Abstract

We present a novel Yb:KGd(WO4)2 oscillator design that generates six beams of temporally delayed, 253 fs, 11 nJ pulses. This allows multifocal nonlinear microscopy to be performed without the need for complicated optical multiplexers. We demonstrate our design with twelve simultaneously acquired two-photon, second-harmonic and/or third-harmonic images generated from six laterally separated foci.

© 2008 Optical Society of America

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  1. V. Andresen, A. Egner, and S. W. Hell, "Time-multiplexed multifocal multiphoton microscope," Opt. Lett. 26, 75-77, (2001).
    [CrossRef]
  2. N. Ji, J. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat Methods 5, 197-202 (2008).
    [CrossRef]
  3. M. Fricke and T. Nielsen, "Two-dimensional imaging without scanning by multifocal multiphoton microscopy," Appl. Opt. 44,2984-2988 (2005).
    [CrossRef] [PubMed]
  4. M. Straub and S. W. Hell, "Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging," Bioimaging 6. 177-185 (1998).
    [CrossRef]
  5. K. Bahlmann, P. T. C. 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, 10991-10998 (2007).
    [CrossRef] [PubMed]
  6. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
    [CrossRef]
  7. A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
    [CrossRef]
  8. D. N. Fittinghoff and J. Squier, "Time-decorrelated multifocal array for multiphoton microscopy and micromachining," Opt. Lett. 25, 1213-1215 (2000).
    [CrossRef]
  9. K. H. Kim, C. Buehler, K. Bahlmann, T. Ragan, W. 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, 11658-11678 (2007).
    [CrossRef] [PubMed]
  10. W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. Squier "Simultaneous imaging of multiple focal planes using a two-photon scanning microscope," Opt. Lett. 32, 1731-1733 (2007).
    [CrossRef] [PubMed]
  11. R. Carriles, K. E. Sheetz, E. E. Hoover, and J. Squier, "Simultaneous multifocal, multiphoton, photon counting microscopy," Opt. Express 16, 10364-10371 (2008).
    [CrossRef] [PubMed]
  12. A. Major, R. Cisek, and V. Barzda, "Femtosecond Yb: KGd (WO4) 2 laser oscillator pumped by a high power fiber-coupled diode laser module," Opt. Express 14, 12163-12168 (2006).
    [CrossRef] [PubMed]
  13. F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
    [CrossRef]
  14. D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
    [CrossRef]
  15. J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
    [CrossRef]
  16. Q. T. Nguyen and D. Kleinfeld, "Positive feedback in a brainstem tactile sensorimotor loop," Neuron 45, 447-457 (2005).
    [CrossRef] [PubMed]
  17. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  18. F. Helmchen and W. Denk, "New developments in multiphoton microscopy," Curr. Opin. Neurobiol. 12, 593-601 (2002).
    [CrossRef] [PubMed]

2008

N. Ji, J. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat Methods 5, 197-202 (2008).
[CrossRef]

R. Carriles, K. E. Sheetz, E. E. Hoover, and J. Squier, "Simultaneous multifocal, multiphoton, photon counting microscopy," Opt. Express 16, 10364-10371 (2008).
[CrossRef] [PubMed]

2007

2006

2005

Q. T. Nguyen and D. Kleinfeld, "Positive feedback in a brainstem tactile sensorimotor loop," Neuron 45, 447-457 (2005).
[CrossRef] [PubMed]

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

2002

F. Helmchen and W. Denk, "New developments in multiphoton microscopy," Curr. Opin. Neurobiol. 12, 593-601 (2002).
[CrossRef] [PubMed]

2001

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

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

2000

1998

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

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
[CrossRef]

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

1992

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Amir, W.

Andresen, V.

Bahlmann, K.

Barzda, V.

Bellve, K.

Betzig, E.

N. Ji, J. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat Methods 5, 197-202 (2008).
[CrossRef]

Bewersdorf, J.

Brabec, T.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Brakenhoff, G. J.

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

Buehler, C.

Buist, A. H.

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

Carriles, R.

Cisek, R.

Curley, P. F.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Denk, W.

F. Helmchen and W. Denk, "New developments in multiphoton microscopy," Curr. Opin. Neurobiol. 12, 593-601 (2002).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Durfee, C. G.

Egner, A.

Fantini, S.

Fermann, M. E.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Fittinghoff, D. N.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

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

Fricke, M.

Heffer, E. L.

Hell, S. W.

Helmchen, F.

F. Helmchen and W. Denk, "New developments in multiphoton microscopy," Curr. Opin. Neurobiol. 12, 593-601 (2002).
[CrossRef] [PubMed]

Hofer, M.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Hoover, E. E.

Ji, N.

N. Ji, J. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat Methods 5, 197-202 (2008).
[CrossRef]

Kim, K. H.

Kirber, M.

Kleinfeld, D.

Q. T. Nguyen and D. Kleinfeld, "Positive feedback in a brainstem tactile sensorimotor loop," Neuron 45, 447-457 (2005).
[CrossRef] [PubMed]

Kosicki, B.

Krausz, F.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Lee, W. A.

Magee, J.

N. Ji, J. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat Methods 5, 197-202 (2008).
[CrossRef]

Major, A.

Mazur, E.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

McGonagle, W.

Muller, M.

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

Müller, M.

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

Nedivi, E.

Nguyen, Q. T.

Q. T. Nguyen and D. Kleinfeld, "Positive feedback in a brainstem tactile sensorimotor loop," Neuron 45, 447-457 (2005).
[CrossRef] [PubMed]

Nielsen, T.

Ober, M. H.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Pick, R.

Planchon, T. A.

Ragan, T.

Reich, R.

Schaffer, C. B.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

Schmidt, A. J.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Sheetz, K. E.

So, P. T. C.

Spielmann, C.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Squier, J.

R. Carriles, K. E. Sheetz, E. E. Hoover, and J. Squier, "Simultaneous multifocal, multiphoton, photon counting microscopy," Opt. Express 16, 10364-10371 (2008).
[CrossRef] [PubMed]

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

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

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

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

Straub, M.

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

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Wintner, E.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

Appl. Opt.

Bioimaging

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

Curr. Opin. Neurobiol.

F. Helmchen and W. Denk, "New developments in multiphoton microscopy," Curr. Opin. Neurobiol. 12, 593-601 (2002).
[CrossRef] [PubMed]

IEEE J. Quantum Electron.

F. Krausz, M. E. Fermann, T. Brabec, P. F. Curley, M. Hofer, M. H. Ober, C. Spielmann, E. Wintner, and A. J. Schmidt "Femtosecond solid-state lasers," IEEE J. Quantum Electron. 28, 2097-2121 (1992).
[CrossRef]

IEEE J. Sel. Top. Quantum Electron.

D. N. Fittinghoff, C. B. Schaffer, E. Mazur, and J. Squier, "Time-decorrelated multifocal micromachining and trapping," IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

J. Microsc.

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

Nat Methods

N. Ji, J. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat Methods 5, 197-202 (2008).
[CrossRef]

Neuron

Q. T. Nguyen and D. Kleinfeld, "Positive feedback in a brainstem tactile sensorimotor loop," Neuron 45, 447-457 (2005).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Rev. Sci. Instrum.

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

Science

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

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Figures (8)

Fig. 1.
Fig. 1.

Block diagram showing the simplification realized with the Yb:KGW multibeam laser design for nonlinear microscopy.

Fig. 2.
Fig. 2.

Schematic representation of Yb:KGW oscillator layout. Mirrors are labeled by M1–M10, Gire-Tournois interferometer mirrors by GTI, and semiconductor saturable absorber mirror by SESAM. The laser cavity footprint is 125 cm by 50 cm.

Fig. 3.
Fig. 3.

Schematic representation of multifocal scan optics. Achromatic doublets are labeled L1–L5. Magnified inset shows beam configuration on back pupil of objective required for scanning. Central beams are omitted for clarity.

Fig. 4.
Fig. 4.

Four corners of an 88 µm by 88 µm scan area for the six-foci system. Image shows overlaid CCD screen captures of two-photon fluorescence from Rhodamine-6G sample. Objective was a Zeiss A-plan 40×, 0.65 NA.

Fig. 5.
Fig. 5.

Graphical portrayal of foci geometry in a sample. Axial dimension (z) is stretched relative to lateral dimension for clarity. The depth of focus is indicated by curved black lines on the focusing beam and the offset of the center of focus is marked by black dot. Beams are numbered top to bottom as arrayed in the CCD image capture.

Fig. 6.
Fig. 6.

Schematic representation of the experimental setup (not to scale): D, dichroic mirror; HS, harmonic separator; CS, 190 µm-thick coverslip; F, interference and/or color glass filters; FM, flipper mirrors; FL, focusing lenses (f=50 mm); PD, photodiode; PMT, photomultiplier tube; CCD, CCD camera; OBJ, objective lens. The photodiode is used to provide a laser clock for electronic demultiplexing. The flipper mirrors are used to obtain white light images of the sample region of interest. Details of the multibeam laser and microscope scan optics are in Figs. 2 and 3 respectively.

Fig. 7.
Fig. 7.

Twelve simultaneously acquired images of trigeminal nerve from the adult mouse sectioned at 50 µm. Panel (a) shows a white light image of the region of interest; field of view denoted by black box. Panels (b) and (c) are SHG and THG images respectively generated at each of the six foci; beam numbers between panels correspond to numbering shown in Fig. 6. Images are 128×128 pixels; photon count scales shown with image 1 apply to all images in the respective column. A Zeiss A-plan 40×, 0.65 NA objective was used for excitation and a New Focus Aspheric lens, 60x, 0.65 NA was used for collection; the acquisition time was 10 s.

Fig. 8.
Fig. 8.

Eight of twelve simultaneously acquired images of a corn starch granule surrounded by fluorescent microspheres; image pairs 5 and 6 do not differ from 4. All beams except beam 2 are blocked just prior to the objective lens. Panel (a) shows a white light image of the region of interest. Panels (b) and (c) are SHG and TPEF images, respectively, attributed to the first four foci; beam numbers between panels correspond to numbering shown in Fig. 6. Images are 128×128 pixels, Zeiss A-plan 40×, 0.65 NA objectives were used for excitation and collection; the acquisition time was 10 s.

Tables (1)

Tables Icon

Table 1. Axial resolution and axial offset for each of the six beams. Axial offset is relative to the first focusing (shallowest) beam (beam 4).

Metrics