Abstract

We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.

© 2012 OSA

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  1. J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol. 2(6), 444–456 (2001).
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  4. B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  7. A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  21. E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
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    [CrossRef] [PubMed]

2010 (6)

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

W. Inami, K. Nakajima, A. Miyakawa, and Y. Kawata, “Electron beam excitation assisted optical microscope with ultra-high resolution,” Opt. Express 18(12), 12897–12902 (2010).
[CrossRef] [PubMed]

F. J. G. de Abajo, “Optical excitations in electron microscopy,” Rev. Mod. Phys. 82(1), 209–275 (2010).
[CrossRef]

2009 (1)

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

2008 (3)

P. J. Fisher, W. S. Wessels, A. B. Dietz, and F. G. Prendergast, “Enhanced biological cathodoluminescence,” Opt. Commun. 281(7), 1901–1908 (2008).
[CrossRef]

D. Ripper, H. Schwarz, and Y. D. Stierhof, “Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration,” Biol. Cell 100(2), 109–123 (2008).
[CrossRef] [PubMed]

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

2006 (2)

L. N. Dem’yanets, L. E. Li, and T. G. Uvarova, “Hydrothermal synthesis and cathodoluminescence of ZnO crystalline powders and coatings,” J. Cryst. Growth 287(1), 23–27 (2006).
[CrossRef]

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

2005 (1)

W. Baumeister, “A voyage to the inner space of cells,” Protein Sci. 14(1), 257–269 (2005).
[CrossRef] [PubMed]

2004 (1)

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

2003 (2)

R. Y. Tsien, “Imagining imaging’s future,” Nat. Rev. Mol. Cell Biol. 5(Suppl), SS16–SS21 (2003).
[PubMed]

A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
[CrossRef] [PubMed]

2001 (1)

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol. 2(6), 444–456 (2001).
[CrossRef] [PubMed]

1996 (1)

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

1976 (1)

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

1940 (1)

U. Fano, “A theory on cathode luminescence,” Phys. Rev. 58(6), 544–553 (1940).
[CrossRef]

Agard, D.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Alfici, R.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Anaby, D.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Baumeister, W.

W. Baumeister, “A voyage to the inner space of cells,” Protein Sci. 14(1), 257–269 (2005).
[CrossRef] [PubMed]

A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
[CrossRef] [PubMed]

Beery, R.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Bergan, J.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

Boulanger, J.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Brandt, O.

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

Carlton, P. M.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Cohen, D.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Cohen, O.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Czerniak, A.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Dani, A.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

de Abajo, F. J. G.

F. J. G. de Abajo, “Optical excitations in electron microscopy,” Rev. Mod. Phys. 82(1), 209–275 (2010).
[CrossRef]

de Jonge, N.

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

Dem’yanets, L. N.

L. N. Dem’yanets, L. E. Li, and T. G. Uvarova, “Hydrothermal synthesis and cathodoluminescence of ZnO crystalline powders and coatings,” J. Cryst. Growth 287(1), 23–27 (2006).
[CrossRef]

Demers, H.

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

Dietz, A. B.

P. J. Fisher, W. S. Wessels, A. B. Dietz, and F. G. Prendergast, “Enhanced biological cathodoluminescence,” Opt. Commun. 281(7), 1901–1908 (2008).
[CrossRef]

Drouin, D.

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

Dulac, C.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

Earnest, T.

A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
[CrossRef] [PubMed]

Fano, U.

U. Fano, “A theory on cathode luminescence,” Phys. Rev. 58(6), 544–553 (1940).
[CrossRef]

Fernández-Suárez, M.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

Fisher, P. J.

P. J. Fisher, W. S. Wessels, A. B. Dietz, and F. G. Prendergast, “Enhanced biological cathodoluminescence,” Opt. Commun. 281(7), 1901–1908 (2008).
[CrossRef]

Fujii, K.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Fujita, Y.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Glaeser, R.

A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
[CrossRef] [PubMed]

Hein, B.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Hell, S. W.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Hough, P. V. C.

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

Huang, B.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

Ilany, J.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Inami, W.

Jahn, U.

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

Jakobs, S.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Karasik, A.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Kawata, Y.

Kenworthy, A.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol. 2(6), 444–456 (2001).
[CrossRef] [PubMed]

Kervrann, C.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Kimura, E.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Kimura, M.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Kner, P.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Ledbeter, M. C.

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

Levit, S.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Li, L. E.

L. N. Dem’yanets, L. E. Li, and T. G. Uvarova, “Hydrothermal synthesis and cathodoluminescence of ZnO crystalline powders and coatings,” J. Cryst. Growth 287(1), 23–27 (2006).
[CrossRef]

Lippincott-Schwartz, J.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol. 2(6), 444–456 (2001).
[CrossRef] [PubMed]

Matsuda, A.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

McKinney, W. R.

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

Menniger, J.

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

Miyakawa, A.

Moos, H. W.

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

Morito, S.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Nakajima, K.

Nakamura, M.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Nakayama, Y.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Niitsuma, J.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Ohba, T.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Oikawa, H.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Peckys, D. B.

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

Ploog, K.

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

Poirier-Demers, N.

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

Pollack, R. E.

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

Prendergast, F. G.

P. J. Fisher, W. S. Wessels, A. B. Dietz, and F. G. Prendergast, “Enhanced biological cathodoluminescence,” Opt. Commun. 281(7), 1901–1908 (2008).
[CrossRef]

Ripper, D.

D. Ripper, H. Schwarz, and Y. D. Stierhof, “Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration,” Biol. Cell 100(2), 109–123 (2008).
[CrossRef] [PubMed]

Sali, A.

A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
[CrossRef] [PubMed]

Sato, M.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Schwartz, I.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Schwarz, H.

D. Ripper, H. Schwarz, and Y. D. Stierhof, “Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration,” Biol. Cell 100(2), 109–123 (2008).
[CrossRef] [PubMed]

Sedat, J. W.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Sekiguchi, T.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Senthilkumar, K.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Senthilkumar, O.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Shabtai, M.

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Shao, L.

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Snapp, E.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol. 2(6), 444–456 (2001).
[CrossRef] [PubMed]

Stierhof, Y. D.

D. Ripper, H. Schwarz, and Y. D. Stierhof, “Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration,” Biol. Cell 100(2), 109–123 (2008).
[CrossRef] [PubMed]

Suzuki, H.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Ting, A. Y.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

Tsien, R. Y.

R. Y. Tsien, “Imagining imaging’s future,” Nat. Rev. Mol. Cell Biol. 5(Suppl), SS16–SS21 (2003).
[PubMed]

Ushiki, T.

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Uvarova, T. G.

L. N. Dem’yanets, L. E. Li, and T. G. Uvarova, “Hydrothermal synthesis and cathodoluminescence of ZnO crystalline powders and coatings,” J. Cryst. Growth 287(1), 23–27 (2006).
[CrossRef]

Wessels, W. S.

P. J. Fisher, W. S. Wessels, A. B. Dietz, and F. G. Prendergast, “Enhanced biological cathodoluminescence,” Opt. Commun. 281(7), 1901–1908 (2008).
[CrossRef]

Westphal, V.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Willig, K. I.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Wurm, C. A.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Yamauchi, K.

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

Yang, H.

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

Zhuang, X.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

Arch. Histol. Cytol. (1)

E. Kimura, T. Sekiguchi, H. Oikawa, J. Niitsuma, Y. Nakayama, H. Suzuki, M. Kimura, K. Fujii, and T. Ushiki, “Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy,” Arch. Histol. Cytol. 67(3), 263–270 (2004).
[CrossRef] [PubMed]

Biol. Cell (1)

D. Ripper, H. Schwarz, and Y. D. Stierhof, “Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration,” Biol. Cell 100(2), 109–123 (2008).
[CrossRef] [PubMed]

Biophys. J. (1)

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

J. Cryst. Growth (1)

L. N. Dem’yanets, L. E. Li, and T. G. Uvarova, “Hydrothermal synthesis and cathodoluminescence of ZnO crystalline powders and coatings,” J. Cryst. Growth 287(1), 23–27 (2006).
[CrossRef]

Nat. Rev. Mol. Cell Biol. (3)

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol. 2(6), 444–456 (2001).
[CrossRef] [PubMed]

R. Y. Tsien, “Imagining imaging’s future,” Nat. Rev. Mol. Cell Biol. 5(Suppl), SS16–SS21 (2003).
[PubMed]

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

Nature (1)

A. Sali, R. Glaeser, T. Earnest, and W. Baumeister, “From words to literature in structural proteomics,” Nature 422(6928), 216–225 (2003).
[CrossRef] [PubMed]

Neuron (1)

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution imaging of chemical synapses in the brain,” Neuron 68(5), 843–856 (2010).
[CrossRef] [PubMed]

Opt. Commun. (1)

P. J. Fisher, W. S. Wessels, A. B. Dietz, and F. G. Prendergast, “Enhanced biological cathodoluminescence,” Opt. Commun. 281(7), 1901–1908 (2008).
[CrossRef]

Opt. Express (1)

Phys. Rev. (1)

U. Fano, “A theory on cathode luminescence,” Phys. Rev. 58(6), 544–553 (1940).
[CrossRef]

Phys. Rev. B Condens. Matter (1)

J. Menniger, U. Jahn, O. Brandt, H. Yang, and K. Ploog, “Identification of optical transitions in cubic and hexagonal GaN by spatially resolved cathodoluminescence,” Phys. Rev. B Condens. Matter 53(4), 1881–1885 (1996).
[CrossRef] [PubMed]

Phys. Status Solidi B (1)

K. Senthilkumar, O. Senthilkumar, K. Yamauchi, M. Sato, S. Morito, T. Ohba, M. Nakamura, and Y. Fujita, “Preparation of ZnO nanoparticles for bio-imaging applications,” Phys. Status Solidi B 246(4), 885–888 (2009).
[CrossRef]

PLoS ONE (1)

A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P. M. Carlton, P. Kner, D. Agard, and J. W. Sedat, “Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones,” PLoS ONE 5(9), e12768 (2010).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (1)

P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976).
[CrossRef] [PubMed]

Protein Sci. (1)

W. Baumeister, “A voyage to the inner space of cells,” Protein Sci. 14(1), 257–269 (2005).
[CrossRef] [PubMed]

Rev. Mod. Phys. (1)

F. J. G. de Abajo, “Optical excitations in electron microscopy,” Rev. Mod. Phys. 82(1), 209–275 (2010).
[CrossRef]

Thyroid (1)

O. Cohen, R. Beery, S. Levit, J. Ilany, I. Schwartz, M. Shabtai, D. Anaby, D. Cohen, R. Alfici, A. Czerniak, and A. Karasik, “Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation,” Thyroid 16(10), 997–1001 (2006).
[CrossRef] [PubMed]

Ultramicroscopy (1)

N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers,” Ultramicroscopy 110(9), 1114–1119 (2010).
[CrossRef] [PubMed]

Other (2)

D. C. Joy, Monte Carlo Modeling for Electron Microscopy and Microanalysis (Oxford Univ. Press, 1995).

G. Arnold, ed., Cathodoluminescence and Its Application in the Planetary Sciences, 1st ed. (Springer, 2009).

Supplementary Material (1)

» Media 1: MOV (3703 KB)     

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Figures (5)

Fig. 1
Fig. 1

Principle of the D-EXA microscope. Specimens are placed on the thin film and a focused electron beam excites luminescence in the specimens directly through the thin film. The electron beam can be focused to a few tens of nanometers in diameter in the specimen. The thin film is also used to separate vacuum from the environment of the specimen such as air or liquid. The dynamic behavior of living cells is thus imaged with nanometer-scale resolution.

Fig. 2
Fig. 2

Structure of the D-EXA microscope. (a) Prototype of the D-EXA microscope we have developed. (b) Schematic of the D-EXA microscope. A scanning electron microscope is used for excitation and scanning the electron beam. A fluorescence microscope is used to collect the luminescence from the specimens.

Fig. 3
Fig. 3

Luminescence images of 100 nm ZnO nanoparticles. (a) Fluorescence image excited with UV light in a conventional epi-fluorescence microscope. (b) Luminescence image acquired with the D-EXA microscope. The acceleration voltage was 5 kV and probe current was 1 nA. (d, e) Line profiles of luminescence intensity distributions between the arrows in Figs. 3(a) and 3(b) respectively. In Fig. 3(b), the ZnO nanoparticles can be clearly distinguished from each other. (c) Luminescence image of isolated ZnO nanoparticles acquired with the D-EXA microscope. (f) Line profile of one ZnO nanoparticle between the arrows in Fig. 3(c). The full width at half maximum is 100 nm.

Fig. 4
Fig. 4

Observation results of the movements of 100 nm ZnO nanoparticles in a water solution. (Media 1) (a)-(e) Time lapse images of the movements of ZnO nanoparticles. Since the electron beam was focused on the surface of the film, ZnO nanoparticles moving away from the surface of the film disappeared in the next image, as shown by the dashed circle, whereas ZnO nanoparticles moving closer to the surface of the film appeared, as shown within the solid circles. (f)-(j) Magnified images of the areas indicated by the squares in Figs. 4(a)-4(e). The ZnO nanoparticle indicated by the arrow moved along the surface of the film. (k) An analysis of the movement of the ZnO nanoparticle. The nanoparticle moved randomly with the change in luminescence intensity. The change in the intensity showed that the nanoparticle moved up and down in the direction away from the film plane within the focal plane of the electron beam.

Fig. 5
Fig. 5

Observation results of living cells using the D-EXA microscope. (a) A culture dish for the D-EXA microscope. Cells were directly cultured on the Si3N4 membrane. (b) A luminescence image of living MARCO-expressing CHO cells in culture solution without any treatments. The intracellular granules indicated with arrows are observed as white sports and the cell membranes are observed as light-gray contrast against the dark-gray background. The acceleration voltage was 5 kV and probe current was 1 nA. (c) A phase contrast microscope image of the living cells. It represents the same area as Fig. 5(b).

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