SBIR-STTR Award

The objective of our application is to develop hardware and procedures that will allow researchers to practically utilize phase plates in the back focal plane of the objective lens in a Transmission Electron Microscope. Use of precisely positioned phase plates can increase resolution in electron tomography, and also increase throughput when using the single-particle approach. Our specific aim is to provide a phase plate system that will include a modified objective aperture holder that incorporates (1) a piezo positioning system, (2) a heater to avoid contamination, and (3) an electrical feed-through for use with electrostatic phase plates. Simply exchanging the original objective aperture holder with the Hummingbird phase plate holder will allow nearly any cryo-EM to be equipped with a phase plate. The cost of this holder will be substantially lower than any other technology that can yield a similar improvement in imaging of frozen-hydrated specimens. In close collaboration with the Resource for the Visualization of Biological Complexity (RVBC) at the Wadsworth Center's NIH Biotechnological Resource, we will develop a new phase plate holder, demonstrate its use, and publish the results. It is our intent to develop and commercialize a cost effective system that will enable these techniques to become widely available to the biology research community. This proposal will develop tools that allow biologists to get a clearer picture of the structure and function of the cell. This knowledge is fundamental to innumerable areas of medical research and practice.

Public Health Relevance Statement:


Project narrative:
This proposal will develop tools that allow biologists to get a clearer picture of the structure and function of the cell. This knowledge is fundamental to innumerable areas of medical research and practice.

Project Terms:
3-D structure; 3-dimensional structure; 3D structure; Area; Back; Biological; Biology; Cell Function; Cell Process; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Chemicals; Collaborations; Collection; Communities; Computer Programs; Computer software; Computers; Data Collection; Dehydration; Devices; Dorsum; Electron Microscope; Electron Microscopy; Electronics; Electrons; Electrostatics; Elements; Fixation; Freezing; Heating; Image; Imagery; In Situ; Investigators; Knowledge; Manuals; Medical Research; Method LOINC Axis 6; Methodology; Methods and Techniques; Methods, Other; NIH; National Institutes of Health; National Institutes of Health (U.S.); Negative Beta Particle; Negatrons; Optics; Organelles; Phase; Position; Positioning Attribute; Procedures; Production; Publishing; Research; Research Personnel; Research Resources; Research Specimen; Researchers; Resolution; Resources; SBIR; SBIRS (R43/44); Sampling; Series; Small Business Innovation Research; Small Business Innovation Research Grant; Software; Specimen; Staining method; Stainings; Stains; Structure; Subcellular Process; System; System, LOINC Axis 4; TEM; Techniques; Technology; Time; Transmission; Transmission Electron Microscopy; United States National Institutes of Health; Visualization; Work; Writing; abstracting; base; body water dehydration; computer program/software; cost; cost effective; electron tomography; experience; experiment; experimental research; experimental study; feeding; frontier; imaging; irradiation; lens; macromolecule; particle; product development; prototype; research study; sample fixation; three dimensional structure; tomography; tool; transmission process

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.

Public Health Relevance:
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.