Abstract

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 μJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 μm were observed in real-time with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

© 2012 OSA

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2011

2010

2008

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef] [PubMed]

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J 2(3), 143–155 (2008).
[CrossRef] [PubMed]

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett. 33(16), 1819–1821 (2008).
[CrossRef] [PubMed]

T.-M. Liu, M.-C. Chan, I.-H. Chen, S. H. Chia, and C.-K. Sun, “Miniaturized multiphoton microscope with a 24Hz frame-rate,” Opt. Express 16(14), 10501–10506 (2008).
[CrossRef] [PubMed]

2006

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

C.-Y. Chung, K.-C. Cho, C.-C. Chang, C. H. Lin, W. C. Yen, and S. J. Chen, “Adaptive-optics system with liquid-crystal phase-shift interferometer,” Appl. Opt. 45(15), 3409–3414 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

2005

2004

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2000

1999

1998

A. Van Orden, N. P. Machara, P. M. Goodwin, and R. A. Keller, “Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate,” Anal. Chem. 70(7), 1444–1451 (1998).
[CrossRef] [PubMed]

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

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

T. Wilson, M. A. A. Neil, and R. Juškaitis, “Real-time three-dimensional imaging of macroscopic structures,” J. Microsc. 191(2), 116–118 (1998).
[CrossRef] [PubMed]

1997

1996

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

1986

Anselmi, F.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

Athey, B.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Aubé, B.

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Be`gue, A.

E. Papagiakoumou, A. Be`gue, O. Schwartz, D. Oron, and V. Emiliani, “Shaped two-photon excitation deep inside scattering tissue,” submitted for publication.

Becker, D. L.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Bègue, A.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bewersdorf, J.

Bliton, A. C.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Brakenhoff, G. J.

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

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Buehler, C.

Buist, A. H.

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

Campagnola, P. J.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Chan, M.-C.

Chang, C.-C.

Chang, C.-Y.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Chang, N.-S.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Chen, I.-H.

Chen, S. J.

Chen, S.-J.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Cheng, L.-C.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Chia, S. H.

Cho, K.-C.

Chu, K. K.

Chung, C.-Y.

Côté, D.

Dana, H.

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

de Sars, V.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

Deutsch, M.

Dong, C. Y.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Durst, M. E.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Egner, A.

Ekwobi, C.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Emiliani, V.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

E. Papagiakoumou, A. Be`gue, O. Schwartz, D. Oron, and V. Emiliani, “Shaped two-photon excitation deep inside scattering tissue,” submitted for publication.

Fraser, S. E.

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J 2(3), 143–155 (2008).
[CrossRef] [PubMed]

Freund, I.

Girirajan, T. P.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Glückstad, J.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

Goodwin, P. M.

A. Van Orden, N. P. Machara, P. M. Goodwin, and R. A. Keller, “Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate,” Anal. Chem. 70(7), 1444–1451 (1998).
[CrossRef] [PubMed]

Hell, S. W.

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Isacoff, E. Y.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

Juškaitis, R.

T. Wilson, M. A. A. Neil, and R. Juškaitis, “Real-time three-dimensional imaging of macroscopic structures,” J. Microsc. 191(2), 116–118 (1998).
[CrossRef] [PubMed]

M. A. A. Neil, R. Juškaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

Keller, P. J.

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef] [PubMed]

Keller, R. A.

A. Van Orden, N. P. Machara, P. M. Goodwin, and R. A. Keller, “Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate,” Anal. Chem. 70(7), 1444–1451 (1998).
[CrossRef] [PubMed]

Kim, D.

Kim, K. H.

Koninck, P. D.

Li, Y.-C.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Liebling, M.

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J 2(3), 143–155 (2008).
[CrossRef] [PubMed]

Lien, C.-H.

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

Lim, D.

Lin, C. H.

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Liu, T.-M.

Machara, N. P.

A. Van Orden, N. P. Machara, P. M. Goodwin, and R. A. Keller, “Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate,” Anal. Chem. 70(7), 1444–1451 (1998).
[CrossRef] [PubMed]

Mason, M. D.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Mertz, J.

Müller, M.

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

Neil, M. A. A.

T. Wilson, M. A. A. Neil, and R. Juškaitis, “Real-time three-dimensional imaging of macroscopic structures,” J. Microsc. 191(2), 116–118 (1998).
[CrossRef] [PubMed]

M. A. A. Neil, R. Juškaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

Norris, T.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Oron, D.

Pagès, S.

Papagiakoumou, E.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

E. Papagiakoumou, A. Be`gue, O. Schwartz, D. Oron, and V. Emiliani, “Shaped two-photon excitation deep inside scattering tissue,” submitted for publication.

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pick, R.

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Schwartz, O.

E. Papagiakoumou, A. Be`gue, O. Schwartz, D. Oron, and V. Emiliani, “Shaped two-photon excitation deep inside scattering tissue,” submitted for publication.

Shank, C. V.

A. Vaziri and C. V. Shank, “Ultrafast widefield optical sectioning microscopy by multifocal temporal focusing,” Opt. Express 18(19), 19645–19655 (2010).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Shoham, S.

Shroff, H.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Silberberg, Y.

So, P. T.

So, P. T. C.

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Squier, J.

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

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Stelzer, E. H. K.

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef] [PubMed]

Sun, C.-K.

Tal, E.

Tang, J.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

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T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Therrien, O. D.

Thrasivoulou, C.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Van Orden, A.

A. Van Orden, N. P. Machara, P. M. Goodwin, and R. A. Keller, “Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate,” Anal. Chem. 70(7), 1444–1451 (1998).
[CrossRef] [PubMed]

Vaziri, A.

A. Vaziri and C. V. Shank, “Ultrafast widefield optical sectioning microscopy by multifocal temporal focusing,” Opt. Express 18(19), 19645–19655 (2010).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Vermot, J.

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J 2(3), 143–155 (2008).
[CrossRef] [PubMed]

Wade, M. H.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, M. A. A. Neil, and R. Juškaitis, “Real-time three-dimensional imaging of macroscopic structures,” J. Microsc. 191(2), 116–118 (1998).
[CrossRef] [PubMed]

M. A. A. Neil, R. Juškaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

Xu, C.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Yen, W. C.

Zhu, G.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Anal. Chem.

A. Van Orden, N. P. Machara, P. M. Goodwin, and R. A. Keller, “Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate,” Anal. Chem. 70(7), 1444–1451 (1998).
[CrossRef] [PubMed]

Appl. Opt.

Biomed. Opt. Express

Biophys. J.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Curr. Opin. Neurobiol.

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008).
[CrossRef] [PubMed]

HFSP J

J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J 2(3), 143–155 (2008).
[CrossRef] [PubMed]

J. Microsc.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

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

T. Wilson, M. A. A. Neil, and R. Juškaitis, “Real-time three-dimensional imaging of macroscopic structures,” J. Microsc. 191(2), 116–118 (1998).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

J. Opt. Soc. Am. B

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Methods

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Opt. Commun.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Proc. Natl. Acad. Sci. U.S.A.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Science

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Other

Y.-C. Li, L.-C. Cheng, C.-H. Lien, C.-Y. Chang, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned generation,” submitted for publication.

E. Papagiakoumou, A. Be`gue, O. Schwartz, D. Oron, and V. Emiliani, “Shaped two-photon excitation deep inside scattering tissue,” submitted for publication.

R. M. Mazo, Brownian Motion: Fluctuations, Dynamics, and Applications (Oxford University Press, 2009).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

Supplementary Material (2)

» Media 1: MOV (706 KB)     
» Media 2: MOV (622 KB)     

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Figures (6)

Fig. 1
Fig. 1

Optical setup of the widefield multiphoton microscope based on spatiotemporal focusing.

Fig. 2
Fig. 2

Spatial resolution of the widefield multiphoton excitation microscope: (a) axial resolutions under different laser powers at 10, 20, 30, and 40 mW are 2.5, 2.8, 3.1, and 3.4 μm, respectively; and, (b) the point spread function showing 0.5 μm FWHM.

Fig. 3
Fig. 3

TPEF intensities of the PMMA thin film doped with R6G dye as a function of time under different excitation powers. Circles: ultrafast oscillator; squares: ultrafast amplifier.

Fig. 4
Fig. 4

(a) Brownian motion of 1.0 μm fluorescent beads at 100 Hz frame rate (Media 1). The excitation laser power is 40 mW with a 9 ms exposure time. (b) Displacements of the bead at the top of Fig. 4(a) along the x and y axes as a function of time recorded at 10 ms intervals from Media 1.

Fig. 5
Fig. 5

(a) Brownian motions of 0.5 μm fluorescent beads at 100 Hz frame rate with an exposure time of 9 ms (Media 2). (b) Sequentially images captured from Fig. 5(a).

Fig. 6
Fig. 6

SHG images of chicken tendon: (a) 10 Hz frame rate at the full pixel number of 1000 × 1000 pixels and exposure time of 100 ms, (b) 100 Hz frame rate at 4x4 binning with pixel number of 250 × 250 pixels and exposure time of 9 ms, (c) image of Fig. 6(a) red square region at 100 Hz frame rate, 256 × 256 pixels, and 9 ms exposure time, and (d) image of Fig. 6(a) red square region at 10 Hz frame rate by accumulating 10 shots in one frame.

Equations (2)

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F W H M z k 1 + τ k 2 τ l + k 3 M N A 2 ,
Δ d r m s = 6 D × Δ t ,

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