawbacks and cryo-electron microscopy is no exception. One drawback is that the density of the specimens being viewed are very close to the density of the embedding medium (sugar solution or vitreous ice), and the resulting images are inherently low in contrast. Due to the inherently poor signal to noise ratio produced by these types of specimens, defocus phase contrast is used to enhance the signal. In addition, working at very low temperatures is more demanding than working with room temperature specimens so special precautions have to be followed to ensure success.

The technique of preparing specimens in this fashion was once only practiced by a small group of laboratories worldwide with the ultimate goal of achieving near atomic resolution for a wide variety of biological macromolecular assemblies, single particles and 2D crystalline structures. The past 25 years have seen a steady progression from a variety of ‘home made’ cryo-plunging devices, to state of the art commercially available instruments that provide consistent results through automation and climate control for the specimen prior to the freezing process. In addition, the manual beam blanking methods originally used for producing a low electron dose image of the specimen on photographic film has now given way to sophisticated low dose software programs for controlling the electron microscope, which can be done in a totally automated fashion from a remote location on high sensitivity, large format CCD and direct detection cameras. In some instances, data sets that used to take months or even years to collect can now be done in a fully automated fashion in a matter of hours, and a technique that was once limited to the masters is now accessible to all.

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