Abstract

High-resolution mosaic imaging is performed for the first time to our knowledge with a multifocal, multiphoton, photon-counting imaging system. We present a novel design consisting of a home-built femtosecond Yb-doped KGdWO4 laser with an optical multiplexer, which is coupled with a commercial Olympus IX-71 microscope frame. Photon counting is performed using single-element detectors and an inexpensive electronic demultiplexer and counters.

© 2009 Optical Society of America

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References

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  1. J. Bewersdorf, R. Pick, and S. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23, 655-657 (1998).
    [CrossRef]
  2. M. Straub and S. Hell, “Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6, 177-185 (1998).
  3. A. Egner and S. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17, 1192-1201 (2000).
    [CrossRef]
  4. A. Buist, M. Müller, J. Squier, and G. Brakenhoff, “Real time two-photon absorption microscopy using multi-point excitation,” J. Microsc. (Oxford) 192, 217-226 (1998).
    [CrossRef]
  5. K. Bahlmann, P. 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 (2007).
    [CrossRef] [PubMed]
  6. V. Andresen, A. Egner, and S. Hell, “Time-multiplexed multifocal multiphoton microscope,” Opt. Lett. 26, 75-77 (2001).
    [CrossRef]
  7. K. Kim, C. Buehler, K. Bahlmann, T. Ragan, W. Lee, E. Nedivi, E. Heffer, S. Fantini, and P. So, “Multifocal multiphoton microscopy based on multianode photomultiplier tubes,” Opt. Express 15, 11658-11678 (2007).
    [CrossRef] [PubMed]
  8. D. Fittinghoff and J. Squier, “Time-decorrelated multifocal array for multiphoton microscopy and micromachining,” Opt. Lett. 25, 1213-1215 (2000).
    [CrossRef]
  9. R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
    [CrossRef] [PubMed]
  10. J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
    [CrossRef] [PubMed]
  11. S. Kumar, C. Dunsby, P. De Beule, D. Owen, U. Anand, P. Lanigan, R. Benninger, D. Davis, M. Neil, P. Anand, C. Benham, A. Naylor, and P. French, “Multifocal multiphoton excitation and time correlated single photon counting detecting for 3-D fluorescence lifetime imaging,” Opt. Express 15, 12548-12561 (2007).
    [CrossRef] [PubMed]
  12. D. Fittinghoff, C. Schaffer, E. Mazur, and J. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
    [CrossRef]
  13. J. Jureller, H. Kim, and N. Scherer, “Stochastic scanning multiphoton multifocal microscopy,” Opt. Express 14, 3406-3414(2006).
    [CrossRef] [PubMed]
  14. L. Sacconi, E. Froner, R. Antolini, M. Taghizadeh, A. Choudhury, and F. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28, 1918-1920 (2003).
    [CrossRef] [PubMed]
  15. W. Amir, R. Carriles, E. Hoover, T. Planchon, C. Durfee, and J. Squier, “Simultaneous imaging of multiple focal planes using a two-photon scanning microscope,” Opt. Lett. 32, 1731-1733 (2007).
    [CrossRef] [PubMed]
  16. R. Carriles, K. Sheetz, E. Hoover, J. Squier, and V. Barzda, “Simultaneous multifocal, multiphoton, photon counting microscopy,” Opt. Express 16, 10364-10371 (2008).
    [CrossRef] [PubMed]
  17. K. Sheetz, E. Hoover, R. Carriles, D. Kleinfeld, and J. Squier, “Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator,” Opt. Express 16, 17574-17584 (2008).
    [CrossRef] [PubMed]
  18. 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]
  19. B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
    [CrossRef]
  20. M. Müller, Introduction to Confocal Fluorescence Microscopy (SPIE, 2006).

2008 (3)

2007 (6)

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

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

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

K. Bahlmann, P. 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 (2007).
[CrossRef] [PubMed]

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

2006 (2)

2003 (1)

2001 (2)

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

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

2000 (2)

1998 (3)

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

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

M. Straub and S. Hell, “Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6, 177-185 (1998).

Amir, W.

Anand, P.

Anand, U.

Andresen, V.

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

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

Anselmetti, D.

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

Antolini, R.

Bahlmann, K.

Barzda, V.

Bellve, K.

Benham, C.

Benninger, R.

Bewersdorf, J.

Brakenhoff, G.

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

Buehler, C.

Buist, A.

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

Canfield, B.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Carriles, R.

Chandler, E.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Choudhury, A.

Cisek, R.

Davis, D.

De Beule, P.

Dunsby, C.

Durfee, C.

Egner, A.

Fantini, S.

Fittinghoff, D.

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

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

French, P.

Froner, E.

Gunzer, M.

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

Heffer, E.

Hell, S.

Hoover, E.

Hrin, A.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Husu, H.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Jureller, J.

Kauranen, M.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Kim, H.

Kim, K.

Kirber, M.

Kleinfeld, D.

Kontio, J.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Kosicki, B.

Kumar, S.

Lanigan, P.

Lee, W.

Major, A.

Martini, J.

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

Mazur, E.

D. Fittinghoff, C. 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.

Merkle, T.

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

Müller, M.

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

M. Müller, Introduction to Confocal Fluorescence Microscopy (SPIE, 2006).

Naylor, A.

Nedivi, E.

Neil, M.

Neumann, J.

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

Niemi, T.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Niesner, R.

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

Owen, D.

Palmisano, R.

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

Pavone, F.

Pick, R.

Planchon, T.

Ragan, T.

Reich, R.

Rytkönen, T.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Sacconi, L.

Schaffer, C.

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

Scherer, N.

Schmied, K.

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

Sheetz, K.

So, P.

Spiecker, H.

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

Squier, J.

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

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

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

W. Amir, R. Carriles, E. Hoover, T. Planchon, C. 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. Fittinghoff, C. Schaffer, E. Mazur, and J. Squier, “Time-decorrelated multifocal micromachining and trapping,” IEEE J. Sel. Top. Quantum Electron. 7, 559-566 (2001).
[CrossRef]

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

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

Straub, M.

M. Straub and S. Hell, “Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6, 177-185 (1998).

Taghizadeh, M.

Toensing, K.

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

Viheriälä, J.

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Bioimaging (1)

M. Straub and S. Hell, “Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging,” Bioimaging 6, 177-185 (1998).

Biophys. J. (1)

R. Niesner, V. Andresen, J. Neumann, H. Spiecker, and M. Gunzer, “The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging,” Biophys. J. 93, 2519-2529 (2007).
[CrossRef] [PubMed]

IEEE J. Sel. Top. Quantum Electron. (1)

D. Fittinghoff, C. 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. (Oxford) (1)

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

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

J. Struct. Biol. (1)

J. Martini, K. Schmied, R. Palmisano, K. Toensing, D. Anselmetti, and T. Merkle, “Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time,” J. Struct. Biol. 158, 401-409 (2007).
[CrossRef] [PubMed]

New J. Phys. (1)

B. Canfield, H. Husu, J. Kontio, J. Viheriälä, T. Rytkönen, T. Niemi, E. Chandler, A. Hrin, J. Squier, and M. Kauranen, “Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots,” New J. Phys. 10, 013001 (2008).
[CrossRef]

Opt. Express (7)

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

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

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]

J. Jureller, H. Kim, and N. Scherer, “Stochastic scanning multiphoton multifocal microscopy,” Opt. Express 14, 3406-3414(2006).
[CrossRef] [PubMed]

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

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

K. Bahlmann, P. 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 (2007).
[CrossRef] [PubMed]

Opt. Lett. (5)

Other (1)

M. Müller, Introduction to Confocal Fluorescence Microscopy (SPIE, 2006).

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Figures (8)

Fig. 1
Fig. 1

Schematic representation of Yb:KGW oscillator layout. Mirrors are labeled by M1–M4; L1 and L2, 40 mm singlet lenses; GTI, Gires–Tournois interferometer; OC, output coupler; SESAM, semiconductor saturable absorber mirror.

Fig. 2
Fig. 2

Schematic representation of the optical multiplexer and scan optics, not to scale: HWP, half-wave plate; QWP, quarter-wave plate; BS, polarizing beam splitter; PD, photodiode; SM, scan mirrors; L1 and L3, 5 cm ; L2, 70 cm ; L4, 10 cm ; L5, 21 cm ; L6, 18 cm lenses. M1 is a flip mirror, redirecting the beam to a f 4 f telescope to further expand the beam, bypassing the scan mirrors. M2 is a silver mirror on a kinematic mount to direct the expanded beam into the IX-71.

Fig. 3
Fig. 3

Pulse sequence from both the photodiode and the PMT. The photodiode registers twice the laser frequency, as the interlaced pulse train contains two pulse trains of 56 MHz at orthogonal polarizations, designated by the dashed and solid curves. The PMT signal, with some variation in timing within a bin, is meant to simulate a MPEF response. The signal counters increment when a PMT pulse occurs between one clock cycle and the next.

Fig. 4
Fig. 4

Detection and demultiplexing electronics: PMT, photomultiplier tube; PA, pulse amplifier; PS, power supply; CP, comparator; PD, photodiode; SM, scan mirror driver; PC, personal computer. Each separate PMT signal is directed to a different DE2 board.

Fig. 5
Fig. 5

Crystalline cellulose imaged with (a), (b) epi-SHG at orthogonal polarizations, and (c), (d) transmission THG at orthogonal polarizations. Top and bottom image pairs are generated from the same excitation polarization. All images are scaled in photon counts.

Fig. 6
Fig. 6

Epi-SHG images of gold nandisks in (a) and (b) when the second beam focus is translated in depth by 13 μm . Images in (c) and (d) are generated when the piezoelectric stage is moved 13 μm in depth. Top and bottom image pairs are generated from the same excitation polarization. All images are scaled in units of photon counts.

Fig. 7
Fig. 7

Example of mosaic imaging. Nine individually acquired 30 μm × 30 μm epi-SHG images are shown in (a). The corresponding “stitched” together image is shown in (c). An epi-SHG image for the orthogonal excitation polarization is shown in (b). Transmission THG images are shown in (d) and (e). Left and right image pairs are generated from the same excitation polarization. All images are scaled in photon counts.

Fig. 8
Fig. 8

Epi-SHG mosaic images of crystalline cellulose imaged at 1.35 NA, imaged simultaneously at two orthogonal polarizations. These images are also composed of nine individual scans.

Equations (2)

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FWHM lateral = 0.61 λ NA ,
FWHM axial = 2 n λ NA 2 .

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