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Laboratory-Based Cryogenic Soft X-Ray Tomography with Correlative Cryo-Light and Electron Microscopy

Published online by Cambridge University Press:  18 January 2013

David B. Carlson ,
Jeff Gelb ,
Vadim Palshin  and
James E. Evans
David B. Carlson
Affiliation:
University of California, Davis, Department of Molecular and Cellular Biology, 1 Shields Avenue, Davis, CA 95616, USA
Jeff Gelb
Affiliation:
Xradia, Inc., 4385 Hopyard Road, Suite 100, Pleasanton, CA 94588, USA
Vadim Palshin
Affiliation:
Xradia, Inc., 4385 Hopyard Road, Suite 100, Pleasanton, CA 94588, USA
James E. Evans*
Affiliation:
University of California, Davis, Department of Molecular and Cellular Biology, 1 Shields Avenue, Davis, CA 95616, USA
*
*Corresponding author. E-mail: [email protected]
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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 subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 μm thick. Structures as small as 90 nm can be detected in tomographic reconstructions 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 enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

Keywords

compact light sourcelaboratory light sourceX-ray tomographycorrelative microscopycryogenic soft X-ray microscope
Type
Software, Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 22 - 29
Copyright
Copyright © Microscopy Society of America 2013

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Footnotes

†

Current Address: Pacific Northwest National Laboratory, Environmental Molecular Sciences Laboratory, 3335 Q Avenue, Richland, WA 99354, USA

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