Project Summary/Abstract X-ray microscopy has newly developed into an important new cellular imaging technique for visualizing and measuring intact cells in three dimensions. The development of these systems, enabled by advances in x-ray optic and detector technology, has been primarily driven at synchrotron light source facilities. The first laboratory x-ray microscopes for cellular imaging were introduced by the proposal’s co-PI in 2009 and utilized the absorption contrast of “water window” (285-540eV) x-rays for imaging. The system enabled unstained cellular tomography for the first time in the laboratory. However, the system operational energy is too low for most mammalian cells, which are larger in diameter than eukaryotic (yeast, bacteria, etc.) cells that the water window enables. We propose to develop a new 2.7 keV x-ray laboratory cellular microscope to provide fast (~30 minute) and high contrast complete 3D imaging of unstained cells of up to 80um in diameter and their organelles at 30nm resolution. The system operates using Zernike phase contrast at 2.7 keV energy x-rays, which provides higher contrast than even absorption contrast of water window x-rays. Additionally, the system will enable several major advantages over water window x-ray microscopy, including: much larger cell imaging (80µm vs. 10µm), larger depth- of-field for higher 3D resolution, and practical benefits (more stable x-ray source and larger working distance to incorporate correlative techniques). The microscope uses the company’s patented high brightness x-ray source and proprietary x-ray optic technology. The proposed Phase II 24-month project is to develop a complete prototype 2.7 keV Zernike phase contrast system for cellular imaging and to experimentally verify its performance.