At the frontier of biological electron microscopy, there is a strong emphasis on imaging the 3-D structure of cells, organelles, and macromolecules in their "native" state. This is facilitated by the use of frozen-hydrated specimens which have not been subjected to chemical fixation, dehydration or stains. The resolution is currently limited to about 4 nm by electron irradiation damage. The use of in-focus phase contrast imaging in the transmission electron microscope (TEM) - accomplished by incorporating a phase plate in the back focal plane of the objective lens - can increase resolution in electron tomography, and also increase throughput. Although these improvements have recently been confirmed by preliminary experiments, and the benefits of phase plates have been known for decades, technical difficulties have always inhibited their use. The three main difficulties are: keeping the phase plate centered on the electron optical axis, avoiding contamination and allowing easy phase plate replacement in the microscope. In Phase I we have developed and tested a working prototype of a phase plate holder with precision positioning system, a heater and an electrical feed-through for use with electrostatic phase plates. Exchanging the original objective aperture holder of the TEM with the phase plate holder allows nearly any cryo-EM to be equipped with a phase plate. The cost of this holder is substantially lower than any other technology that can yield a similar improvement in imaging of frozen- hydrated specimens. In Phase II we will refine this concept into a commercial product and add auto-centering software as well as a load-lock to the holder, allowing replacement of the phase plates without breaking the vacuum in the microscope column. This latter addition will eliminate the need to break the vacuum to insert the phase plate, and makes this product into a complete solution to all technical issues that have traditionally prevented scientists from more routinely using this characterization technique.
Phase plate holder for transmission electron microscopy Relevance The phase plate holder that will be developed and commercialized in this project allows routine transmission electron microscopy characterization of biological structures at unprecedented resolution and contrast levels. The routine use of phase plates will allow the acquisition of levels of details in 3D tomographic images of non-crystalline materials to increase by a large leap. This TEM imaging ability is used to gain a comprehensive understanding of cellular structures and allows direct correlation between structure and function.