Project Summary/Abstract The National Center for X-ray Tomography (NCXT) provides biomedical researchers with access to unique technologies for visualizing and quantifying intact, fully hydrated cells in 3D. The NCXT designed, built, and now operates a soft x-ray microscope (XM-2) at the Advanced Light Source, Berkeley. XM-2 is currently the only microscope of its kind in the Nation. The unique characteristics of soft x-rays make them an excellent illumination source in a microscope designed to images cells. Soft x-rays penetrate large cells easily, allowing researchers to image mammalian cells in great detail. Moreover, soft x-rays produce image contrast without the need to use stains or other contrast agents; procedures that could damage the delicate sub-cellular structures and organization. XM-2 is equipped with a cryogenic specimen stage that can rotate freely through 360 degrees, allowing the specimen to be imaged from different perspectives. The specimen stage allows researchers to image cells by tomographic methods. In tomography 2D images are taken from many different perspectives. The resultant `stack' of projection images is used to calculate a 3D reconstruction of the specimen. In effect, we following the same principles as used in clinical Computed Tomography (CT), but on individual cells rather than a whole body. This funding will support the day-to-day maintenance, upgrades, and user operation of XM-2, together with associated specimen preparation laboratories and ongoing upgrades to our software/computing resources. This support will ensure biomedical researchers have access to a unique cell imaging technology. During the funding cycle, we will achieve the following specific aims: Aim 1: Enable user access to biological soft x-ray tomography (SXT) at the ALS. The work of this aim will ensure users can bring cells to NCXT and leave with soft x-ray tomographic reconstructions of their specimens. Significant consideration will be striving to make the Resource as user-friendly as possible, and therefore accessible to the broadest possible research community as possible, not just to imaging specialists. Aim 2: Maintain and calibrate XM-2, the NCXT operated soft x-ray microscope at the ALS. As with all synchrotron beamlines, XM-2 is a sophisticated instrument that requires regular maintenance and calibration. We will take a programmatic approach to ensure XM-2 is robust, reliable, and operates with minimum unscheduled downtime. Aim 3: Implement upgrades and enhancements to ensure the NCXT remains `state-of-the-art.' Over the timescale of the funding cycles, new hardware, software, and algorithms will become available, and some existing components will reach their end-of-life. We will, therefore, carry out upgrades and enhancements to maintain the NCXT at the `state-of-the-art.'