Project Summary X-ray fluorescence microscopes (XFM) offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential information for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With NIH support, we have developed approaches to rapidly freeze cells for the best preservation of trace metal content as well as cellular ultrastructure, and have shown that cryo x-ray fluorescence microscopy can be used to effectively mitigate radiation damage limitations in the Bionanoprobe, an instrument operated at the Advanced Photon Source (APS) at Argonne and open to researchers based on peer-reviewed, no-cost beamtime proposals. In order to complement XFM’s ability to quantitatively image trace element distributions, we have developed high throughput x-ray ptychography (a scanned coherent beam imaging method) to go beyond lens limits and simultaneously obtain 18 nm resolution images of frozen hydrated eukaryotic cells, complementing XFM by providing a high resolution view of cellular ultrastructure. We propose here to develop and validate cryo confocal light microscopy of Bionanoprobe-mounted samples to complement XFM with the capability to image selectively labeled proteins, and to move ptychography from 2D to 3D imaging. To validate these approaches and work from the beginning on a crucial biomedical research project, we will do this in the context of ongoing research in the use of DNA-conjugated nanoparticles containing titanium and/or gadolinium that are meant to target mitochondria for the treatment and imaging of prostrate, breast, and other cancers. In this way, we will develop the methods needed to fully realize the investment NIH has already made in the Bionanoprobe, and build upon Argonne’s investment in increasing its available access time at a new experimental station at the APS.