Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.

© 2009 Optical Society of America

1. S. Rice, "New ideas for guiding the evolution of a quantum system," Science 258, 412-413 (1992). [CrossRef] [PubMed]
2. W. Warren, H. Rabitz, and M. Dahleh, "Coherent control of quantum dynamics - the dream is alive," Science 259, 1581-1589 (1993). [CrossRef] [PubMed]
3. M. Dantus and V. Lozovoy, "Experimental coherent laser control of physicochemical processes," Chem. Rev. 104, 1813-1859 (2004). [CrossRef] [PubMed]
4. P. Nuernberger, G. Vogt, T. Brixner, and G. Gerber, "Femtosecond quantum control of molecular dynamics in the condensed phase," Phys. Chem. Chem. Phys. 9, 2470-2497 (2007). [CrossRef] [PubMed]
5. W. Denk, J. Strickler, and W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990). [CrossRef] [PubMed]
6. W. Zipfel, R. Williams, and W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1368-1376 (2003). [CrossRef]
7. N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002). [CrossRef] [PubMed]
8. D. Meshulach and Y. Silberberg, "Coherent quantum control of two-photon transitions by a femtosecond laser pulse," Nature 396, 239-242 (1998). [CrossRef]
9. I. Pastirk, J. D. Cruz, K. Walowicz, V. Lozovoy, and M. Dantus, "Selective two-photon microscopy with shaped femtosecond pulses," Opt. Express 11, 1695-1701 (2003). [CrossRef] [PubMed]
10. J. D. Cruz, I. Pastirk, M. Comstock, and M. Dantus, "Multiphoton intrapulse interference 8. Coherent control through scattering tissue," Opt. Express 12, 4144-4149 (2004). [CrossRef]
11. J. D. Cruz, I. Pastirk, M. Comstock, V. Lozovoy, and M. Dantus, "Use of coherent control methods through scattering biological tissue to achieve functional imaging," Proc. Natl. Acad. Sci. USA 101, 16,996-17,001 (2004).
12. J. P. Ogilvie, D. Debarre, X. Solinas, J.-L. Martin, E. Beaurepaire, and M. Joffre, "Use of coherent control for selective two-photon fluorescence microscopy in live organisms," Opt. Express 14, 759-766 (2006). [CrossRef] [PubMed]
13. P. Tournois, "Acousto-optic programmable dispersive filter for adaptive compensation of group delay time dispersion in laser systems," Opt. Commun. 140, 245-249 (1997). [CrossRef]
14. F. Verluise, V. Laude, Z. Cheng, C. Spielmann, and P. Tournois, "Amplitude and phase control of ultrashort pulses by use of an acousto-optic programmable dispersive filter: pulse compression and shaping," Opt. Lett. 25, 575-577 (2000). [CrossRef]
15. E. Frumker and Y. Silberberg, "Femtosecond pulse shaping using a two-dimensional liquid-crystal spatial light modulator," Opt. Lett. 32, 1384-1386 (2007). [CrossRef] [PubMed]
16. J. Vaughan, T. Hornung, T. Feurer, and K. Nelson, "Diffraction-based femtosecond pulse shaping with a twodimensional spatial light modulator," Opt. Lett. 30, 323-325 (2005). [CrossRef] [PubMed]
17. E. Frumker, D. Oron, D. Mandelik, and Y. Silberberg, "Femtosecond pulse-shape modulation at kilohertz rates," Opt. Lett. 29, 890-892 (2004). [CrossRef] [PubMed]
18. C. Dorrer, F. Salin, F. Verluise, and J. Huignard, "Programmable phase control of femtosecond pulses by use of a nonpixelated spatial light modulator," Opt. Lett. 23, 709-711 (1998). [CrossRef]
19. J. Vaughan, T. Feurer, K. Stone, and K. Nelson, "Analysis of replica pulses in femtosecond pulse shaping with pixelated devices," Opt. Express 14, 1314-1328 (2006). [CrossRef] [PubMed]
20. B. J. Sussman, R. Lausten, and A. Stolow, "Focusing of light following a 4-f pulse shaper: Considerations for quantum control," Phys. Rev. A 77, 043,416 (2008). [CrossRef]
21. A. Monmayrant and B. Chatel, "New phase and amplitude high resolution pulse shaper," Rev. Sci. Instrum. 75, 2668-2671 (2004). [CrossRef]
22. M. M. Salour, "Quantum interference effects in 2-photon spectroscopy," Rev. Mod. Phys. 50, 667-681 (1978). [CrossRef]
23. K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, "Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases," J. Phys. Chem. A 106, 9369-9373 (2002). [CrossRef]
24. V. Lozovoy, I. Pastirk, K. Walowicz, and M. Dantus, "Multiphoton intrapulse interference. II. Control of twoand three-photon laser induced fluorescence with shaped pulses," J. Chem. Phys. 118, 3187-3196 (2003). [CrossRef]
25. K. Naganuma, K. Mogi, and H. Yamada, "General method for ultrashort light-pulse chirp measurement," IEEE J. Quant. Electron. 25, 1225-1233 (1989). [CrossRef]
26. J. P. Ogilvie, K. J. Kubarych, A. Alexandrou, and M. Joffre, "Fourier transform measurement of two-photon excitation spectra: applications to microscopy and optimal control," Opt. Lett. 30, 911-913 (2005). [CrossRef] [PubMed]
27. J. D. Cruz, I. Pastirk, V. Lozovoy, K. Walowicz, and M. Dantus, "Multiphoton intrapulse interference 3: Probing microscopic chemical environments," J. Phys. Chem. A 108, 53-58 (2004). [CrossRef]
28. M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, "Multiphoton intrapulse interference 6; binary phase shaping," Opt. Express 12, 1061-1066 (2004). [CrossRef] [PubMed]
29. V. Lozovoy, B. Xu, J. Shane, and M. Dantus, "Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields," Phys. Rev. A 74, 041,805 (2006). [CrossRef]
30. A. Larson and A. Yeh, "Ex vivo characterization of sub-10-fs pulses," Opt. Lett. 31, 1681-1683 (2006). [CrossRef] [PubMed]
31. Y. Coello, V. Lozovoy, T. Gunaratne, B. Xu, I. Borukhovich, C. Tseng, T. Weinacht, and M. Dantus, "Interference without an interferometer: a different approach to measuring, compressing, and shaping ultrashort laser pulses," J. Opt. Soc. Am. B 25, A140-A150 (2008). [CrossRef]
32. B. von Vacano, T. Buckup, and M. Motzkus, "Shaper-assisted collinear SPIDER: fast and simple broadband pulse compression in nonlinear microscopy," J. Opt. Soc. Am. B 24, 1091-1100 (2007). [CrossRef]
33. V. Lozovoy, B. Xu, Y. Coello, and M. Dantus, "Direct measurement of spectral phase for ultrashort laser pulses," Opt. Express 16, 592-597 (2008). [CrossRef] [PubMed]
34. I. Davis, C. Girdham, and P. O’Farrell, "A nuclear gfp that marks nuclei in living drosophila embryos - maternal supply overcomes a delay in the appearance of zygotic fluorescence," Developmental Biology 170, 726-729 (1995). [CrossRef] [PubMed]
35. E. Wieschaus and C. Nusslein-Volhard, "Looking at embryos" in "Drosophila, a practical approach", D.B. Roberts, ed. (Oxford University Press, Oxford, 1998), pp. 179-214.

Chem. Rev.

• M. Dantus and V. Lozovoy, "Experimental coherent laser control of physicochemical processes," Chem. Rev. 104, 1813-1859 (2004). [CrossRef] [PubMed]

Developmental Biology

• I. Davis, C. Girdham, and P. O’Farrell, "A nuclear gfp that marks nuclei in living drosophila embryos - maternal supply overcomes a delay in the appearance of zygotic fluorescence," Developmental Biology 170, 726-729 (1995). [CrossRef] [PubMed]

IEEE J. Quant. Electron.

• K. Naganuma, K. Mogi, and H. Yamada, "General method for ultrashort light-pulse chirp measurement," IEEE J. Quant. Electron. 25, 1225-1233 (1989). [CrossRef]

J. Chem. Phys.

• V. Lozovoy, I. Pastirk, K. Walowicz, and M. Dantus, "Multiphoton intrapulse interference. II. Control of twoand three-photon laser induced fluorescence with shaped pulses," J. Chem. Phys. 118, 3187-3196 (2003). [CrossRef]

J. Opt. Soc. Am. B

• Y. Coello, V. Lozovoy, T. Gunaratne, B. Xu, I. Borukhovich, C. Tseng, T. Weinacht, and M. Dantus, "Interference without an interferometer: a different approach to measuring, compressing, and shaping ultrashort laser pulses," J. Opt. Soc. Am. B 25, A140-A150 (2008). [CrossRef]
• B. von Vacano, T. Buckup, and M. Motzkus, "Shaper-assisted collinear SPIDER: fast and simple broadband pulse compression in nonlinear microscopy," J. Opt. Soc. Am. B 24, 1091-1100 (2007). [CrossRef]

J. Phys. Chem. A

• J. D. Cruz, I. Pastirk, V. Lozovoy, K. Walowicz, and M. Dantus, "Multiphoton intrapulse interference 3: Probing microscopic chemical environments," J. Phys. Chem. A 108, 53-58 (2004). [CrossRef]
• K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, "Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases," J. Phys. Chem. A 106, 9369-9373 (2002). [CrossRef]

Nat. Biotechnol.

• W. Zipfel, R. Williams, and W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1368-1376 (2003). [CrossRef]

Nature

• N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002). [CrossRef] [PubMed]
• D. Meshulach and Y. Silberberg, "Coherent quantum control of two-photon transitions by a femtosecond laser pulse," Nature 396, 239-242 (1998). [CrossRef]

Opt. Commun.

• P. Tournois, "Acousto-optic programmable dispersive filter for adaptive compensation of group delay time dispersion in laser systems," Opt. Commun. 140, 245-249 (1997). [CrossRef]

Opt. Express

• J. Vaughan, T. Feurer, K. Stone, and K. Nelson, "Analysis of replica pulses in femtosecond pulse shaping with pixelated devices," Opt. Express 14, 1314-1328 (2006). [CrossRef] [PubMed]
• J. P. Ogilvie, D. Debarre, X. Solinas, J.-L. Martin, E. Beaurepaire, and M. Joffre, "Use of coherent control for selective two-photon fluorescence microscopy in live organisms," Opt. Express 14, 759-766 (2006). [CrossRef] [PubMed]
• I. Pastirk, J. D. Cruz, K. Walowicz, V. Lozovoy, and M. Dantus, "Selective two-photon microscopy with shaped femtosecond pulses," Opt. Express 11, 1695-1701 (2003). [CrossRef] [PubMed]
• J. D. Cruz, I. Pastirk, M. Comstock, and M. Dantus, "Multiphoton intrapulse interference 8. Coherent control through scattering tissue," Opt. Express 12, 4144-4149 (2004). [CrossRef]
• M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, "Multiphoton intrapulse interference 6; binary phase shaping," Opt. Express 12, 1061-1066 (2004). [CrossRef] [PubMed]
• V. Lozovoy, B. Xu, Y. Coello, and M. Dantus, "Direct measurement of spectral phase for ultrashort laser pulses," Opt. Express 16, 592-597 (2008). [CrossRef] [PubMed]

Opt. Lett.

• J. P. Ogilvie, K. J. Kubarych, A. Alexandrou, and M. Joffre, "Fourier transform measurement of two-photon excitation spectra: applications to microscopy and optimal control," Opt. Lett. 30, 911-913 (2005). [CrossRef] [PubMed]
• A. Larson and A. Yeh, "Ex vivo characterization of sub-10-fs pulses," Opt. Lett. 31, 1681-1683 (2006). [CrossRef] [PubMed]
• F. Verluise, V. Laude, Z. Cheng, C. Spielmann, and P. Tournois, "Amplitude and phase control of ultrashort pulses by use of an acousto-optic programmable dispersive filter: pulse compression and shaping," Opt. Lett. 25, 575-577 (2000). [CrossRef]
• E. Frumker and Y. Silberberg, "Femtosecond pulse shaping using a two-dimensional liquid-crystal spatial light modulator," Opt. Lett. 32, 1384-1386 (2007). [CrossRef] [PubMed]
• J. Vaughan, T. Hornung, T. Feurer, and K. Nelson, "Diffraction-based femtosecond pulse shaping with a twodimensional spatial light modulator," Opt. Lett. 30, 323-325 (2005). [CrossRef] [PubMed]
• E. Frumker, D. Oron, D. Mandelik, and Y. Silberberg, "Femtosecond pulse-shape modulation at kilohertz rates," Opt. Lett. 29, 890-892 (2004). [CrossRef] [PubMed]
• C. Dorrer, F. Salin, F. Verluise, and J. Huignard, "Programmable phase control of femtosecond pulses by use of a nonpixelated spatial light modulator," Opt. Lett. 23, 709-711 (1998). [CrossRef]

Phys. Chem. Chem. Phys.

• P. Nuernberger, G. Vogt, T. Brixner, and G. Gerber, "Femtosecond quantum control of molecular dynamics in the condensed phase," Phys. Chem. Chem. Phys. 9, 2470-2497 (2007). [CrossRef] [PubMed]

Phys. Rev. A

• B. J. Sussman, R. Lausten, and A. Stolow, "Focusing of light following a 4-f pulse shaper: Considerations for quantum control," Phys. Rev. A 77, 043,416 (2008). [CrossRef]
• V. Lozovoy, B. Xu, J. Shane, and M. Dantus, "Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields," Phys. Rev. A 74, 041,805 (2006). [CrossRef]

Proc. Natl. Acad. Sci. USA

• J. D. Cruz, I. Pastirk, M. Comstock, V. Lozovoy, and M. Dantus, "Use of coherent control methods through scattering biological tissue to achieve functional imaging," Proc. Natl. Acad. Sci. USA 101, 16,996-17,001 (2004).

Rev. Mod. Phys.

• M. M. Salour, "Quantum interference effects in 2-photon spectroscopy," Rev. Mod. Phys. 50, 667-681 (1978). [CrossRef]

Rev. Sci. Instrum.

• A. Monmayrant and B. Chatel, "New phase and amplitude high resolution pulse shaper," Rev. Sci. Instrum. 75, 2668-2671 (2004). [CrossRef]

Science

• W. Denk, J. Strickler, and W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990). [CrossRef] [PubMed]
• S. Rice, "New ideas for guiding the evolution of a quantum system," Science 258, 412-413 (1992). [CrossRef] [PubMed]
• W. Warren, H. Rabitz, and M. Dahleh, "Coherent control of quantum dynamics - the dream is alive," Science 259, 1581-1589 (1993). [CrossRef] [PubMed]

Other

• E. Wieschaus and C. Nusslein-Volhard, "Looking at embryos" in "Drosophila, a practical approach", D.B. Roberts, ed. (Oxford University Press, Oxford, 1998), pp. 179-214.

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