Abstract

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

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    [CrossRef]
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  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]
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    [CrossRef] [PubMed]
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2008

2007

2006

2005

2004

M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, "Multiphoton intrapulse interference 6; binary phase shaping," Opt. Express 12, 1061-1066 (2004).
[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]

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]

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]

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

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

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).

2003

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

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]

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]

2002

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]

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

2000

1998

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]

D. Meshulach and Y. Silberberg, "Coherent quantum control of two-photon transitions by a femtosecond laser pulse," Nature 396, 239-242 (1998).
[CrossRef]

1997

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]

1995

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]

1993

W. Warren, H. Rabitz, and M. Dahleh, "Coherent control of quantum dynamics - the dream is alive," Science 259, 1581-1589 (1993).
[CrossRef] [PubMed]

1992

S. Rice, "New ideas for guiding the evolution of a quantum system," Science 258, 412-413 (1992).
[CrossRef] [PubMed]

1990

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

1989

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

1978

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

Alexandrou, A.

Beaurepaire, E.

Borukhovich, I.

Brixner, T.

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]

Buckup, T.

Chatel, B.

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

Cheng, Z.

Coello, Y.

Comstock, M.

Cruz, J. D.

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).

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]

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]

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]

Dahleh, M.

W. Warren, H. Rabitz, and M. Dahleh, "Coherent control of quantum dynamics - the dream is alive," Science 259, 1581-1589 (1993).
[CrossRef] [PubMed]

Dantus, M.

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]

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]

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]

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]

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

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).

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]

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]

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]

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]

Davis, I.

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]

Debarre, D.

Denk, W.

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

Dorrer, C.

Dudovich, N.

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

Feurer, T.

Frumker, E.

Gerber, G.

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]

Girdham, C.

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]

Gunaratne, T.

Hornung, T.

Huignard, J.

Joffre, M.

Kubarych, K. J.

Larson, A.

Laude, V.

Lausten, R.

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]

Lozovoy, V.

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]

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]

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]

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]

M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, "Multiphoton intrapulse interference 6; binary phase shaping," Opt. Express 12, 1061-1066 (2004).
[CrossRef] [PubMed]

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).

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

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]

Lozovoy, V. V.

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]

Mandelik, D.

Martin, J.-L.

Meshulach, D.

D. Meshulach and Y. Silberberg, "Coherent quantum control of two-photon transitions by a femtosecond laser pulse," Nature 396, 239-242 (1998).
[CrossRef]

Mogi, K.

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

Monmayrant, A.

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

Motzkus, M.

Naganuma, K.

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

Nelson, K.

Nuernberger, P.

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]

O’Farrell, P.

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]

Ogilvie, J. P.

Oron, D.

E. Frumker, D. Oron, D. Mandelik, and Y. Silberberg, "Femtosecond pulse-shape modulation at kilohertz rates," Opt. Lett. 29, 890-892 (2004).
[CrossRef] [PubMed]

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

Pastirk, I.

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).

M. Comstock, V. Lozovoy, I. Pastirk, and M. Dantus, "Multiphoton intrapulse interference 6; binary phase shaping," Opt. Express 12, 1061-1066 (2004).
[CrossRef] [PubMed]

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]

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]

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]

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]

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]

Rabitz, H.

W. Warren, H. Rabitz, and M. Dahleh, "Coherent control of quantum dynamics - the dream is alive," Science 259, 1581-1589 (1993).
[CrossRef] [PubMed]

Rice, S.

S. Rice, "New ideas for guiding the evolution of a quantum system," Science 258, 412-413 (1992).
[CrossRef] [PubMed]

Salin, F.

Salour, M. M.

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

Shane, J.

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]

Silberberg, Y.

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]

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Supplementary Material (1)

» Media 1: AVI (7315 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

Top (a) and side (b) views of the pulse shaper. A spatial light modulator (SLM) is placed in the Fourier plane of a grating (GR)-cylindrical mirror (CM) combination in a folded 4-f configuration. The 2D phase mask is used in the x-dimension as a phase shaper and in the y-dimension as a ruled grating used in Littrow configuration. A galvanometer mounted mirror (G) switches the light beam between the two areas of the SLM with two different phase shapes.

Fig. 2.
Fig. 2.

2D masks as viewed in the beam propagation direction. In panel (a), a mask with uniform grating length is shown and the resulting angular chirp is shown in (c). The angular chirp was corrected by using a grating length directly proportional to the wavelength as shown in (b). Panel (d) schematically illustrates that the angular chirp is removed due to the scaled grating.

Fig. 3.
Fig. 3.

Experimental setup. CM: coupling mirror, G: galvanometer-mounted mirror, 2D-SLM: spatial light modulator, DM: dichroic mirror, F: filter, PMT: photomultiplier tube.

Fig. 4.
Fig. 4.

(a) Trace obtained from a chirp scan measurement for a 0.8NA 40× water immersion objective in the case of a residual third-order phase. (b) Second order interferometric autocorrelation trace (line) and the intensity autocorrelation trace (circles) corresponding to a near transform limited pulse (~14 fs) at the focus of the microscope objective. (c) Laser spectrum (L.S.) at the focus of the microscope objective and Spectral phases (R.S.-red shifted and B.S-blue shifted) for the two different pulse shapes used in the imaging experiments. The “red shifted” spectral phase is anti-symmetric with respect to frequency corresponding to λ=840 nm and the “blue shifted” spectral phase is anti-symmetric with respect to frequency corresponding to λ=780 nm. (d) Two-photon spectra measured at the focus of the microscope objective for a near transform limited pulse (TL), “blue shifted” spectral phase and “red shifted” spectral phase in (c).

Fig. 5.
Fig. 5.

(a and b) Images of 100 nm beads located (a) at the center and (b) at the edge of the field of view respectively. The scale bars correspond to 1 µm. Each image is obtained by merging the two images acquired by the red-shifted (shown in green) and the blue-shifted (shown in blue) pulses. (c), (d): radial intensity point spread functions measured from the bead images shown in (a) and (b). FWHM is 0.49 µm. (e), (f): axial intensity point spread functions measured from the same beads. FWHM is 2 µm.

Fig. 6.
Fig. 6.

Multiplexed in vivo imaging of an eGFP-expressing Drosophila embryo using (a) blue-shifted pulse shaping for preferential excitation of the endogenous fluorescence at 780 nm and (b) red-shifted pulse shaping for preferential eGFP excitation at 840 nm. Dorsal side is up. (c), (d): linear combinations of images (a) and (b) for separating the two fluorescent components (spectral unmixing). (e), (f): images obtained by combining (c) and (d) at two different stages of embryo development. Tissue extension is visible at the embryo posterior pole during germ band extension (white arrow). (g), (h): Kymographs (space-time projections) along the dotted red lines indicated in (c) and (d). These projections reveal the correlated motion of cells (h) and underlying yolk structures (g) during extension movements. (i) and (Media 1) 3D+t movie of the posterior region of another embryo imaged from the dorsal side, during germ band extension. One 3D image was recorded every minute. Scale bars: 50 µm (X) and 5 min (time).

Equations (7)

Equations on this page are rendered with MathJax. Learn more.

S= g(2) (ω)𝓔(2)(ω)2 dω2π
(2)(ω)= (t)2 eiωt dt
(2)(ω)= (ω)(ωω)2π
(2)(ω)=(ω')(ωω')exp(iΦω(ω'))'2π
(2)(ω)(ω)(ωω)2π=φ=0(2)(ω)
Φ2ω1(ω)=φ(ω)+φ(2ω1ω)=0
φ(ω)=16φ(ω1).(ωω1)3

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