HIGH PRESSURE CRYOCOOLING FOR BIOLOGICAL X-RAY DIFFRACTION MICROSCOPY

Cornell University, Ithaca NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have developed cryogenic x-ray diffraction microscopy (cryo-XDM) for imaging hydrated, unstained biological samples and recently reported the first frozen-hydrated images of D. radiodurans with 8 keV x-rays at a resolution of 30 to 50 nm. Having shown the feasability of cryo-XDM in biological imaging, we hope to improve the resolution toward 10 nm to visualize fine cellular structures, possibly at the molecular level. So far, one of the main challenges in reaching this goal has been the ice condition. During the plunge-freezing stage, samples can become contaminated with crystalline ice which can result in increased noise levels preventing the phasing algorithm from finding a solution. To solve this problem we plan to utilize high-pressure freezing samples to preserve them in the amorphous ice state in collaboration with Dr. S. Gruner and Dr. C.U. Kim at Cornell. We request 1 day beamtime to test the feasibility of applying high-pressure freezing for biological XDM.