Abstract

High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.

© 2015 Optical Society of America

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References

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]

2014 (4)

D. B. Peckys and N. de Jonge, “Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells,” Microsc. Microanal. 20(2), 346–365 (2014).
[Crossref] [PubMed]

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Y. Nawa, W. Inami, S. Lin, Y. Kawata, S. Terakawa, C.-Y. Fang, and H.-C. Chang, “Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation,” ChemPhysChem 15(4), 721–726 (2014).
[Crossref] [PubMed]

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]

2013 (4)

H. T. Miyazaki, T. Kasaya, T. Takemura, N. Hanagata, T. Yasuda, and H. Miyazaki, “Diffraction-unlimited optical imaging of unstained living cells in liquid by electron beam scanning of luminescent environmental cells,” Opt. Express 21(23), 28198–28218 (2013).
[Crossref] [PubMed]

T. Hoshino and K. Mabuchi, “Closed-looped in situ nano processing on a culturing cell using an inverted electron beam lithography system,” Biochem. Biophys. Res. Commun. 432(2), 345–349 (2013).
[Crossref] [PubMed]

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

2012 (4)

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (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]

2011 (1)

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

2010 (2)

2007 (1)

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

2004 (2)

J. V. Rocheleau, W. S. Head, and D. W. Piston, “Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response,” J. Biol. Chem. 279(30), 31780–31787 (2004).
[Crossref] [PubMed]

T. Yasui, Y. Tohno, and T. Araki, “Determination of collagen fiber orientation in human tissue by use of polarization measurement of molecular second-harmonic-generation light,” Appl. Opt. 43(14), 2861–2867 (2004).
[Crossref] [PubMed]

1993 (1)

G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993).
[Crossref] [PubMed]

1987 (1)

M. Kubista, B. Aakerman, and B. Norden, “Characterization of Interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy,” Biochemistry 26(14), 4545–4553 (1987).
[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]

Aakerman, B.

M. Kubista, B. Aakerman, and B. Norden, “Characterization of Interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy,” Biochemistry 26(14), 4545–4553 (1987).
[Crossref] [PubMed]

Adamo, C.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Allouche, F.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Aloni, S.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Alterini, R.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Andreoli, F.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Araki, T.

Basile, V.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Bhattacharya, D.

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012).
[Crossref] [PubMed]

Bischak, C. G.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Casana, Y.

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012).
[Crossref] [PubMed]

Chang, H.-C.

Y. Nawa, W. Inami, S. Lin, Y. Kawata, S. Terakawa, C.-Y. Fang, and H.-C. Chang, “Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation,” ChemPhysChem 15(4), 721–726 (2014).
[Crossref] [PubMed]

Chiba, A.

Choi, W.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Clarkson, J. D.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Dassari, R. R.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

de Jonge, N.

D. B. Peckys and N. de Jonge, “Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells,” Microsc. Microanal. 20(2), 346–365 (2014).
[Crossref] [PubMed]

de Kock, C. P. J.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Ebihara, T.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Endo, H.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Fang, C.-Y.

Y. Nawa, W. Inami, S. Lin, Y. Kawata, S. Terakawa, C.-Y. Fang, and H.-C. Chang, “Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation,” ChemPhysChem 15(4), 721–726 (2014).
[Crossref] [PubMed]

Fang-Yen, C.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Feld, M. S.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Fu, D.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Fujimoto, T.

G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993).
[Crossref] [PubMed]

Fujita, K.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Fusi, F.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Ginsberg, N. S.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Giustozzi, G.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Hanagata, N.

Head, W. S.

J. V. Rocheleau, W. S. Head, and D. W. Piston, “Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response,” J. Biol. Chem. 279(30), 31780–31787 (2004).
[Crossref] [PubMed]

Heilemann, M.

M. Heilemann, “Fluorescence microscopy beyond the diffraction limit,” J. Biotechnol. 149(4), 243–251 (2010).
[Crossref] [PubMed]

Hetherington, C. L.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Hirano, K.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Hoshino, T.

T. Hoshino and K. Mabuchi, “Closed-looped in situ nano processing on a culturing cell using an inverted electron beam lithography system,” Biochem. Biophys. Res. Commun. 432(2), 345–349 (2013).
[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]

Howard, H. H.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Inami, W.

Jamme, F.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Kasaya, T.

Kascakova, S.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Kawata, S.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Kawata, Y.

Kaz, D. M.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Kinoshita, T.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Koike, H.

G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993).
[Crossref] [PubMed]

Kubista, M.

M. Kubista, B. Aakerman, and B. Norden, “Characterization of Interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy,” Biochemistry 26(14), 4545–4553 (1987).
[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]

Lin, S.

Lodder, J. C.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Louise Groot, M.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Mabuchi, K.

T. Hoshino and K. Mabuchi, “Closed-looped in situ nano processing on a culturing cell using an inverted electron beam lithography system,” Biochem. Biophys. Res. Commun. 432(2), 345–349 (2013).
[Crossref] [PubMed]

Mansvelder, H. D.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Marti, X.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Maruyama, Y.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Matsudaira, P.

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012).
[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]

Mirsaidov, U. M.

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012).
[Crossref] [PubMed]

Miyakawa, A.

Miyake, A.

Miyazaki, H.

Miyazaki, H. T.

Monici, M.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

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]

Motohashi, H.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Nakajima, K.

Nawa, Y.

Negrean, A.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Ning, G.

G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993).
[Crossref] [PubMed]

Nishihara, S.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Nishiyama, H.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Norden, B.

M. Kubista, B. Aakerman, and B. Norden, “Characterization of Interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy,” Biochemistry 26(14), 4545–4553 (1987).
[Crossref] [PubMed]

Ogawa, K.

G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993).
[Crossref] [PubMed]

Ogletree, D. F.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Oh, S.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Okada, M.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Ono, A.

Pallu, S.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Palonpon, A. F.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Pantalone, D.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Park, Y.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Peckys, D. B.

D. B. Peckys and N. de Jonge, “Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells,” Microsc. Microanal. 20(2), 346–365 (2014).
[Crossref] [PubMed]

Piston, D. W.

J. V. Rocheleau, W. S. Head, and D. W. Piston, “Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response,” J. Biol. Chem. 279(30), 31780–31787 (2004).
[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]

Ramesh, R.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Réfrégiers, M.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Rigacci, L.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Rocheleau, J. V.

J. V. Rocheleau, W. S. Head, and D. W. Piston, “Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response,” J. Biol. Chem. 279(30), 31780–31787 (2004).
[Crossref] [PubMed]

Romano, G.

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

Rouam, V.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Sato, C.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Sato, M.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Schlom, D. G.

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Smith, N. I.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Sodeoka, M.

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

Suga, M.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Takemura, T.

Terakawa, S.

Testa Silva, G.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Tohno, Y.

Tsuji, N. M.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Uemura, T.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Villette, S.

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Watanabe, Y.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Witte, S.

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

Yamamoto, M.

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Yaqoob, Z.

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Yasuda, T.

Yasui, T.

Zheng, H.

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012).
[Crossref] [PubMed]

ACS Nano (1)

D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013).
[Crossref] [PubMed]

Appl. Opt. (1)

Biochem. Biophys. Res. Commun. (1)

T. Hoshino and K. Mabuchi, “Closed-looped in situ nano processing on a culturing cell using an inverted electron beam lithography system,” Biochem. Biophys. Res. Commun. 432(2), 345–349 (2013).
[Crossref] [PubMed]

Biochemistry (1)

M. Kubista, B. Aakerman, and B. Norden, “Characterization of Interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy,” Biochemistry 26(14), 4545–4553 (1987).
[Crossref] [PubMed]

Biol. Cell (1)

F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

Biophys. J. (1)

S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012).
[Crossref] [PubMed]

Cell Tissue Res. (1)

G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993).
[Crossref] [PubMed]

ChemPhysChem (1)

Y. Nawa, W. Inami, S. Lin, Y. Kawata, S. Terakawa, C.-Y. Fang, and H.-C. Chang, “Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation,” ChemPhysChem 15(4), 721–726 (2014).
[Crossref] [PubMed]

Clin. Gastroenterol. Hepatol. (1)

D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007).
[Crossref] [PubMed]

J. Biol. Chem. (1)

J. V. Rocheleau, W. S. Head, and D. W. Piston, “Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response,” J. Biol. Chem. 279(30), 31780–31787 (2004).
[Crossref] [PubMed]

J. Biotechnol. (1)

M. Heilemann, “Fluorescence microscopy beyond the diffraction limit,” J. Biotechnol. 149(4), 243–251 (2010).
[Crossref] [PubMed]

Microsc. Microanal. (1)

D. B. Peckys and N. de Jonge, “Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells,” Microsc. Microanal. 20(2), 346–365 (2014).
[Crossref] [PubMed]

Opt. Express (3)

Proc. Natl. Acad. Sci. U.S.A. (4)

M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012).
[Crossref] [PubMed]

S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011).
[Crossref] [PubMed]

U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012).
[Crossref] [PubMed]

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]

Ultramicroscopy (1)

K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014).
[PubMed]

Other (1)

A. Rose, Advances in Electronics vol.1 (Academic Press Inc. New York, 1948).

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Figures (5)

Fig. 1
Fig. 1 Stained adiposomes in MC3T3-E1 cells observed with the D-EXA, confocal, and phase contrast. (a) Raw D-EXA microscope image without processing. Granules were observed with high spatial resolution. The D-EXA image is primarily autofluorescence. (b) Confocal image of adiposomes. (c) Phase-contrast microscope image. (d, e, f) Magnified image of the area indicated with white rectangle shown in (a), (b), and (c), respectively. Adiposomes were observed as a bright spots in (d) and (e), and they were observed as dark spots in (f). In (d), particles other than adiposomes were also imaged by autofluorescence, as indicated by arrows. D-EXA and confocal images have pseudocolour.
Fig. 2
Fig. 2 Actin filaments, mitochondria, and nuclei observed with a D-EXA microscope, epifluorescence, and phase-contrast. D-EXA and epifluorescence images are in pseudocolour. In the epifluorescence images, mitochondrial, actin filaments, and nuclei are red, green, and blue, respectively. (a-d) Comparison of actin filament images. (a) D-EXA image. Filamentous structures were clearly observed. Granules were also observed without staining, but with autofluorescence excited by electron beam irradiation, as indicated with the arrowhead. (b, c) Epifluorescence image (d) Phase contrast image. (e-h) Comparison of mitochondria images. (e) D-EXA image. Mitochondria were imaged as indicated with the arrows. The arrowhead shows a granule observed without staining, but with autofluorescence excited by an electron beam. (f, g) Epi-fluorescence image (h) Phase contrast image. (i-l) Comparison of nuclei images. (i) D-EXA image. DNA dense area in nucleus was observed indicated with arrows in (i), (k), and (l). (j, k) Epi-fluorescence image (l) Phase contrast image.
Fig. 3
Fig. 3 D-EXA imaging of live MC3T3-E1 cells. (a) D-EXA image in pseudocolour without processing. Cells were observed without staining. The nucleus, nucleus membrane, intracellular granules, mitochondria, and DNA dense regions in the nucleus are identified. Only cells closely attached to the substrate were observed. (b) Phase contrast image. (c) D-EXA image of other cells with high magnification. Fine structures inside nucleus and membrane are observed.
Fig. 4
Fig. 4 Bleaching in the D-EXA imaging. (a) Second frame image of Fig. 3(a). Components inside cells were observed clearly. (b) Magnified area indicated with white rectangle of the first frame image. (c) Magnified area of the second frame image. (d) CL intensity distributions indicated with lines in fig. (b) and (c). CL from cell membrane is more decreased than CL from organelles during image acquisition.
Fig. 5
Fig. 5 Imaging of fixed cells for evaluation of spatial resolution. (a) D-EXA image in pseudocolour without processing. The outline of the nucleus and fine particles are observed. (b) Phase contrast image. (c) CL intensity distribution of the particle indicated between white arrows in (a). The FWHM of the Gaussian fit is 105 nm.

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