X-ray protein crystallography is currently the primary methodology used for determining the 3D structure of protein molecules at near-atomic or atomic resolution. However, many biological samples such as whole cells, cellular organelles and other important protein molecules are difficult or impossible to crystallize and hence their structures are not accessible by crystallography. Overcoming these limitations requires employment of different techniques and methods such as nuclear magnetic resonance and cryo-electron microscopy. A very promising approach currently under rapid development is X-ray diffraction microscopy (i.e. X-ray crystallography without crystals) in which the X-ray diffraction pattern of a non-crystalline specimen is measured and then directly phased by an iterative algorithm. Since its first experimental demonstration in 1999, X-ray diffraction microscopy has been successfully applied to 2D and 3D imaging of non-crystalline specimens such as whole cells. The highest resolution achieved thus far is 7 nm, while the ultimate resolution is limited by the X-ray wavelengths and radiation damage to the specimens. Due to its applicability to thick specimens and its high-resolution imaging capability, X-ray diffraction microscopy can be used to bridge the gap between light and electron microscopy and provide quantitative 3D structural information of whole cells at 10 nm resolution. During the next 4 years of this project, we propose to 1) minimize the mean phase error (i.e. the image ambiguity) in phasing the 3D X-ray diffraction patterns of whole cells;2) experimentally study the ultimate 3D resolution attainable from frozen-hydrated whole cells affected by radiation damage;3) test the hypothesis that X-ray diffraction microscopy can image the 3D intracellular structure of frozen-hydrated cells at a resolution of 10 nm;and 4) localize specific multiprotein complexes in Caulobacter and yeast. We are confident that our multidisciplinary team with expertise in physics, biology and instrumentation will help to more rapidly validate this novel imaging technique for quantitative 3D imaging of the intracellular structure at 10 nm resolution and the localization of multiprotein complexes inside whole cells. Cryo X-ray diffraction microscopy will be demonstrated to quantitatively image the 3D intracellular structure of frozen-hydrated yeast cells to a resolution of 10 nm. This novel imaging technique will be applied to the localization of multiprotein complexes inside whole cells.