Home > Nanotechnology Columns > FEI > Vitrified samples preserve structure and context of biomolecular machines
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Marc Storms TEM Product Group FEI Company |
Abstract:
High resolution transmission electron microscopy (TEM) of vitrified samples allows scientists to visualize molecular scale biological structures in pristine condition and fully hydrated native context. It has become a powerful tool for understanding the form and function of the molecular machines that constitute living systems. A new automated tool (Vitrobot™ Mark IV, FEI Company, Hillsboro, OR) addresses the difficulties of manual vitrification, allowing operators at any skill level to prepare vitrified samples quickly and reliably.
July 21st, 2007
Vitrified samples preserve structure and context of biomolecular machines
Vitrified samples preserve structure and context of biomolecular machines
By Marc Storms, FEI Company
High resolution transmission electron microscopy (TEM) of vitrified samples allows scientists to visualize molecular scale biological structures in pristine condition and fully hydrated native context. It has become a powerful tool for understanding the form and function of the molecular machines that constitute living systems. A new automated tool (Vitrobot™ Mark IV, FEI Company, Hillsboro, OR) addresses the difficulties of manual vitrification, allowing operators at any skill level to prepare vitrified samples quickly and reliably.
A TEM focuses high energy electrons that have passed through the sample into a real magnified image that is projected on a phosphor viewing screen or other imaging device. In some samples TEMs can resolve individual atoms, though in most biological materials their resolution is considerably less. Still, they can resolve features of large biological molecules, such as the tertiary and quaternary structure of proteins and protein complexes. This capability has proven to be a powerful complement to the atomic level resolution of such techniques as nuclear magnetic resonance (NMR) and X-ray diffraction (XRD). A primary benefit of TEM is its ability to look at individual molecules in their native context, whereas NMR and XRD can only determine the average characteristics of large collections of presumably identical molecules in highly unnatural context—crystallized or in purified suspension. Advanced TEM imaging techniques, such as electron tomography, can combine multiple two dimensional images to build detailed three-dimensional models of biological structures with resolutions down to a few nanometers.
TEMs require a high vacuum along the beam path—which includes the sample—to prevent scattering of beam electrons by gas molecules. The sample must be thin enough to transmit electrons (100 nm or less) and must be compatible with the vacuum environment—clean, dry and non-volatile. A number of drying and chemical fixing techniques have been developed to prepare biological samples for the vacuum environment of the TEM, but all induce significant structural changes, especially when examined at the molecular level. Freezing techniques avoid the introduction of foreign materials, but the formation of ice crystals can easily destroy delicate biological structures. Vitrification freezes the sample so quickly that water molecules do not have time to crystallize, instead forming a vitreous (amorphous) solid that does little or no damage to the sample structure. The low temperature of the vitrified sample also reduces the damage caused by beam electrons during observations, permitting more or longer exposures at higher beam currents for better quality images.
Vitrified samples are, quite literally, frozen in time, allowing researchers to investigate time based phenomena such as the structural dynamics of flexible proteins or the aggregation and dissociation of protein complexes. By measuring the variability within a set of images, each capturing the shape of a molecule at an instant in time, scientists can calculate the range of motion and the intra molecular forces operating in flexible proteins. Similarly, a collection of images might provide a freeze frame sequence of the assembly of a protein complex or conformational changes during antigen binding . Careful control of the time interval between mixing and vitrification permits investigations of the reaction dynamics of protein interactions and other biological processes.
Vitrified TEM samples are prepared from a water based suspension of the sample material. A TEM grid dipped in the suspension is covered by a thin film. The grid is blotted to remove excess fluid and the remaining film is vitrified by plunging the grid rapidly into super cooled liquid propane or ethane. Although simple in concept, the process has a number of variables that are critical to success and difficult to control precisely during manual execution, including temperature, humidity, number of blotting actions, blotting pressure, and drain time. After freezing, the sample must be carefully handled as it is transferred from the vitrification medium to the cryogenically cooled sample holder of the TEM.
The new tool completely automates the vitrification process and provides precise control of all critical parameters. Small sample volumes can be applied directly to the grid by a pipette through a port in the preparation chamber. The application time and the wait time between application and blotting can be controlled. The system also permits repetitive application and blotting, enabling precise timing in dynamic experiments investigating interactions among separately introduced components. The number of blotting actions and the duration of each action are software controlled. Automated shutter control allows precisely timed injection of the sample grid into the vitrification medium. The coolant container and grid holder are then lowered together to the transfer position where the grid can be safely moved to a transfer box within the nitrogen cooled environment. The careful control provided by the automated tool guarantees fast, reliable, reproducible sample preparation regardless of the skill level of the operator. Routine preparation of vitrified samples will be an important enabler in the continuing effort to understand the molecular mechanisms of biological systems.
About the Author
Marc Storms is a member of FEI's TEM product group where he specializes in life science applications. He can be reached at .
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