Abstract

We demonstrate selective fluorescence excitation of specific molecular species in live organisms by using coherent control of two-photon excitation. We have acquired quasi-simultaneous images in live fluorescently-labeled Drosophila embryos by rapid switching between appropriate pulse shapes. Linear combinations of these images demonstrate that a high degree of fluorophore selectivity is attainable through phase-shaping. Broadband phase-shaped excitation opens up new possibilities for single-laser, multiplex, in-vivo fluorescence microscopy.

© 2006 Optical Society of America

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Biophys. J. (1)

S. Huang, A. A. Heikal, W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

J. Cell Biol. (1)

D. P. Kiehart, C. G. Galbraith, K. A. Edwards, W. L. Rickoll, R. A. Montague, "Multiple forces contribute to cell sheet morphogenesis for dorsal closure in drosophila," J. Cell Biol. 149, 471-490 (2000).
[CrossRef] [PubMed]

J. Chem. Phys. (1)

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

J. of Microscopy (2)

E. Spiess, F. Bestvater, A. Heckel-Pompey, K. Toth, M. Hacker, G. Stobrawa, T. Feurer, C. Wotzlaw, U. Berchner-Pfannschmidt, T. Porwol, et al., "Two-photon excitation and emission spectra of the green fluorescent protein variants eCFP, eGFP and eYFP," J. of Microscopy 217, 200-204 (2005).
[CrossRef]

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Muller, A. L. Gaeta, "Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy," J. of Microscopy 193, 179-181 (1999).
[CrossRef]

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

J. Phys. Chem. A (2)

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, M. Dantus, "Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases," J. Phys. Chem. A 106, 9369-9373 (2002).
[CrossRef]

J. M. Dela Cruz, I. Pastirk, V. V. Lozovoy, K. A. Walowicz, M. Dantus, "Multiphoton intrapulse interference 3: Probing microscopic chemical environments," J. Phys. Chem. A 108, 53-58 (2004).
[CrossRef]

Nat. Biotech. (1)

W. R. Zipfel, Williams, R. M., W. W. Webb, "Nonlinear magic: Multiphoton microscopy in the biosciences," Nat. Biotech. 21, 1369-1377 (2003).
[CrossRef]

Natl. Acad. Sci (1)

J. M. Dela Cruz, I. Pastirk, M. Comstock, V. V. Lozovoy, M. Dantus, "Use of coherent control methods through scattering biological tissue to achieve functional imaging," Proc. Natl. Acad. Sci. 101, 16996-17001 (2004).
[CrossRef] [PubMed]

Natl. Acad. Sci. (2)

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, W. W. Webb, "Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy," Proc. Natl. Acad. Sci. 93, 10763-10768 (1996).
[CrossRef] [PubMed]

W. Supatto, D. Débarre, B. Moulia, E. Brouzés, J.-L. Martin, E. Farge, E. Beaurepaire, "In vivo modulation of morphogenetic movements in drosophila embryos with femtosecond laser pulses," Proc. Natl. Acad. Sci. 102, 1047-1052 (2005).
[CrossRef] [PubMed]

Nature (2)

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

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

Opt. Comm. (1)

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

Opt. Express (3)

Opt. Lett. (6)

Phys. Rev. Lett. (1)

R. S. Judson, H. Rabitz, "Teaching lasers to control molecules," Phys. Rev. Lett. 68, 1500-1503 (1992).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. (1)

W. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci. 100, 7075-7080 (2003).
[CrossRef] [PubMed]

Protein localization by fluorescence mic (1)

I. Davis, "Visualizing fluorescence in drosophila - optical detection in thick specimens". In Protein localization by fluorescence microscopy: A practical approach, Edited by Oxford University Press, 133-162 (2000).

Science (1)

A. Assion, T. Baumert, M. Bergt, T. Brixner, B. Kiefer, V. Seyfried, M. Strehle, G. Gerber, "Control of chemical reactions by feedback-optimized phase-shaped femtosecond laser pulses," Science 282, 919-922 (1998).
[CrossRef] [PubMed]

Other (1)

E. Wieschaus, C. Nusslein-Volhard, "Drosophila: A practical approach", Oxford, Oxford University Press (1998).

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Figures (4)

Fig. 1.
Fig. 1.

(a) 2PEF ratio of eGFP/yolk, as measured with a narrow-(b) Experimental setup. BS: beamsplitter, PC: prism compressor, PMT: photomultiplier tube. The interferometer, shown within the dashed box, was used only for characterizing the shaped pulses and was otherwise bypassed. (c) Spectral amplitude (solid line) for the pulses used in the experiments, as well as the applied sinusoidal phase for the red (dash-dot) and blue (dotted) phase-shaped pulses. (d) Corresponding SH spectra for the TL (solid), red-shaped (dash-dot) and blue-shaped (dotted) pulses. The TL amplitude has been reduced by a factor of two for easier comparison with the other spectra. The applied phases for the red and blue-shaped pulses were sinusoidal as defined in the text, with parameters A = 25π rad, γ = 0.09 rad/THz for both pulses. The offset values used for the red and blue-tuned pulses were δ = -1.85 rad and δ = -0.1 rad respectively.

Fig. 2.
Fig. 2.

2PEF images of an eGFP labeled Drosophila embryo. (a) Blue-tuned excitation. (b) Red-tuned excitation. (c) Transform limited pulse. These three images are normalized to the fluorescence signal of the vitelline membrane. (d) Linear combination of A and B to isolate the eGFP fluorescence. (e) Linear combination of A and B to isolate the yolk fluorescence. (f) Composite image of C and D to illustrate the good separation between eGFP and yolk fluorescence.

Fig. 3.
Fig. 3.

2PEF signal profiles for the shaped pulses through the section indicated in Fig. 2(C), showing significantly different signal levels in the eGFP and yolk.

Fig. 4.
Fig. 4.

A) 2PEF spectra of yolk and eGFP measured in vivo under 820-nm excitation by recording the descanned epidetected fluorescence filtered using a 15-nm tunable interferential filter (S-60, Schott) . Also shown are the wavelength ranges selected by the emission filters. B1) 2PEF images using blue-filter for TL and B2) blue-shaped pulses. C1) 2PEF images using green-filter for TL and C2) red-shaped (right) pulses. D1) X-profile for TL and D2) shaped pulses with filters at location indicated in B1.

Equations (2)

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E ( 2 ) ( ω ) 2 E ( ω ) E ( ω ω ) exp [ i { φ ( ω ) + φ ( ω ω ) } ] 2
Γ = R blue R red R blue + R red

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