PNNL

Abstract

Here we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal sub-cellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 micrometers thick. Structures as small as 90 nm can be detected in tomographic reconstructions at roughly 70 nm spatial resolution following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft x-ray microscopy and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft x-ray microscope will therefore enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.