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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: James.Evans@PNNL.Gov
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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

References

Adam, J.F., Moy, J.P. & Susini, J. (2005). Table-top water window transmission X-ray microscopy: Review of the key issues, and conceptual design of an instrument for biology. Rev Sci Instrum 76(9), 091301. CrossRefGoogle Scholar
Adrian, M., Dubochet, J., Lepault, J. & McDowall, A.W. (1984). Cryo-electron microscopy of viruses. Nature 308(5954), 32–36.CrossRefGoogle ScholarPubMed
Al-Amoudi, A., Norlen, L.P. & Dubochet, J. (2004). Cryo-electron microscopy of vitreous sections of native biological cells and tissues. J Struct Biol 148(1), 131–135.Google Scholar
Baker, L.A. & Rubinstein, J.L. (2010). Radiation damage in electron cryomicroscopy. Methods Enzymol 481, 371–388.CrossRefGoogle ScholarPubMed
Berglund, M., Rymell, L., Peuker, M., Wilhein, T. & Hertz, H.M. (2000). Compact water-window transmission X-ray microscopy. J Microsc 197(3), 268–273.Google Scholar
Bertilson, M., von Hofsten, O., Vogt, U., Holmberg, A. & Hertz, H.M. (2009). High-resolution computed tomography with a compact soft X-ray microscope. Opt Express 17(13), 11057–11065.Google Scholar
Carlson, D.B. & Evans, J.E. (2011). Low cost cryo-light microscopy stage fabrication for correlated light/electron microscopy. J Vis Exp 52, e2909. Google Scholar
Chichon, F.J., Rodriguez, M.J., Pereiro, E., Chiappi, M., Perdiguero, B., Guttmann, P., Werner, S., Rehbein, S., Schneider, G., Esteban, M. & Carrascosa, J.L. (2012). Cryo X-ray nano-tomography of vaccinia virus infected cells. J Struct Biol 177(2), 202–211.Google Scholar
Egerton, R.F., Li, P. & Malac, M. (2004). Radiation damage in the TEM and SEM. Micron 35(6), 399–409.Google Scholar
Hagen, C., Guttmann, P., Klupp, B., Werner, S., Rehbein, S., Mettenleiter, T.C., Schneider, G. & Grunewald, K. (2012). Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J Struct Biol 177(2), 193–201.Google Scholar
Hanssen, E., Knoechel, C., Klonis, N., Abu-Bakar, N., Deed, S., LeGros, M., Larabell, C. & Tilley, L. (2011). Cryo transmission X-ray imaging of the malaria parasite, P. falciparum . J Struct Biol 173(1), 161–168.Google Scholar
Henderson, R. (1995). The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q Rev Biophys 28(2), 171–193.CrossRefGoogle ScholarPubMed
Hertz, H.M., von Hofsten, O., Bertilson, M., Vogt, U., Holmberg, A., Reinspach, J., Martz, D., Selin, M., Christakou, A.E., Jerlstrom-Hultqvist, J. & Svard, S. (2012). Laboratory cryo soft X-ray microscopy. J Struct Biol 177(2), 267–272.CrossRefGoogle ScholarPubMed
Horne, S.F., Silterra, J. & Holber, W. (2009). A compact soft X-ray microscope using an electrode-less Z-pinch source. J Phys Conf Ser 186, 12028. Google Scholar
Howells, M.R., Beetz, T., Chapman, H.N., Cui, C., Holton, J.M., Jacobsen, C.J., Kirz, J., Lima, E., Marchesini, S., Miao, H., Sayre, D., Shapiro, D.A., Spence, J.C. & Starodub, D. (2009). An assessment of the resolution limitation due to radiation-damage in X-ray diffraction microscopy. J Electron Spectros Relat Phenomena 170(1-3), 4–12.CrossRefGoogle ScholarPubMed
Ingerman, E., Perkins, E.M., Marina, M., Mears, J.A., McCaffery, J.M., Hinshaw, J.E. & Nunnari, J. (2005). Dnm1 forms spirals that are structurally tailored to fit mitochondria. J Cell Biol 170(7), 1021–1027.Google Scholar
Jacobsen, C., Kirz, J. & Williams, S. (1992). Resolution in soft X-ray microscopes. Ultramicroscopy 47, 55–79.Google Scholar
Kirz, J., Jacobsen, C. & Howells, M. (1995). Soft X-ray microscopes and their biological applications. Q Rev Biophys 28(1), 33–130.CrossRefGoogle ScholarPubMed
Kneip, S., McGuffey, C., Martins, J.L., Martins, S.F., Bellei, C., Chvykov, V., Dollar, F., Fonseca, R., Huntington, C., Kalintchenko, G., Maksimchuk, A., Mangles, S.P.D., Matsuoka, T., Nagel, S.R., Palmer, C.A.J., Schreiber, J., Ta Phuoc, K., Thomas, A.G.R., Yanovsky, V., Silva, L.O., Krushelnick, K. & Najmudin, Z. (2010). Bright spatially coherent synchrotron X-rays from a table-top source. Nat Phys 6, 980–983.CrossRefGoogle Scholar
Larabell, C.A. & Le Gros, M.A. (2004). X-ray tomography generates 3-D reconstructions of the yeast, saccharomyces cerevisiae, at 60-nm resolution. Mol Biol Cell 15(3), 957–962.Google Scholar
Larabell, C.A. & Nugent, K.A. (2010). Imaging cellular architecture with X-rays. Curr Opin Struct Biol 20(5), 623–631.Google Scholar
Le Gros, M.A., McDermott, G., Uchida, M., Knoechel, C.G. & Larabell, C.A. (2009). High-aperture cryogenic light microscopy. J Microsc 235(1), 1–8.Google Scholar
Li, Z. & Jensen, G.J. (2009). Electron cryotomography: A new view into microbial ultrastructure. Curr Opin Microbiol 12(3), 333–340.Google Scholar
McDermott, G., Le Gros, M.A., Knoechel, C.G., Uchida, M. & Larabell, C.A. (2009). Soft X-ray tomography and cryogenic light microscopy: The cool combination in cellular imaging. Trends Cell Biol 19(11), 587–595.CrossRefGoogle ScholarPubMed
Milne, J.L. & Subramaniam, S. (2009). Cryo-electron tomography of bacteria: Progress, challenges and future prospects. Nat Rev Microbiol 7(9), 666–675.Google Scholar
Muller, W.G., Heymann, J.B., Nagashima, K., Guttmann, P., Werner, S., Rehbein, S., Schneider, G. & McNally, J.G. (2012). Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography. J Struct Biol 177(2), 179–192.CrossRefGoogle ScholarPubMed
Nelson, J., Huang, X., Steinbrener, J., Shapiro, D., Kirz, J., Marchesini, S., Neiman, A.M., Turner, J.J. & Jacobsen, C. (2010). High-resolution X-ray diffraction microscopy of specifically labeled yeast cells. Proc Natl Acad Sci USA 107(16), 7235–7239.Google Scholar
Niemann, B., Rudolph, D. & Schmahl, G. (1976). X-ray microscopy with synchrotron radiation. Appl Optics 15(8), 1883–1884.CrossRefGoogle ScholarPubMed
Parkinson, D.Y., McDermott, G., Etkin, L.D., Le Gros, M.A. & Larabell, C.A. (2008). Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography. J Struct Biol 162(3), 380–386.CrossRefGoogle ScholarPubMed
Reimer, L. (1998). Scanning Electron Microscopy: Physics of Image Formation and Microanalysis. Heidelberg, Germany: Springer-Verlag.Google Scholar
Sartori, A., Gatz, R., Beck, F., Rigort, A., Baumeister, W. & Plitzko, J.M. (2007). Correlative microscopy: Bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol 160(2), 135–145.Google Scholar
Schmahl, G., Rudolph, D., Guttmann, P., Schneider, G., Thieme, J. & Niemann, B. (1995). Phase-contrast studies of biological specimens with the X-ray microscope at Bessy. Rev Sci Instrum 66(2), 1282–1286.CrossRefGoogle Scholar
Schneider, G., Guttmann, P., Heim, S., Rehbein, S., Mueller, F., Nagashima, K., Heymann, J.B., Muller, W.G. & McNally, J.G. (2010). Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat Methods 7(12), 985–987.CrossRefGoogle ScholarPubMed
Schneider, G., Guttmann, P., Rehbein, S., Werner, S. & Follath, R. (2012). Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging. J Struct Biol 177(2), 212–223.Google Scholar
Schwartz, C.L., Sarbash, V.I., Ataullakhanov, F.I., McIntosh, J.R. & Nicastro, D. (2007). Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J Microsc 227(Pt 2), 98–109.CrossRefGoogle ScholarPubMed
Uchida, M., McDermott, G., Wetzler, M., Le Gros, M.A., Myllys, M., Knoechel, C., Barron, A.E. & Larabell, C.A. (2009). Soft X-ray tomography of phenotypic switching and the cellular response to antifungal peptoids in Candida albicans . Proc Natl Acad Sci USA 106(46), 19375–19380.Google Scholar
Uchida, M., Sun, Y., McDermott, G., Knoechel, C., Le Gros, M.A., Parkinson, D., Drubin, D.G. & Larabell, C.A. (2011). Quantitative analysis of yeast internal architecture using soft X-ray tomography. Yeast 28(3), 227–236.Google Scholar