Abstract

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes.

© 2010 OSA

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References

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  1. W. Denk, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in “Handbook of Biological Confocal Microscopy,”, 3rd ed., J. B. Pawley, ed. (Springer Science + Business Media, LLC, New York, 2006), chap. 28, pp. 535–549
  2. J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998).
    [CrossRef] [PubMed]
  3. M. Straub and S. W. Hell, “Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6(4), 177–185 (1998).
    [CrossRef]
  4. A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
    [CrossRef]
  5. A. Egner and S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000).
    [CrossRef] [PubMed]
  6. V. Andresen, A. Egner, and S. W. Hell, “Time-multiplexed multifocal multiphoton microscope,” Opt. Lett. 26(2), 75–77 (2001).
    [CrossRef] [PubMed]
  7. K. Bahlmann, P. T. So, M. Kirber, R. Reich, B. Kosicki, W. McGonagle, and K. Bellve, “Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz,” Opt. Express 15(17), 10991–10998 (2007).
    [CrossRef] [PubMed]
  8. K. H. Kim, C. Buehler, K. Bahlmann, T. Ragan, W.-C. A. Lee, E. Nedivi, E. L. Heffer, S. Fantini, and P. T. C. So, “Multifocal multiphoton microscopy based on multianode photomultiplier tubes,” Opt. Express 15(18), 11658–11678 (2007).
    [CrossRef] [PubMed]
  9. D. N. Fittinghoff and J. A. Squier, “Time-decorrelated multifocal array for multiphoton microscopy and micromachining,” Opt. Lett. 25(16), 1213–1215 (2000).
    [CrossRef] [PubMed]
  10. L. Sacconi, E. Froner, R. Antolini, M. R. Taghizadeh, A. Choudhury, and F. S. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28(20), 1918–1920 (2003).
    [CrossRef] [PubMed]
  11. D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
    [CrossRef]
  12. T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
    [CrossRef] [PubMed]
  13. M. Fricke and T. Nielsen, “Two-dimensional imaging without scanning by multifocal multiphoton microscopy,” Appl. Opt. 44(15), 2984–2988 (2005).
    [CrossRef] [PubMed]
  14. S. Kumar, C. Dunsby, P. A. De Beule, D. M. Owen, U. Anand, P. M. P. Lanigan, R. K. P. Benninger, D. M. Davis, M. A. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. W. French, “Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging,” Opt. Express 15(20), 12548–12561 (2007).
    [CrossRef] [PubMed]
  15. N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008) (PMID: 18204458.).
    [CrossRef] [PubMed]
  16. K. E. Sheetz, E. E. Hoover, R. Carriles, D. Kleinfeld, and J. A. Squier, “Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator,” Opt. Express 16(22), 17574–17584 (2008).
    [CrossRef] [PubMed]
  17. W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. A. Squier, “Simultaneous imaging of multiple focal planes using a two-photon scanning microscope,” Opt. Lett. 32(12), 1731–1733 (2007).
    [CrossRef] [PubMed]
  18. R. Carriles, K. E. Sheetz, E. E. Hoover, J. A. Squier, and V. Barzda, “Simultaneous multifocal, multiphoton, photon counting microscopy,” Opt. Express 16(14), 10364–10371 (2008).
    [CrossRef] [PubMed]
  19. E. Chandler, E. Hoover, J. Field, K. Sheetz, W. Amir, R. Carriles, S. Y. Ding, and J. Squier, “High-resolution mosaic imaging with multifocal, multiphoton photon-counting microscopy,” Appl. Opt. 48(11), 2067–2077 (2009).
    [CrossRef] [PubMed]
  20. J. J. Field, R. Carriles, K. E. Sheetz, E. V. Chandler, E. E. Hoover, S. E. Tillo, T. E. Hughes, A. W. Sylvester, D. Kleinfeld, and J. A. Squier, “Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation,” Opt. Express 18(13), 13661–13672 (2010).
    [CrossRef] [PubMed]
  21. G. Labroille, R. S. Pillai, X. Solinas, C. Boudoux, N. Olivier, E. Beaurepaire, and M. Joffre, “Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy,” Opt. Lett. 35(20), 3444–3446 (2010).
    [CrossRef] [PubMed]
  22. E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “Aberration-free optical refocusing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
    [CrossRef] [PubMed]
  23. E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
    [CrossRef]
  24. E. J. Botcherby, M. J. Booth, R. Juskaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
    [CrossRef] [PubMed]
  25. E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time slit scanning microscopy in the meridional plane,” Opt. Lett. 34(10), 1504–1506 (2009).
    [CrossRef] [PubMed]
  26. K. Carlsson and N. Åslund, “Confocal imaging for 3-D digital microscopy,” Appl. Opt. 26(16), 3232–3238 (1987).
    [CrossRef] [PubMed]
  27. P. G. Kazansky, W. Yang, E. Bricchi, J. Bovatsek, and A. Arai, ““quill” writing with ultrashort light pulses in transparent optical materials,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference and Photonic Applications Systems Technologies (Optical Society of America, 2007), p. CThJ2.
  28. D. N. Vitek, D. E. Adams, A. Johnson, P. S. Tsai, S. Backus, C. G. Durfee, D. Kleinfeld, and J. A. Squier, “Temporally focused femtosecond laser pulses for low numerical aperture micromachining through optically transparent materials,” Opt. Express 18(17), 18086–18094 (2010).
    [CrossRef] [PubMed]
  29. D. N. Vitek, E. Block, Y. Bellouard, D. E. Adams, S. Backus, D. Kleinfeld, C. G. Durfee, and J. A. Squier, “Spatio-temporally focused femtosecond laser pulses for nonreciprocal writing into optically transparent materials,” Opt. Express 18(24), 24673–24678 (2010).
    [CrossRef]

2010 (4)

2009 (2)

2008 (5)

2007 (5)

2005 (1)

2003 (1)

2001 (3)

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
[CrossRef]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[CrossRef] [PubMed]

V. Andresen, A. Egner, and S. W. Hell, “Time-multiplexed multifocal multiphoton microscope,” Opt. Lett. 26(2), 75–77 (2001).
[CrossRef] [PubMed]

2000 (2)

1998 (3)

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998).
[CrossRef] [PubMed]

M. Straub and S. W. Hell, “Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6(4), 177–185 (1998).
[CrossRef]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
[CrossRef]

1987 (1)

Adams, D. E.

Amir, W.

Anand, P.

Anand, U.

Andresen, P.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[CrossRef] [PubMed]

Andresen, V.

Antolini, R.

Åslund, N.

Backus, S.

Bahlmann, K.

Barzda, V.

Beaurepaire, E.

Bellouard, Y.

Bellve, K.

Benham, C.

Benninger, R. K. P.

Betzig, E.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008) (PMID: 18204458.).
[CrossRef] [PubMed]

Bewersdorf, J.

Block, E.

Booth, M. J.

Botcherby, E. J.

Boudoux, C.

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
[CrossRef]

Buehler, C.

Buist, A. H.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
[CrossRef]

Carlsson, K.

Carriles, R.

Chandler, E.

Chandler, E. V.

Choudhury, A.

Davis, D. M.

De Beule, P. A.

Ding, S. Y.

Dunsby, C.

Durfee, C. G.

Egner, A.

Fantini, S.

Field, J.

Field, J. J.

Fittinghoff, D. N.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
[CrossRef]

D. N. Fittinghoff and J. A. Squier, “Time-decorrelated multifocal array for multiphoton microscopy and micromachining,” Opt. Lett. 25(16), 1213–1215 (2000).
[CrossRef] [PubMed]

French, P. M. W.

Fricke, M.

M. Fricke and T. Nielsen, “Two-dimensional imaging without scanning by multifocal multiphoton microscopy,” Appl. Opt. 44(15), 2984–2988 (2005).
[CrossRef] [PubMed]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[CrossRef] [PubMed]

Froner, E.

Heffer, E. L.

Hell, S. W.

Hellweg, D.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[CrossRef] [PubMed]

Hoover, E.

Hoover, E. E.

Hughes, T. E.

Ji, N.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008) (PMID: 18204458.).
[CrossRef] [PubMed]

Joffre, M.

Johnson, A.

Juskaitis, R.

Juškaitis, R.

Kim, K. H.

Kirber, M.

Kleinfeld, D.

Kosicki, B.

Kumar, S.

Labroille, G.

Lanigan, P. M. P.

Lee, W.-C. A.

Magee, J. C.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008) (PMID: 18204458.).
[CrossRef] [PubMed]

Mazur, E.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
[CrossRef]

McGonagle, W.

Müller, M.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
[CrossRef]

Naylor, A.

Nedivi, E.

Neil, M. A. A.

Nielsen, T.

M. Fricke and T. Nielsen, “Two-dimensional imaging without scanning by multifocal multiphoton microscopy,” Appl. Opt. 44(15), 2984–2988 (2005).
[CrossRef] [PubMed]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[CrossRef] [PubMed]

Olivier, N.

Owen, D. M.

Pavone, F. S.

Pick, R.

Pillai, R. S.

Planchon, T. A.

Ragan, T.

Reich, R.

Sacconi, L.

Schaffer, C. B.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
[CrossRef]

Sheetz, K.

Sheetz, K. E.

So, P. T.

So, P. T. C.

Solinas, X.

Squier, J.

E. Chandler, E. Hoover, J. Field, K. Sheetz, W. Amir, R. Carriles, S. Y. Ding, and J. Squier, “High-resolution mosaic imaging with multifocal, multiphoton photon-counting microscopy,” Appl. Opt. 48(11), 2067–2077 (2009).
[CrossRef] [PubMed]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
[CrossRef]

Squier, J. A.

J. J. Field, R. Carriles, K. E. Sheetz, E. V. Chandler, E. E. Hoover, S. E. Tillo, T. E. Hughes, A. W. Sylvester, D. Kleinfeld, and J. A. Squier, “Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation,” Opt. Express 18(13), 13661–13672 (2010).
[CrossRef] [PubMed]

D. N. Vitek, D. E. Adams, A. Johnson, P. S. Tsai, S. Backus, C. G. Durfee, D. Kleinfeld, and J. A. Squier, “Temporally focused femtosecond laser pulses for low numerical aperture micromachining through optically transparent materials,” Opt. Express 18(17), 18086–18094 (2010).
[CrossRef] [PubMed]

D. N. Vitek, E. Block, Y. Bellouard, D. E. Adams, S. Backus, D. Kleinfeld, C. G. Durfee, and J. A. Squier, “Spatio-temporally focused femtosecond laser pulses for nonreciprocal writing into optically transparent materials,” Opt. Express 18(24), 24673–24678 (2010).
[CrossRef]

R. Carriles, K. E. Sheetz, E. E. Hoover, J. A. Squier, and V. Barzda, “Simultaneous multifocal, multiphoton, photon counting microscopy,” Opt. Express 16(14), 10364–10371 (2008).
[CrossRef] [PubMed]

K. E. Sheetz, E. E. Hoover, R. Carriles, D. Kleinfeld, and J. A. Squier, “Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator,” Opt. Express 16(22), 17574–17584 (2008).
[CrossRef] [PubMed]

W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. A. Squier, “Simultaneous imaging of multiple focal planes using a two-photon scanning microscope,” Opt. Lett. 32(12), 1731–1733 (2007).
[CrossRef] [PubMed]

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
[CrossRef]

D. N. Fittinghoff and J. A. Squier, “Time-decorrelated multifocal array for multiphoton microscopy and micromachining,” Opt. Lett. 25(16), 1213–1215 (2000).
[CrossRef] [PubMed]

Straub, M.

M. Straub and S. W. Hell, “Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6(4), 177–185 (1998).
[CrossRef]

Sylvester, A. W.

Taghizadeh, M. R.

Tillo, S. E.

Tsai, P. S.

Vitek, D. N.

Wilson, T.

Appl. Opt. (3)

Bioimaging (1)

M. Straub and S. W. Hell, “Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6(4), 177–185 (1998).
[CrossRef]

IEEE J. Quantum Electron. (1)

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. A. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Quantum Electron. 7(4), 559–566 (2001).
[CrossRef]

J. Microsc. (1)

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201(3), 368–376 (2001).
[CrossRef] [PubMed]

J. Microsc. (Oxford) (1)

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. (Oxford) 192(2), 217–226 (1998).
[CrossRef]

J. Opt. Soc. Am. A (1)

Nat. Methods (1)

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008) (PMID: 18204458.).
[CrossRef] [PubMed]

Opt. Commun. (1)

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
[CrossRef]

Opt. Express (9)

E. J. Botcherby, M. J. Booth, R. Juskaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
[CrossRef] [PubMed]

J. J. Field, R. Carriles, K. E. Sheetz, E. V. Chandler, E. E. Hoover, S. E. Tillo, T. E. Hughes, A. W. Sylvester, D. Kleinfeld, and J. A. Squier, “Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation,” Opt. Express 18(13), 13661–13672 (2010).
[CrossRef] [PubMed]

D. N. Vitek, D. E. Adams, A. Johnson, P. S. Tsai, S. Backus, C. G. Durfee, D. Kleinfeld, and J. A. Squier, “Temporally focused femtosecond laser pulses for low numerical aperture micromachining through optically transparent materials,” Opt. Express 18(17), 18086–18094 (2010).
[CrossRef] [PubMed]

D. N. Vitek, E. Block, Y. Bellouard, D. E. Adams, S. Backus, D. Kleinfeld, C. G. Durfee, and J. A. Squier, “Spatio-temporally focused femtosecond laser pulses for nonreciprocal writing into optically transparent materials,” Opt. Express 18(24), 24673–24678 (2010).
[CrossRef]

R. Carriles, K. E. Sheetz, E. E. Hoover, J. A. Squier, and V. Barzda, “Simultaneous multifocal, multiphoton, photon counting microscopy,” Opt. Express 16(14), 10364–10371 (2008).
[CrossRef] [PubMed]

K. E. Sheetz, E. E. Hoover, R. Carriles, D. Kleinfeld, and J. A. Squier, “Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator,” Opt. Express 16(22), 17574–17584 (2008).
[CrossRef] [PubMed]

S. Kumar, C. Dunsby, P. A. De Beule, D. M. Owen, U. Anand, P. M. P. Lanigan, R. K. P. Benninger, D. M. Davis, M. A. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. W. French, “Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging,” Opt. Express 15(20), 12548–12561 (2007).
[CrossRef] [PubMed]

K. Bahlmann, P. T. So, M. Kirber, R. Reich, B. Kosicki, W. McGonagle, and K. Bellve, “Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz,” Opt. Express 15(17), 10991–10998 (2007).
[CrossRef] [PubMed]

K. H. Kim, C. Buehler, K. Bahlmann, T. Ragan, W.-C. A. Lee, E. Nedivi, E. L. Heffer, S. Fantini, and P. T. C. So, “Multifocal multiphoton microscopy based on multianode photomultiplier tubes,” Opt. Express 15(18), 11658–11678 (2007).
[CrossRef] [PubMed]

Opt. Lett. (8)

D. N. Fittinghoff and J. A. Squier, “Time-decorrelated multifocal array for multiphoton microscopy and micromachining,” Opt. Lett. 25(16), 1213–1215 (2000).
[CrossRef] [PubMed]

L. Sacconi, E. Froner, R. Antolini, M. R. Taghizadeh, A. Choudhury, and F. S. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28(20), 1918–1920 (2003).
[CrossRef] [PubMed]

V. Andresen, A. Egner, and S. W. Hell, “Time-multiplexed multifocal multiphoton microscope,” Opt. Lett. 26(2), 75–77 (2001).
[CrossRef] [PubMed]

W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. A. Squier, “Simultaneous imaging of multiple focal planes using a two-photon scanning microscope,” Opt. Lett. 32(12), 1731–1733 (2007).
[CrossRef] [PubMed]

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998).
[CrossRef] [PubMed]

G. Labroille, R. S. Pillai, X. Solinas, C. Boudoux, N. Olivier, E. Beaurepaire, and M. Joffre, “Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy,” Opt. Lett. 35(20), 3444–3446 (2010).
[CrossRef] [PubMed]

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “Aberration-free optical refocusing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
[CrossRef] [PubMed]

E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time slit scanning microscopy in the meridional plane,” Opt. Lett. 34(10), 1504–1506 (2009).
[CrossRef] [PubMed]

Other (2)

P. G. Kazansky, W. Yang, E. Bricchi, J. Bovatsek, and A. Arai, ““quill” writing with ultrashort light pulses in transparent optical materials,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference and Photonic Applications Systems Technologies (Optical Society of America, 2007), p. CThJ2.

W. Denk, D. W. Piston, and W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in “Handbook of Biological Confocal Microscopy,”, 3rd ed., J. B. Pawley, ed. (Springer Science + Business Media, LLC, New York, 2006), chap. 28, pp. 535–549

Supplementary Material (2)

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Figures (6)

Fig. 1.
Fig. 1.

Basic axial scanning microscope system. HWP: Half Wave-plate, PBS: Polarizing Beam-splitter, QWP: Quarter Wave-plate, PM: Piezoelectric Mirror, L1,L2: Matched Lenses, OBJ: Objective, Solid Beam: Normal (collimated) focus, Dotted Beam: Shallower focus, Dashed Beam: Deeper focus

Fig. 2.
Fig. 2.

Two beam remote focusing microscope layout. HWP: Half Wave-plate, PBS: Polarizing Beam-splitter, QWP: QuarterWave-plate, AS: Asphere, PM: Piezoelectric Mirror, L1: 250mm Lens, L2: 500mm Lens, SM: Scan Mirror, L3: 200mm Lens, L4: 200mm Lens, OBJ: Objective

Fig. 3.
Fig. 3.

Combined x-y and x-z THG scan of features created through spatio-temporal femtosecond micromachining in a fused quartz slide. THG images are limited to 300 photon counts, while the white light image is shown in upper right.

Fig. 4.
Fig. 4.

Four beam remote focusing microscope layout. λ/2: Half Wave-plate, PBS: Polarizing Beam-splitter, λ/4: Quarter Wave-plate, AS: Asphere, PM: Piezoelectric Mirror, L1: 400mm Lens, L2: 100mm Lens, L3: 40mm Lens, L4: 40mm Lens, L5: 35mm Lens, L6: 200mm Lens, NDW: Neutral Density Wheel, SM: Scan Mirror, OBJ: Objective

Fig. 5.
Fig. 5.

Single-scan excerpt from a video illustrating simultaneous acquisition of four focal planes, the x and y axes are in microns and the intensity map is in photon counts. This video (Media 1) demonstrates how the focal planes can be programmatically adjusted by sweeping one of the focal planes through the range of the other three. A large dynamic range is achieved by using a frame exposure time of 40 seconds, with an average per-beam power of 14mW in order to avoid damaging the specimen.

Fig. 6.
Fig. 6.

Single-scan excerpt from a movie of a living corn maize specimen demonstrating the simultaneous acquisition of four focal planes, the x and y axes are in microns and the intensity map is in photon counts. In this video (Media 2) the focal planes are adjusted to a static ~ 7µm offset and a live sample is imaged over time. Each frame is captured with a 40 second exposure time, for a 14mW per-beam average power, in order to produce significant image contrast without harming the sample.

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