Abstract

Intracellular structures of HeLa cells are observed using a direct electron beam excitation-assisted fluorescence (D-EXA) microscope. In this microscope, a silicon nitride membrane is used as a culture plate, which typically has a low biocompatibility between the sample and the silicon nitride surface to prevent the HeLa cells from adhering strongly to the surface. In this work, the surface of silicon nitride is modified to allow strong cell attachment, which enables high-resolution observation of intracellular structures and an increased signal-to-noise ratio. In addition, the penetration depth of the electron beam is evaluated using Monte Carlo simulations. We can conclude from the results of the observations and simulations that the surface modification technique is promising for the observation of intracellular structures using the D-EXA microscope.

© 2015 Optical Society of America

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References

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  1. H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
    [Crossref] [PubMed]
  2. T. Ogura, “Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy,” Biochem. Biophys. Res. Commun. 391(1), 198–202 (2010).
    [Crossref] [PubMed]
  3. N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
    [Crossref] [PubMed]
  4. 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).
    [PubMed]
  5. Y. Nawa, W. Inami, A. Chiba, A. Ono, A. Miyakawa, Y. Kawata, S. Lin, and S. Terakawa, “Dynamic and high-resolution live cell imaging by direct electron beam excitation,” Opt. Express 20(5), 5629–5635 (2012).
    [Crossref] [PubMed]
  6. Y. Nawa, W. Inami, A. Miyake, A. Ono, Y. Kawata, S. Lin, and S. Terakawa, “Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation,” Biomed. Opt. Express 5(2), 378–386 (2014).
    [Crossref] [PubMed]
  7. F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
    [Crossref]
  8. Y. Masuda, W. Inami, A. Miyakawa, and Y. Kawata, “Cell culture on hydrophilicity-controlled silicon nitride surfaces,” World J. Microbiol. Biotechnol.under review.
  9. G. Nichols, “Applications of cathodoluminescence spectroscopy and imaging in the characterisation of pharmaceutical materials,” Eur. J. Pharm. Sci. 45(1-2), 19–42 (2012).
    [Crossref] [PubMed]
  10. 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. USA 73(2), 317–321 (1976).
    [Crossref] [PubMed]

2014 (1)

2012 (2)

G. Nichols, “Applications of cathodoluminescence spectroscopy and imaging in the characterisation of pharmaceutical materials,” Eur. J. Pharm. Sci. 45(1-2), 19–42 (2012).
[Crossref] [PubMed]

Y. Nawa, W. Inami, A. Chiba, A. Ono, A. Miyakawa, Y. Kawata, S. Lin, and S. Terakawa, “Dynamic and high-resolution live cell imaging by direct electron beam excitation,” Opt. Express 20(5), 5629–5635 (2012).
[Crossref] [PubMed]

2010 (3)

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).
[PubMed]

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

T. Ogura, “Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy,” Biochem. Biophys. Res. Commun. 391(1), 198–202 (2010).
[Crossref] [PubMed]

2009 (1)

N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
[Crossref] [PubMed]

2004 (1)

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Cattaruzza, F.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

Chiba, A.

Cricenti, A.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

de Jonge, N.

N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
[Crossref] [PubMed]

Flamini, A.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

Girasole, M.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Inami, W.

Kawata, Y.

Kitamura, S.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Koizumi, M.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Kremers, G. J.

N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
[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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Lin, S.

Longo, G.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

Maruyama, Y.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Masuda, Y.

Y. Masuda, W. Inami, A. Miyakawa, and Y. Kawata, “Cell culture on hydrophilicity-controlled silicon nitride surfaces,” World J. Microbiol. Biotechnol.under review.

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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Mezzi, A.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

Mio, K.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Miyakawa, A.

Miyake, 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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Nakajima, K.

Nawa, Y.

Nichols, G.

G. Nichols, “Applications of cathodoluminescence spectroscopy and imaging in the characterisation of pharmaceutical materials,” Eur. J. Pharm. Sci. 45(1-2), 19–42 (2012).
[Crossref] [PubMed]

Nishiyama, H.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Ogura, T.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

T. Ogura, “Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy,” Biochem. Biophys. Res. Commun. 391(1), 198–202 (2010).
[Crossref] [PubMed]

Ono, A.

Peckys, D. B.

N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
[Crossref] [PubMed]

Piston, D. W.

N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
[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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Prosperi, T.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

Sato, C.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Suga, M.

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Terakawa, S.

Biochem. Biophys. Res. Commun. (1)

T. Ogura, “Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy,” Biochem. Biophys. Res. Commun. 391(1), 198–202 (2010).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

Eur. J. Pharm. Sci. (1)

G. Nichols, “Applications of cathodoluminescence spectroscopy and imaging in the characterisation of pharmaceutical materials,” Eur. J. Pharm. Sci. 45(1-2), 19–42 (2012).
[Crossref] [PubMed]

J. Mater. Chem. (1)

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi, and T. Prosperi, “Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces,” J. Mater. Chem. 14(9), 1461–1468 (2004).
[Crossref]

J. Struct. Biol. (1)

H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 169(3), 438–449 (2010).
[Crossref] [PubMed]

Opt. Express (2)

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

N. de Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2159–2164 (2009).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. USA (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. USA 73(2), 317–321 (1976).
[Crossref] [PubMed]

Other (1)

Y. Masuda, W. Inami, A. Miyakawa, and Y. Kawata, “Cell culture on hydrophilicity-controlled silicon nitride surfaces,” World J. Microbiol. Biotechnol.under review.

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Figures (5)

Fig. 1
Fig. 1 Schematic showing the principle of direct electron beam excitation.
Fig. 2
Fig. 2 Phase-contrast microscopy image of HeLa cells (a) on a modified membrane and (b) on an unmodified membrane.
Fig. 3
Fig. 3 Images of fixed HeLa cells using D-EXA microscopy with (a) modified and (b) unmodified silicon nitride, and the same areas using phase-contrast microscopy with (c) modified and (d) unmodified silicon nitride. Granules (solid arrowheads) and fiber-like structures (hollow arrowheads) are indicated. The shafted arrow in (a) and (c) indicate the nucleus. The white squares in (a) and (b) are the areas evaluated in Fig. 4(a) and 4(b), respectively.
Fig. 4
Fig. 4 Photon number profiles of a granule in a HeLa cell on (a) modified and (b) unmodified silicon nitride. (Insets) Respective D-EXA images from which the line scan profiles are obtained.
Fig. 5
Fig. 5 Monte Carlo simulation result of electron trajectories.

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