Abstract

We present label-free and high spatial-resolution imaging for specific cellular structures using an electron-beam excitation-assisted optical microscope (EXA microscope). Images of the actin filament and mitochondria of stained HeLa cells, obtained by fluorescence and EXA microscopy, were compared to identify cellular structures. Based on these results, we demonstrated the feasibility of identifying label-free cellular structures at a spatial resolution of 82 nm. Using numerical analysis, we calculated the imaging depth region and determined the spot size of a cathodoluminescent (CL) light source to be 83 nm at the membrane surface.

© 2016 Optical Society of America

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2016 (2)

R. Suman, G. Smith, K. E. A. Hazel, R. Kasprowicz, M. Coles, P. O’Toole, and S. Chawla, “Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures,” Sci. Rep. 6, 22032 (2016).
[Crossref] [PubMed]

M. Fukuta, W. Inami, A. Ono, and Y. Kawata, “Intensity distribution analysis of cathodoluminescence using the energy loss distribution of electrons,” Ultramicroscopy 160, 225–229 (2016).
[Crossref] [PubMed]

2015 (6)

Y. Masuda, W. Inami, A. Miyakawa, and Y. Kawata, “Cell culture on hydrophilicity-controlled silicon nitride surfaces,” World J. Microbiol. Biotechnol. 31(12), 1977–1982 (2015).
[Crossref] [PubMed]

Y. Masuda, Y. Nawa, W. Inami, and Y. Kawata, “Carboxylic monolayer formation for observation of intracellular structures in HeLa cells with direct electron beam excitation-assisted fluorescence microscopy,” Biomed. Opt. Express 6(8), 3128–3133 (2015).
[Crossref] [PubMed]

T. Furukawa, S. Kanamori, M. Fukuta, Y. Nawa, H. Kominami, Y. Nakanishi, A. Sugita, W. Inami, and Y. Kawata, “Fabrication of bright and thin Zn2SiO4 luminescent film for electron beam excitation-assisted optical microscope,” Opt. Express 23(14), 18630–18637 (2015).
[Crossref] [PubMed]

M. Fukuta, S. Kanamori, T. Furukawa, Y. Nawa, W. Inami, S. Lin, Y. Kawata, and S. Terakawa, “Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope,” Sci. Rep. 5, 16068 (2015).
[Crossref] [PubMed]

W. Inami, M. Fukuta, Y. Masuda, Y. Nawa, A. Ono, S. Lin, Y. Kawata, and S. Terakawa, “A plastic scintillator film for an electron beam-excitation assisted optical microscope,” Opt. Rev. 22(2), 354–358 (2015).
[Crossref]

S. Hayashi and Y. Okada, “Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics,” Mol. Biol. Cell 26(9), 1743–1751 (2015).
[Crossref] [PubMed]

2012 (3)

X. Zhang, M. B. Roeffaers, S. Basu, J. R. Daniele, D. Fu, C. W. Freudiger, G. R. Holtom, and X. S. Xie, “Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy,” ChemPhysChem 13(4), 1054–1059 (2012).
[Crossref] [PubMed]

K. Klein, A. M. Gigler, T. Aschenbrenner, R. Monetti, W. Bunk, F. Jamitzky, G. Morfill, R. W. Stark, and J. Schlegel, “Label-Free Live-Cell Imaging with Confocal Raman Microscopy,” Biophys. J. 102(2), 360–368 (2012).
[Crossref] [PubMed]

W. Inami, J. Fujiwara, F. Masahiro, A. Ono, and Y. Kawata, “Analysis of electron and light scattering in a fluorescent thin film by combination of Monte Carlo simulation and finite-difference time-domain method,” Appl. Phys. Lett. 101(15), 151104 (2012).
[Crossref]

2011 (3)

P. O. Bagnaninchi and N. Drummond, “Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing,” Proc. Natl. Acad. Sci. U.S.A. 108(16), 6462–6467 (2011).
[Crossref] [PubMed]

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13546–13551 (2011).
[Crossref] [PubMed]

P. A. Pellett, X. Sun, T. J. Gould, J. E. Rothman, M.-Q. Xu, I. R. Corrêa, and J. Bewersdorf, “Two-color STED microscopy in living cells,” Biomed. Opt. Express 2(8), 2364–2371 (2011).
[Crossref] [PubMed]

2010 (4)

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (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]

H. Mellor, “The role of formins in filopodia formation,” Biochim. Biophys. Acta 1803(2), 191–200 (2010).
[Crossref] [PubMed]

2009 (1)

J. W. Chan and D. K. Lieu, “Label-free biochemical characterization of stem cells using vibrational spectroscopy,” J. Biophotonics 2(11), 656–668 (2009).
[Crossref] [PubMed]

2008 (4)

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[Crossref] [PubMed]

B. Xi, N. Yu, X. Wang, X. Xu, and Y. A. Abassi, “The application of cell-based label-free technology in drug discovery,” Biotechnol. J. 3(4), 484–495 (2008).
[Crossref] [PubMed]

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

E. I. Galanzha, E. V. Shashkov, V. V. Tuchin, and V. P. Zharov, “In vivo multispectral photoacoustic lymph frow cytometry with natural cell focusing and multicolor nanoparticle probes,” Cytom. A. 73(10), 884–894 (2008).
[Crossref]

2007 (1)

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[Crossref] [PubMed]

2006 (4)

K. Matsuura-Tokita, M. Takeuchi, A. Ichihara, K. Mikuriya, and A. Nakano, “Live imaging of yeast Golgi cisternal maturation,” Nature 441(7096), 1007–1010 (2006).
[Crossref] [PubMed]

D. C. Chan, “Mitochondria: Dynamic Organelles in Disease, Aging, and Development,” Cell 125(7), 1241–1252 (2006).
[Crossref] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

2005 (1)

D. S. Lidke, K. A. Lidke, B. Rieger, T. M. Jovin, and D. J. Arndt-Jovin, “Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors,” J. Cell Biol. 170(4), 619–626 (2005).
[Crossref] [PubMed]

2004 (4)

A. Rustom, R. Saffrich, I. Markovic, P. Walther, and H.-H. Gerdes, “Nanotubular Highways for Intercellular Organelle Transport,” Science 303(5660), 1007–1010 (2004).
[Crossref] [PubMed]

F. Chen and D. Gerion, “Fluorescent CdSe/ZnS Nanocrystal-Peptide Conjugates for Long-term, Nontoxic Imaging and Nuclear Targeting in Living Cells,” Nano Lett. 4(10), 1827–1832 (2004).
[Crossref]

A. El Hichou, M. Addou, A. Bougrine, R. Dounia, J. Ebothé, M. Troyon, and M. Amrani, “Cathodoluminescence properties of undoped and Al-doped ZnO thin films deposited on glass substrate by spray pyrolysis,” Mater. Chem. Phys. 83(1), 43–47 (2004).
[Crossref]

M. Ohara-Imaizumi, C. Nishiwaki, T. Kikuta, S. Nagai, Y. Nakamichi, and S. Nagamatsu, “TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat β-cells,” Biochem. J. 381(1), 13–18 (2004).
[Crossref] [PubMed]

2003 (3)

P. Rorsman and E. Renström, “Insulin granule dynamics in pancreatic beta cells,” Diabetologia 46(8), 1029–1045 (2003).
[Crossref] [PubMed]

T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S. Kojima, J. M. Vasiliev, and G. G. Borisy, “Mechanism of filopodia initiation by reorganization of a dendritic network,” J. Cell Biol. 160(3), 409–421 (2003).
[Crossref] [PubMed]

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref] [PubMed]

2000 (3)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

T. G. Frey and C. A. Mannella, “The internal structure of mitochondria,” Trends Biochem. Sci. 25(7), 319–324 (2000).
[Crossref] [PubMed]

F. L. Riley, “Silicon Nitride and Related Materials,” J. Am. Ceram. Soc. 83(2), 245–265 (2000).
[Crossref]

1998 (1)

N. Hirokawa, “Kinesin and Dynein Superfamily Proteins and the Mechanism of Organelle Transport,” Science 279(5350), 519–526 (1998).
[Crossref] [PubMed]

1994 (1)

Abassi, Y. A.

B. Xi, N. Yu, X. Wang, X. Xu, and Y. A. Abassi, “The application of cell-based label-free technology in drug discovery,” Biotechnol. J. 3(4), 484–495 (2008).
[Crossref] [PubMed]

Addou, M.

A. El Hichou, M. Addou, A. Bougrine, R. Dounia, J. Ebothé, M. Troyon, and M. Amrani, “Cathodoluminescence properties of undoped and Al-doped ZnO thin films deposited on glass substrate by spray pyrolysis,” Mater. Chem. Phys. 83(1), 43–47 (2004).
[Crossref]

Allan, V. J.

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref] [PubMed]

Amrani, M.

A. El Hichou, M. Addou, A. Bougrine, R. Dounia, J. Ebothé, M. Troyon, and M. Amrani, “Cathodoluminescence properties of undoped and Al-doped ZnO thin films deposited on glass substrate by spray pyrolysis,” Mater. Chem. Phys. 83(1), 43–47 (2004).
[Crossref]

Arndt-Jovin, D. J.

D. S. Lidke, K. A. Lidke, B. Rieger, T. M. Jovin, and D. J. Arndt-Jovin, “Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors,” J. Cell Biol. 170(4), 619–626 (2005).
[Crossref] [PubMed]

Aschenbrenner, T.

K. Klein, A. M. Gigler, T. Aschenbrenner, R. Monetti, W. Bunk, F. Jamitzky, G. Morfill, R. W. Stark, and J. Schlegel, “Label-Free Live-Cell Imaging with Confocal Raman Microscopy,” Biophys. J. 102(2), 360–368 (2012).
[Crossref] [PubMed]

Bagnaninchi, P. O.

P. O. Bagnaninchi and N. Drummond, “Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing,” Proc. Natl. Acad. Sci. U.S.A. 108(16), 6462–6467 (2011).
[Crossref] [PubMed]

Basu, S.

X. Zhang, M. B. Roeffaers, S. Basu, J. R. Daniele, D. Fu, C. W. Freudiger, G. R. Holtom, and X. S. Xie, “Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy,” ChemPhysChem 13(4), 1054–1059 (2012).
[Crossref] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Bewersdorf, J.

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Borisy, G. G.

T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S. Kojima, J. M. Vasiliev, and G. G. Borisy, “Mechanism of filopodia initiation by reorganization of a dendritic network,” J. Cell Biol. 160(3), 409–421 (2003).
[Crossref] [PubMed]

Bougrine, A.

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Y. Masuda, W. Inami, A. Miyakawa, and Y. Kawata, “Cell culture on hydrophilicity-controlled silicon nitride surfaces,” World J. Microbiol. Biotechnol. 31(12), 1977–1982 (2015).
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W. Inami, J. Fujiwara, F. Masahiro, A. Ono, and Y. Kawata, “Analysis of electron and light scattering in a fluorescent thin film by combination of Monte Carlo simulation and finite-difference time-domain method,” Appl. Phys. Lett. 101(15), 151104 (2012).
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K. Klein, A. M. Gigler, T. Aschenbrenner, R. Monetti, W. Bunk, F. Jamitzky, G. Morfill, R. W. Stark, and J. Schlegel, “Label-Free Live-Cell Imaging with Confocal Raman Microscopy,” Biophys. J. 102(2), 360–368 (2012).
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M. Fukuta, S. Kanamori, T. Furukawa, Y. Nawa, W. Inami, S. Lin, Y. Kawata, and S. Terakawa, “Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope,” Sci. Rep. 5, 16068 (2015).
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B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
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E. I. Galanzha, E. V. Shashkov, V. V. Tuchin, and V. P. Zharov, “In vivo multispectral photoacoustic lymph frow cytometry with natural cell focusing and multicolor nanoparticle probes,” Cytom. A. 73(10), 884–894 (2008).
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T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S. Kojima, J. M. Vasiliev, and G. G. Borisy, “Mechanism of filopodia initiation by reorganization of a dendritic network,” J. Cell Biol. 160(3), 409–421 (2003).
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T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S. Kojima, J. M. Vasiliev, and G. G. Borisy, “Mechanism of filopodia initiation by reorganization of a dendritic network,” J. Cell Biol. 160(3), 409–421 (2003).
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A. Rustom, R. Saffrich, I. Markovic, P. Walther, and H.-H. Gerdes, “Nanotubular Highways for Intercellular Organelle Transport,” Science 303(5660), 1007–1010 (2004).
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Xu, X.

B. Xi, N. Yu, X. Wang, X. Xu, and Y. A. Abassi, “The application of cell-based label-free technology in drug discovery,” Biotechnol. J. 3(4), 484–495 (2008).
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B. Xi, N. Yu, X. Wang, X. Xu, and Y. A. Abassi, “The application of cell-based label-free technology in drug discovery,” Biotechnol. J. 3(4), 484–495 (2008).
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X. Zhang, M. B. Roeffaers, S. Basu, J. R. Daniele, D. Fu, C. W. Freudiger, G. R. Holtom, and X. S. Xie, “Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy,” ChemPhysChem 13(4), 1054–1059 (2012).
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Zharov, V. P.

E. I. Galanzha, E. V. Shashkov, V. V. Tuchin, and V. P. Zharov, “In vivo multispectral photoacoustic lymph frow cytometry with natural cell focusing and multicolor nanoparticle probes,” Cytom. A. 73(10), 884–894 (2008).
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Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
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Angew. Chem. Int. Ed. Engl. (1)

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Appl. Phys. Lett. (1)

W. Inami, J. Fujiwara, F. Masahiro, A. Ono, and Y. Kawata, “Analysis of electron and light scattering in a fluorescent thin film by combination of Monte Carlo simulation and finite-difference time-domain method,” Appl. Phys. Lett. 101(15), 151104 (2012).
[Crossref]

Biochem. J. (1)

M. Ohara-Imaizumi, C. Nishiwaki, T. Kikuta, S. Nagai, Y. Nakamichi, and S. Nagamatsu, “TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat β-cells,” Biochem. J. 381(1), 13–18 (2004).
[Crossref] [PubMed]

Biochim. Biophys. Acta (1)

H. Mellor, “The role of formins in filopodia formation,” Biochim. Biophys. Acta 1803(2), 191–200 (2010).
[Crossref] [PubMed]

Biomed. Opt. Express (2)

Biophys. J. (1)

K. Klein, A. M. Gigler, T. Aschenbrenner, R. Monetti, W. Bunk, F. Jamitzky, G. Morfill, R. W. Stark, and J. Schlegel, “Label-Free Live-Cell Imaging with Confocal Raman Microscopy,” Biophys. J. 102(2), 360–368 (2012).
[Crossref] [PubMed]

Biotechnol. J. (1)

B. Xi, N. Yu, X. Wang, X. Xu, and Y. A. Abassi, “The application of cell-based label-free technology in drug discovery,” Biotechnol. J. 3(4), 484–495 (2008).
[Crossref] [PubMed]

Cell (1)

D. C. Chan, “Mitochondria: Dynamic Organelles in Disease, Aging, and Development,” Cell 125(7), 1241–1252 (2006).
[Crossref] [PubMed]

ChemPhysChem (1)

X. Zhang, M. B. Roeffaers, S. Basu, J. R. Daniele, D. Fu, C. W. Freudiger, G. R. Holtom, and X. S. Xie, “Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy,” ChemPhysChem 13(4), 1054–1059 (2012).
[Crossref] [PubMed]

Cytom. A. (1)

E. I. Galanzha, E. V. Shashkov, V. V. Tuchin, and V. P. Zharov, “In vivo multispectral photoacoustic lymph frow cytometry with natural cell focusing and multicolor nanoparticle probes,” Cytom. A. 73(10), 884–894 (2008).
[Crossref]

Diabetologia (1)

P. Rorsman and E. Renström, “Insulin granule dynamics in pancreatic beta cells,” Diabetologia 46(8), 1029–1045 (2003).
[Crossref] [PubMed]

J. Am. Ceram. Soc. (1)

F. L. Riley, “Silicon Nitride and Related Materials,” J. Am. Ceram. Soc. 83(2), 245–265 (2000).
[Crossref]

J. Biophotonics (1)

J. W. Chan and D. K. Lieu, “Label-free biochemical characterization of stem cells using vibrational spectroscopy,” J. Biophotonics 2(11), 656–668 (2009).
[Crossref] [PubMed]

J. Cell Biol. (3)

D. S. Lidke, K. A. Lidke, B. Rieger, T. M. Jovin, and D. J. Arndt-Jovin, “Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors,” J. Cell Biol. 170(4), 619–626 (2005).
[Crossref] [PubMed]

T. M. Svitkina, E. A. Bulanova, O. Y. Chaga, D. M. Vignjevic, S. Kojima, J. M. Vasiliev, and G. G. Borisy, “Mechanism of filopodia initiation by reorganization of a dendritic network,” J. Cell Biol. 160(3), 409–421 (2003).
[Crossref] [PubMed]

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
[Crossref] [PubMed]

J. Microsc. (1)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

Mater. Chem. Phys. (1)

A. El Hichou, M. Addou, A. Bougrine, R. Dounia, J. Ebothé, M. Troyon, and M. Amrani, “Cathodoluminescence properties of undoped and Al-doped ZnO thin films deposited on glass substrate by spray pyrolysis,” Mater. Chem. Phys. 83(1), 43–47 (2004).
[Crossref]

Mol. Biol. Cell (1)

S. Hayashi and Y. Okada, “Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics,” Mol. Biol. Cell 26(9), 1743–1751 (2015).
[Crossref] [PubMed]

Nano Lett. (1)

F. Chen and D. Gerion, “Fluorescent CdSe/ZnS Nanocrystal-Peptide Conjugates for Long-term, Nontoxic Imaging and Nuclear Targeting in Living Cells,” Nano Lett. 4(10), 1827–1832 (2004).
[Crossref]

Nat. Methods (2)

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Nature (1)

K. Matsuura-Tokita, M. Takeuchi, A. Ichihara, K. Mikuriya, and A. Nakano, “Live imaging of yeast Golgi cisternal maturation,” Nature 441(7096), 1007–1010 (2006).
[Crossref] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Opt. Rev. (1)

W. Inami, M. Fukuta, Y. Masuda, Y. Nawa, A. Ono, S. Lin, Y. Kawata, and S. Terakawa, “A plastic scintillator film for an electron beam-excitation assisted optical microscope,” Opt. Rev. 22(2), 354–358 (2015).
[Crossref]

Proc. Natl. Acad. Sci. U.S.A. (2)

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13546–13551 (2011).
[Crossref] [PubMed]

P. O. Bagnaninchi and N. Drummond, “Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing,” Proc. Natl. Acad. Sci. U.S.A. 108(16), 6462–6467 (2011).
[Crossref] [PubMed]

Sci. Rep. (2)

R. Suman, G. Smith, K. E. A. Hazel, R. Kasprowicz, M. Coles, P. O’Toole, and S. Chawla, “Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures,” Sci. Rep. 6, 22032 (2016).
[Crossref] [PubMed]

M. Fukuta, S. Kanamori, T. Furukawa, Y. Nawa, W. Inami, S. Lin, Y. Kawata, and S. Terakawa, “Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope,” Sci. Rep. 5, 16068 (2015).
[Crossref] [PubMed]

Science (5)

N. Hirokawa, “Kinesin and Dynein Superfamily Proteins and the Mechanism of Organelle Transport,” Science 279(5350), 519–526 (1998).
[Crossref] [PubMed]

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref] [PubMed]

A. Rustom, R. Saffrich, I. Markovic, P. Walther, and H.-H. Gerdes, “Nanotubular Highways for Intercellular Organelle Transport,” Science 303(5660), 1007–1010 (2004).
[Crossref] [PubMed]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[Crossref] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Trends Biochem. Sci. (1)

T. G. Frey and C. A. Mannella, “The internal structure of mitochondria,” Trends Biochem. Sci. 25(7), 319–324 (2000).
[Crossref] [PubMed]

Ultramicroscopy (2)

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]

M. Fukuta, W. Inami, A. Ono, and Y. Kawata, “Intensity distribution analysis of cathodoluminescence using the energy loss distribution of electrons,” Ultramicroscopy 160, 225–229 (2016).
[Crossref] [PubMed]

World J. Microbiol. Biotechnol. (1)

Y. Masuda, W. Inami, A. Miyakawa, and Y. Kawata, “Cell culture on hydrophilicity-controlled silicon nitride surfaces,” World J. Microbiol. Biotechnol. 31(12), 1977–1982 (2015).
[Crossref] [PubMed]

Other (2)

A. Rose, Advances in Electronics Vol. 1 (Academic Press Inc., 1948).

C. F. Klingshirn, B. K. Meyer, A. Waag, A. Hoffmann, and J. Geurts, Zinc Oxide: From Fundamental Properties Towards Novel Applications Vol. 120 (Springer-Verlag, 2010).

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Figures (7)

Fig. 1
Fig. 1 (a) Schematic of the EXA microscope. The EXA microscope is constructed from optical microscope, culture dish and electron microscope. (b) Enlarged image of the highlighted square region in Fig. 1(a). The spot size of CL excited in the ZnO layer is less than 100 nm at the Si3N4 surface. The biological cell is directly cultured on the Si3N4 substrate. ZnO presents single emission peak at 380 nm. The single peak is due to the band gap emission of the ZnO. (c) CL spectrum of the ZnO luminescent thin film. (d) Surface morphology of the ZnO observed by an atmic force microscope (AFM). The ZnO surface is composed by 50 nm particles. The RMS surface roughness is 3.68 nm.
Fig. 2
Fig. 2 Comparison of CL image. (a) CL image of the Zn2SiO4. RMS variability of CL intensity includes 24.0%. (b) CL image of the ZnO. RMS variability of CL intensity includes 11.3%.
Fig. 3
Fig. 3 AFM image of (a) as-deposited ZnO and (b) after annealing ZnO surface. Particle size of ZnO in both images is 30 to 50 nm. RMS of as deposited ZnO is 3.00 nm and that of after annealing ZnO is 3.68 nm.
Fig. 4
Fig. 4 (a) High resolution EXA microscopic imaging of the cellular granules (b) Intensity profile of the cellular granules highlighted by arrows in Fig. 2(a). The full width at half maximum is 82 nm and signal-to-noise ratio is 10.5.
Fig. 5
Fig. 5 (a) Electric field intensity distribution of CL excited in ZnO. (b) Electric field intensity profile at the Si3N4 surface. The full width at half maximum (FWHM) of electric field intensity at the Si3N4 surface is 83 nm. (c) Electric field intensity profiles for various distances from the Si3N4 surface. In case of d = 30 nm, the peak intensity falls to 50% compared to the peak intensity of d = 0 nm. (d) Dependence of the FWHM of the electric field intensity on the distance from Si3N4 surface. The FWHM of electric field intensity spreads with increasing distance from Si3N4 surface. If the d = 20 nm, the FWHM of electric field intensity is 117 nm. In the EXA microscope, the high resolution imaging region along the depth direction is around 20 nm from Fig. 3(d).
Fig. 6
Fig. 6 (a) Fluorescent microscopic image of the HeLa cells with stained actin filament and mitochondria. (b) EXA microscope image for the HeLa cells. The observation region is the same as Fig. 6(a). The corresponding actin filaments and mitochondria are indicated by arrows and triangles.
Fig. 7
Fig. 7 (a) Phase-contrast microscopic image for label-free HeLa cells. (b) EXA microscope image for label-free HeLa cells. The actin filament, mitochondria, nucleus, filopodia, and intracellular granule are observed with label-free condition.

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