Abstract

Soft-x-ray cryotomography allows quantitative and high-resolution three-dimensional imaging of intact unstained cells. To date, the method relies on synchrotron-radiation sources, which limits accessibility for re searchers. Here we present a laboratory water-window microscope for cryotomography. It is based on a λ=2.48nm liquid-jet laser-plasma source, a normal-incidence multilayer condenser, a 30nm zone-plate objective, and a cryotilt sample holder. We demonstrate high-resolution imaging, as well as quantitative tomographic imaging, of frozen intact cells. The reconstructed tomogram of the intracellular local absorption coefficient shows details down to 100nm.

© 2011 Optical Society of America

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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]

2011 (2)

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

M. Bertilson, O. von Hofsten, H. M. Hertz, and U. Vogt, Opt. Express 19, 11578 (2011).
[CrossRef] [PubMed]

2010 (4)

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

H. N. Chapman and K. A. Nugent, Nat. Photon. 4, 833(2010).
[CrossRef]

A. Sakdinawat and D. Attwood, Nat. Photon. 4, 840(2010).
[CrossRef]

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

2009 (3)

2008 (2)

2007 (2)

C. Messaoudi, T. Boudier, C. Sorzano, and S. Marco, BMC Bioinf. 8, 288 (2007).
[CrossRef]

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

2005 (1)

P. A. C. Jansson, U. Vogt, and H. M. Hertz, Rev. Sci. Instrum. 76, 043503 (2005).
[CrossRef]

2003 (1)

C. A. Larabell and M. A. Le Gros, Mol. Biol. Cell 15, 957(2003).
[CrossRef] [PubMed]

2002 (1)

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

2000 (2)

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

Abu-Bakar, N.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Aoki, S.

M. Hoshino and S. Aoki, Appl. Phys. Express 1, 067005(2008).
[CrossRef]

Attwood, D.

A. Sakdinawat and D. Attwood, Nat. Photon. 4, 840(2010).
[CrossRef]

Baumeister, W.

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Benk, M.

Berglund,

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

Bergmann, K.

Bertilson, M.

Boudier, T.

C. Messaoudi, T. Boudier, C. Sorzano, and S. Marco, BMC Bioinf. 8, 288 (2007).
[CrossRef]

Chapman, H. N.

H. N. Chapman and K. A. Nugent, Nat. Photon. 4, 833(2010).
[CrossRef]

Deed, S.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Frangakis, A. S.

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Gerisch, G.

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Guttmann, P.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Hanssen, E.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Heim, S.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Hell, S. W.

S. W. Hell, Nat. Meth. 6, 24 (2009).
[CrossRef]

Hertz,

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

Hertz, H. M.

M. Bertilson, O. von Hofsten, H. M. Hertz, and U. Vogt, Opt. Express 19, 11578 (2011).
[CrossRef] [PubMed]

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

O. von Hofsten, M. Bertilson, J. Reinspach, A. Holmberg, H. M. Hertz, and U. Vogt, Opt. Lett. 34, 2631 (2009).
[CrossRef] [PubMed]

M. Bertilson, O. von Hofsten, U. Vogt, A. Holmberg, and H. M. Hertz, Opt. Express 17, 11057 (2009).
[CrossRef] [PubMed]

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

P. A. C. Jansson, U. Vogt, and H. M. Hertz, Rev. Sci. Instrum. 76, 043503 (2005).
[CrossRef]

Heymann, J.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Hofsten, O. V.

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

Holmberg, A.

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

M. Bertilson, O. von Hofsten, U. Vogt, A. Holmberg, and H. M. Hertz, Opt. Express 17, 11057 (2009).
[CrossRef] [PubMed]

O. von Hofsten, M. Bertilson, J. Reinspach, A. Holmberg, H. M. Hertz, and U. Vogt, Opt. Lett. 34, 2631 (2009).
[CrossRef] [PubMed]

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

Hoshino, M.

M. Hoshino and S. Aoki, Appl. Phys. Express 1, 067005(2008).
[CrossRef]

Jansson, P. A. C.

P. A. C. Jansson, U. Vogt, and H. M. Hertz, Rev. Sci. Instrum. 76, 043503 (2005).
[CrossRef]

Johansson, G. A.

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

Klonis, N.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Knoechel, C.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Larabell, C.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Larabell, C. A.

C. A. Larabell and M. A. Le Gros, Mol. Biol. Cell 15, 957(2003).
[CrossRef] [PubMed]

Le Gros, M. A.

C. A. Larabell and M. A. Le Gros, Mol. Biol. Cell 15, 957(2003).
[CrossRef] [PubMed]

LeGros, M.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Lindblom, M.

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

Marco, S.

C. Messaoudi, T. Boudier, C. Sorzano, and S. Marco, BMC Bioinf. 8, 288 (2007).
[CrossRef]

McNally, J.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Medalia, O.

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Messaoudi, C.

C. Messaoudi, T. Boudier, C. Sorzano, and S. Marco, BMC Bioinf. 8, 288 (2007).
[CrossRef]

Mueller, F.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Müller, W.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Nagashima, K.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Nicastro, D.

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Niemann, B.

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Nugent, K. A.

H. N. Chapman and K. A. Nugent, Nat. Photon. 4, 833(2010).
[CrossRef]

Peuker,

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

Rehbein, S.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

Reinspach, J.

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

O. von Hofsten, M. Bertilson, J. Reinspach, A. Holmberg, H. M. Hertz, and U. Vogt, Opt. Lett. 34, 2631 (2009).
[CrossRef] [PubMed]

Rudolph, D.

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Rymell,

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

Sakdinawat, A.

A. Sakdinawat and D. Attwood, Nat. Photon. 4, 840(2010).
[CrossRef]

Schäfer, D.

Schmahl, G.

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Schneider, G.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Sorzano, C.

C. Messaoudi, T. Boudier, C. Sorzano, and S. Marco, BMC Bioinf. 8, 288 (2007).
[CrossRef]

Stollberg, H.

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

Takman, P. A. C.

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

Tilley, L.

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Vogt, U.

von Hofsten, O.

Weber, I.

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Weiss, D.

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Wilhein,

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

Wilhein, T.

Appl. Phys. Express (1)

M. Hoshino and S. Aoki, Appl. Phys. Express 1, 067005(2008).
[CrossRef]

BMC Bioinf. (1)

C. Messaoudi, T. Boudier, C. Sorzano, and S. Marco, BMC Bioinf. 8, 288 (2007).
[CrossRef]

J. Microsc. (2)

Berglund, Rymell, Peuker, Wilhein, and Hertz, J. Microsc. 197, 268 (2000).
[PubMed]

P. A. C. Takman, H. Stollberg, G. A. Johansson, A. Holmberg, M. Lindblom, and H. M. Hertz, J. Microsc. 226, 175 (2007).
[CrossRef] [PubMed]

J. Struct. Biol. (1)

E. Hanssen, C. Knoechel, N. Klonis, N. Abu-Bakar, S. Deed, M. LeGros, C. Larabell, and L. Tilley, J. Struct. Biol. 173, 161 (2011).
[CrossRef]

Microelectron. Eng. (1)

J. Reinspach, M. Lindblom, O. V. Hofsten, M. Bertilson, H. M. Hertz, and A. Holmberg, Microelectron. Eng. 87, 1583(2010).
[CrossRef]

Mol. Biol. Cell (1)

C. A. Larabell and M. A. Le Gros, Mol. Biol. Cell 15, 957(2003).
[CrossRef] [PubMed]

Nat. Meth. (2)

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. Heymann, W. Müller, and J. McNally, Nat. Meth. 7, 985 (2010).
[CrossRef]

S. W. Hell, Nat. Meth. 6, 24 (2009).
[CrossRef]

Nat. Photon. (2)

A. Sakdinawat and D. Attwood, Nat. Photon. 4, 840(2010).
[CrossRef]

H. N. Chapman and K. A. Nugent, Nat. Photon. 4, 833(2010).
[CrossRef]

Opt. Express (2)

Opt. Lett. (2)

Rev. Sci. Instrum. (1)

P. A. C. Jansson, U. Vogt, and H. M. Hertz, Rev. Sci. Instrum. 76, 043503 (2005).
[CrossRef]

Science (1)

O. Medalia, I. Weber, A. S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, Science 298, 1209 (2002).
[CrossRef] [PubMed]

Ultramicroscopy (1)

D. Weiss, G. Schneider, B. Niemann, P. Guttmann, D. Rudolph, and G. Schmahl, Ultramicroscopy 84, 185(2000).
[CrossRef] [PubMed]

Supplementary Material (2)

» Media 1: MOV (3809 KB)     
» Media 2: MOV (2632 KB)     

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Figures (3)

Fig. 1
Fig. 1

Microscope arrangement. Soft x rays ( λ = 2 . 48 nm ) from a liquid-jet laser-plasma source illuminate the specimen via a multilayer condenser. The specimen is imaged onto a CCD with a high-resolution zone plate. The cryotilt holder allows tomographic imaging of hydrated biological specimens.

Fig. 2
Fig. 2

Frozen hydrated cells imaged in the laboratory x-ray microscope. (a) Yeast cells. (b) Human B-cell. Both images show considerable intracellular structure and detail with natural contrast.

Fig. 3
Fig. 3

Laboratory soft x-ray cryotomography of a kidney cell. (a)–(f) Sections through the tomogram at indicated depths (Media 1). Scale bars are 3 μm , and the darker gray corresponds to a higher LAC. (g) Surface rendering of segmented intracellular features (Media 2). Colors indicate nucleus (yellow), vacuoles (blue), and other organelles with higher LAC (green or red).

Metrics