Development of Cryogenic X-ray Microscope using for Imaging Biological Specimens with 25nm Resolution in 3D

With conventional oil and gas resources expected to be fully depleted within ~50 years, investment into renewable energy sources such as biofuels are critical. Optimizing biofuel production efficiency is key, and current approaches include identifying or engineering plants and microbes e.g. yeast, bacteria, etc.) that convert the sugars of the starting plant material to usable biofuel alcohols. Cellular structures are a key biophysical measurement that must be made of plants and microbes, as ultrastructure can influence enzyme function and energy conversion pathways and directly impacts usable-vs-unfavorable biomass content. The proposed cryogenic x-ray microscope will provide fast and high contrast complete three-dimensional imaging of cryogenically preserved cells that are hydrated and unstained cells to enable accurate quantitative measurement of cellular structures. Unlike conventional approaches, the x-ray microscope will use 2.7 keV energy x-rays and image based on phase contrast of x-rays rather than relying on the absorption of x-rays. The proposed system will enable volumetric measurements of subcellular components of plant cells and microbes to directly link such features as organelle sizes, cell wall thickness, number of mitochondria, etc. to biofuel production efficiency and thereby guide bioengineering or selection of plants and cells used in the process. This proposed microscope will employ the company’s patented high brightness x-ray source and proprietary x-ray optic technology. The goal of Phase I is to validate a proposed approach to enable imaging cryogenically preserved samples in the system. Cryogenic capabilities are important to ensure the sample is representative of its biological state and is not radiation damaged required for high resolution imaging of cells). The approach will adapt, for x-ray microscopy, the successful protein crystallography approach of using a cryostream to maintain a cryogenic sample imaging environment after the sample is vitrified and transferred. During Phase I, we will characterize the impact of a cryostream on vibration and drift during imaging and verify our hypothesis that appropriate mechanical design with rigid sample mounting and optimized placement of the nitrogen gas flow can still enable high resolution cellular tomography in a non-vacuum environment. The final Phase II x-ray microscope prototype will be based on the mechanical design developed during Phase I. Not only could this x-ray microscope become an essential characterization tool in biofuel research, but its resolution and high contrast for soft materials will be extremely valuable in mammalian cellular research and for industrial polymeric blend and composite research. The system will have broad applicability to pharmaceutical, basic biological e.g. cellular division, pathology, etc.), and polymer research because of its high resolution, speed, and ability to measure organic/polymeric materials without requiring staining, which is commonly required by electron microscopy and can result in artifacts or modification from its native state.