Abstract

Selective two-photon excitation of fluorescent probe molecules using phase-only modulated ultrashort 15-fs laser pulses is demonstrated. The spectral phase required to achieve the maximum contrast in the excitation of different probe molecules or identical probe molecules in different micro-chemical environments is designed according to the principles of multiphoton intrapulse interference (MII). The MII method modulates the probabilities with which specific spectral components in the excitation pulse contribute to the two-photon absorption process due to the dependence of the absorption on the power spectrum of E2(t) [13]. Images obtained from a number of samples using the multiphoton microscope are presented.

© 2003 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  13. C540 is 3-(2'-Benzothiazolyl)-7-diethylaminocoumarin. R6G is a mixture of rhodamine 590 (2-[6-(ethylamino)-3-(ethylimino)- 2,7-dimethyl-3H-xanthen-9-yl]-ethylester, monohydrochloride), DOCI (3,3'-Diethyloxacarbocyanine Iodide) and DCM 4-Dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H -pyran).
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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Adv. Funct. Mat. (1)

T. Watanabe, M. Akiyama, K. Totani, S.M. Kuebler, F. Stellacci, W. Wenseleers, K. Braun, S.R. Marder, J.W. Perry, "Photoresponsive hydrogel microstructure fabricated by two-photon initiated polymerization," Adv. Funct. Mat. 12, 611-614 (2002).
[CrossRef]

Am. J. Physiol. -Cell Physiol. (1)

K.W. Dunn, R.M. Sandoval, K.J. Kelly, P.C. Dagher, G.A. Tanner, S.J. Atkinson, R.L. Bacallao, B.A. Molitoris, "Functional studies of the kidney of living animals using multicolor two-photon microscopy," Am. J. Physiol. -Cell Physiol. 283, C905-C916 (2002).
[PubMed]

Annu. Rev. Biomed. Eng. (1)

P.T.C. So, C.Y. Dong, B.R. Masters, K.M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. Eng. 2, 399-429 (2000).
[CrossRef]

Appl. Phys. B- Lasers Opt. (2)

R. Wolleschensky, T. Feurer, R. Sauerbrey, I. Simon, "Characterization and optimization of a laserscanning microscope in the femtosecond regime," Appl. Phys. B- Lasers Opt. 67, 87-94 (1998).
[CrossRef]

D. Yelin, D. Oron, E. Korkotian, M. Segal, Y. Silbergerg, "Third-harmonic microscopy with a titaniumsapphire laser," Appl. Phys. B- Lasers Opt. 74, S97-S101 (2002).
[CrossRef]

Appl. Phys. Lett. (1)

C. Xu, W. Denk, "Two photon optical beam induced current imaging throught backside of integrated circuits," Appl. Phys. Lett. 71, 2578-2580 (1997).
[CrossRef]

Biophys. J. (1)

K.A. Kasischke, H. Vishwasrao, A.A. Heikal, W.W. Webb, "Two-photon redox-fluorimetry: A new functional imaging technique for visualizing energy metabolism in brain tissue," Biophys. J. 82, 2420 (2002).

Cell (1)

J.W. Wang, A.M. Wong, J. Flores, L.B. Vosshall, R. Axel, "Two-photon calcium imaging reveals an odorevoked map of activity in the fly brain," Cell 112, 271-282 (2003).
[CrossRef] [PubMed]

Chem. Phys. Lett. (1)

T.H. Tran-Thi, T. Gustavsson, C. Prayer, S. Pommeret, J.T. Hynes, "Primary ultrafast events preceding the photoinduced proton transfer from pyranine to water," Chem. Phys. Lett. 329, 421-430 (2000).
[CrossRef]

Exp. Physiol. (1)

T.G. Oertner, "Functional imaging of single synapses in brain slices," Exp. Physiol. 87, 733-736 (2002).
[CrossRef] [PubMed]

J. Appl. Phys. (1)

D.L. Osborn, S.R. Leone, "Spectral and intensity dependence of spatially resolved two- photon conductivity defects on a GaAsP photodiode," J. Appl. Phys. 89, 626-633 (2001).
[CrossRef]

J. Chem. Phys. (1)

V.V. Lozovoy, I. Pastirk, K.A. Walowicz, M. Dantus, "Multiphoton intrapulse interference. 2. Control of two- and three-photon laser induced fluorescence with shaped pulses," J. Chem. Phys. 118, 3187-3196 (2003).
[CrossRef]

J. Microsc. -Oxf. (1)

K. Konig, "Multiphoton microscopy in life sciences," J. Microsc. -Oxf. 200, 83-104 (2000).
[CrossRef]

J. Phys. Chem. A (3)

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 (in press) (2003).

T.H. Tran-Thi, C. Prayer, P. Millie, P. Uznanski, J.T. Hynes, "Substituent and solvent effects on the nature of the transitions of pyrenol and pyranine. Identification of an intermediate in the excited-state protontransfer reaction," J. Phys. Chem. A 106, 2244-2255 (2002).
[CrossRef]

Nature (2)

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

T. Brixner, N.H. Damrauer, P. Niklaus, G. Gerber, "Photoselective adaptive femtosecond quantum control in the liquid phase," Nature 414, 57-60 (2001).
[CrossRef] [PubMed]

Opt. Lett. (1)

V.V. Lozovoy, I. Pastirk, K.A. Walowicz, M. Dantus, "Multiphoton intrapulse interference. 4. Characterization of the phase of ultrashort laser pulses.," Opt. Lett. submitted

Pflugers Arch. (1)

C.R. Rose, Y. Kovalchuk, J. Eilers, A. Konnerth, "Two-photon Na+ imaging in spines and fine dendrites of central neurons," Pflugers Arch. 439, 201-207 (1999).
[CrossRef]

Photochem. Photobiol. (1)

V. Shafirovich, A. Dourandin, N.P. Luneva, C. Singh, F. Kirigin, N.E. Geacintov, "Multiphoton nearinfrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine," Photochem. Photobiol. 69, 265-274 (1999).
[CrossRef] [PubMed]

Phys. Rev. A (1)

B. Broers, L.D. Noordam, H.B.V. Vandenheuvell, "Diffraction and focusing of spectral energy in multiphoton processes," Phys. Rev. A 46, 2749-2756 (1992)
[CrossRef] [PubMed]

Phys. Rev. Lett. (1)

M. Dyba, S.W. Hell, "Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. (1)

M.W. Berns, Z. Wang, A. Dunn, V. Wallace, V. Venugopalan, "Gene inactivation by multiphoton-targeted photochemistry," Proc. Natl. Acad. Sci. 97, 9504-9507 (2000).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

A.M. Weiner, "Femtosecond pulse shaping using spatial light modulators," Rev. Sci. Instrum. 71, 1929-1960 (2000).
[CrossRef]

Science (4)

W. Denk, J.H. Strickler, W.W. Webb, "2-Photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

D.R. Larson, W.R. Zipfel, R.M. Williams, S.W. Clark, M.P. Bruchez, F.W. Wise, W.W. Webb, "Watersoluble quantum dots for multiphoton fluorescence imaging in vivo," Science 300, 1434-1436 (2003).
[CrossRef] [PubMed]

D.A. VandenBout, W.T. Yip, D.H. Hu, D.K. Fu, T.M. Swager, P.F. Barbara, "Discrete intensity jumps and intramolecular electronic energy transfer in the spectroscopy of single conjugated polymer molecules," Science 277, 1074-1077 (1997).
[CrossRef]

S. Maiti, J.B. Shear, R.M. Williams, W.R. Zipfel, W.W. Webb, "Measuring serotonin distribution in live cells with three-photon excitation," Science 275, 530-532 (1997).
[CrossRef] [PubMed]

Other (1)

C540 is 3-(2'-Benzothiazolyl)-7-diethylaminocoumarin. R6G is a mixture of rhodamine 590 (2-[6-(ethylamino)-3-(ethylimino)- 2,7-dimethyl-3H-xanthen-9-yl]-ethylester, monohydrochloride), DOCI (3,3'-Diethyloxacarbocyanine Iodide) and DCM 4-Dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H -pyran).

Supplementary Material (4)

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Figures (5)

Fig. 1.
Fig. 1.

Schematic experimental setup for selective two-photon microscopy. Femtosecond laser pulses are compressed and sent to the pulse shaper. The modulated beam is then focused on the microscope slide with the specimen. The two-photon induced fluorescence is collected by a microscope objective and imaged on a CCD.

Fig. 2.
Fig. 2.

(2.02 MB) Experimental results and theoretical predictions of selective two-photon excitation of HTPS solutions at different pH. For this image the pulses were shaped with α=1.5π, γ=20 fs, and δ scanned from 0 to 4π. The movie shows the changes in the measured (black dots) and calculated (green line) contrast ratio as a function of the phase function parameter δ. (a) The laser spectrum (black line) and phase (green line) of the laser pulse. (b) Calculated power spectrum (green) of E2(t) phase modulated pulse at specific phase δ and for TL pulse (black line). Absorption spectra of HTPS at pH 10 (red line) and pH 6 (blue line) (c) Measured (black dots) and predicted (green line and big green dot) contrast ratio.

Fig. 3.
Fig. 3.

(1.28 MB) Experimental demonstration of pH-sensitive selective two-photon microscopy. The sample being imaged has an acidic (left side of the frame at pH 6) and a basic (right side of the frame at pH 10) region, both labeled with HPTS. (a) Image of the sample obtained with transform-limited pulses. The diagrams on the right show the spectrum of the 21-fs laser pulses, centered at 842 nm, and the spectral phase of the pulse (blue dashed line or red dotted line, that maximize pH 6 or pH 10 fluorescence, respectively). (b) Image of the same sample and location obtained with pulses that have been optimized for selective excitation of HPTS in an acidic micro-environment. Notice that only the left region shows significant two-photon excitation. For this image α=1.5π, γ=20 fs, and δ=0.75π. (c) Image of the same sample and location obtained with pulses that have been optimized for selective excitation of HPTS in a basic micro-environment. Notice that only the right region shows significant two-photon excitation. For this image α=1.5π, γ=20 fs, and δ=0.25π. The movie shows an experiment where the phase function parameter δ is scanned from 0 to 4π. Selective two-photon excitation from the two pH regions in the sample is observed at specific values of δ.

Fig. 4.
Fig. 4.

(2.4 MB) Selective two-photon microscopy of pieces of PMMA doped with different fluorescent probes. (a) Image showing two-photon induced fluorescence from both pieces top-C540 doped PMMA and bottom-R6G doped PMMA, obtained with 17 fs transform-limited pulses centered at 790 nm. (b) Image obtained with pulses optimized for selective C540 excitation. For this image the pulses were shaped with α=1.5π, γ=20 fs, and δ=0.31π. (c) Image obtained with pulses optimized for selective R6G excitation. For this image the pulses were shaped with α=1.5π, γ=20 fs, and δ=0.74π. The movie shows an experiment where the phase function parameter δ is scanned from 0 to 2π. Selective two-photon excitation from the two PMMA pieces (left is C540 and right is R6G) is observed at specific values of δ.

Fig. 5.
Fig. 5.

(738 kB) Selective two-photon microscopy of 10 µm blue and 15 µm green fluorescent polystyrene microspheres. (a) Image showing two-photon induced fluorescence from both microspheres, obtained with 15 fs transform-limited pulses centered at 790 nm. (b) Image obtained with pulses optimized for selective excitation of the blue microsphere. For this image the pulses were shaped with α=2.5π, γ=10 fs, and δ=0.75π. (c) Image obtained with pulses optimized for selective excitation of the green microsphere. For this image the pulses were shaped with α=2.5π, γ=10 fs, and δ=1.25π. The movie shows an experiment where the phase function parameter δ is scanned from 0 to 2π. Selective two-photon excitation from the two microspheres is observed at specific values of δ.

Equations (3)

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ϕ ( Ω ) = α Cos ( γ Ω δ ) ,
E ( 2 ) ( Δ ) = E ( Δ 2 + Ω ) E ( Δ 2 Ω ) d Ω
S ( 2 ) g ( 2 ) ( Δ ) E ( 2 ) ( Δ ) 2 d Δ ,

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