Abstract

We report on third-harmonic generation (THG) of biomolecular solutions at the fluid/glass interface as a means to probe resonant contributions to their nonlinear absorption spectra that could serve as contrast mechanisms for functional imaging. Our source was 100fs laser pulses whose center wavelength varied from 760 to 1000nm. We find evidence of a two-photon resonance in the ratio of third-order susceptibilities, χsample(3)(3ω)χglass(3), for aqueous solutions of Rhodamine B, Fura-2, and hemoglobin and a three-photon resonance in χsample(3)(3ω)χglass(3) for solutions of bovine serum albumin. Consistent with past work, we find evidence of a one-photon resonance of χsample(3)(3ω)χglass(3) for water, while confirming a lack of resonant enhancement for benzene. At physiological concentrations, hemoglobin in different ligand-binding states could be distinguished on the basis of features of its THG spectrum.

© 2006 Optical Society of America

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2004

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
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D. Debarre, W. Supatto, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, "Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos," Opt. Lett. 29, 2881-2883 (2004).
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S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, "Studies of C(2)/C(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914-3922 (2004).

D. L. Wokosin, C. M. Loughrey, and G. L. Smith, "Characterization of a range of Fura dyes with two-photon excitation," Biophys. J. 86, 1726-1738 (2004).

V. Shcheslavskiy, G. I. Petrov, S. Saltiel, and V. V. Yakovlev, "Quantitative characterization of aqueous solutions probed by the third-harmonic generation microscopy," J. Struct. Biol. 147, 42-49 (2004).
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2003

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, "Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses," Biochem. Biophys. Res. Commun. 11, 592-596 (2003).

E. Chaigneau, M. Oheim, E. Audinat, and S. Charpak, "Two-photon imaging of capillary blood flow in olfactory bulb glomeruli," Proc. Natl. Acad. Sci. U.S.A. 100, 13081 -13086 (2003).
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C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, "Multiharmonic-generation biopsy of skin," Opt. Lett. 28, 2488-2490 (2003).

D. A. Akimov, A. A. Ivanov, M. V. Alfimov, E. P. Grabchak, A. A. Shtykova, A. N. Petrov, A. A. Podshivalov, and A. M. Zheltikov, "J-aggregation visualized with two-photon resonant third-harmonic generation," J. Raman Spectrosc. 34, 1007-1012 (2003).
[CrossRef]

S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, and C.-K. Sun, "In vivo developmental biology study using noninvasive multi-harmonic generation microscopy," Opt. Express 11, 3093-3099 (2003).

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
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S. O. Konorov, D. A. Akimov, A. A. Ivanov, M. V. Alfimov, S. Botti, R. Ciardi, L. D. Deominicis, L. S. Asilyan, A. A. Podshivalov, D. A. Sidorov-Biryukov, R. Fantoni, and A. M. Zheltikov, "Femtosecond optical harmonic generation as a non-linear spectroscopic probe for carbon nanotubes," J. Raman Spectrosc. 34, 1018-1024 (2003).
[CrossRef]

V. Shcheslavskiy, G. Petrov, and V. V. Yakovlev, "Nonlinear optical susceptibility measurements of solutions using third-harmonic generation on the interface," Appl. Phys. Lett. 82, 3982-3984 (2003).
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B. Halle and M. Davidovic, "Biomolecular hydration: from water dynamics to hydrodynamics," Proc. Natl. Acad. Sci. U.S.A. 100, 12135-12140 (2003).
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K. Murayama, B. Yuan, Y. Ozaki, M. Tomida, and S. Era, "Near-infrared spectroscopy for liquids of microliter volume using capillaries with wall transmission," Analyst (Cambridge, U.K.) 128, 957-959 (2003).

2002

H. EI-Kashef, "Study of the refractive properties of laser dye solvents: toluene, carbon disulphide, chloroform, and benzene," Opt. Mater. (Amsterdam, Neth.) 20, 81-86 (2002).
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J. L. Dela Cruz and G. J. Blanchard, "The influence of chromophore structure on intermolecular interactions. A study of selected rhodamines in polar protic and aprotic solvents," J. Phys. Chem. A 106, 10718-10724 (2002).
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R. Barille, L. Canioni, S. Rivet, L. Sarger, and G. Rivoire, "Nonlinearity measurements of thin films by third-harmonic-generation microscopy," Phys. Rev. E 66, 067062, 1-4 (2002).

D. Yelin, D. Oron, E. Korkotian, M. Segal, and Y. Silberberg, "Third-harmonic microscopy with a titanium-sapphire laser," Appl. Phys. B 74, 97-101 (2002).

J. M. Schins, T. Schrama, J. Squier, G. J. Brakenhoff, and M. Müller, "Determination of material properties by use of third-harmonic generation microscopy," J. Opt. Soc. Am. B 19, 1627-1634 (2002).

R. D. Schaller, J. C. Johnson, K. R. Wilson, L. F. Lee, L. H. Haber, and R. J. Saykally, "Nonlinear chemical imaging nanomicroscopy: from second and third harmonic generation to multiplex (broad-bandwidth) sum frequency generation near-field scanning optical microscopy," J. Phys. Chem. B 106, 5143-5154 (2002).
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J. M. Resa, C. Gonzalez, S. O. de Landaluce, and J. Lanz, "Densities, excess molar volumes, and refractive indices of ethyl acetate and aromatic hydrocarbon binary mixtures," J. Chem. Thermodyn. 34, 995-1004 (2002).

S. Yaltkaya and R. Aydin, "Experimental investigation of temperature effect on the refractive index of dye laser solutions," Turk. J. Phys. 26, 41-47 (2002).

D. Arosio, H. E. Kwansa, H. Gering, G. Piszczek, and E. Bucci, "Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites," Biopolymers 63, 1-11 (2002).
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2001

M. L. Ferrer, R. Duchowicz, B. Carrasco, J. G. de la Torre, and A. U. Acuñ, "The conformation of serum albumin in solution: a combined phosphorescence depolarization-hydrodymic modeling study," Biophys. J. 80, 2422-2430 (2001).

L. Canioni, S. Rivet, L. Sarger, R. Barille, P. Vacher, and P. Voisin, "Imaging of Ca2+ intracellular dynamics with a third-harmonic generation microscope," Opt. Lett. 26, 515-517 (2001).

A. N. Naumov, D. A. Sidorov-Biryukov, A. B. Fedotov, and A. M. Zheltikov, "Third-harmonic generation in focused beams as a method of 3D microscopy of a laser-produced plasma," Opt. Spectrosc. 90, 778-783 (2001).
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A. Schulzgen, Y. Kawabe, E. Hanamura, A. Yamanaka, P. A. Blanche, J. Lee, H. Sato, M. Naito, N. T. Dan, S. Uchida, Y. Tanabe, and N. Peyghambarian, "Two-photon resonant third-harmonic generation in La2CuO4," Phys. Rev. Lett. 86, 3164-3167 (2001).
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A. B. Schumacher, J. S. Dodge, M. A. Carnahan, R. A. Kaindl, D. S. Chemla, and L. L. Miller, "Parity-forbidden excitation of Si2CuO2Cl2 revealed by optical third-harmonic spectroscopy," Phys. Rev. Lett. 87, 1-4 (2001).
[CrossRef]

2000

C. Bosshard, U. Gubler, P. Kaatz, W. Mazerant, and U. Meier, "Non-phase-matched optical third-harmonic generation in noncentrosymmetric media: cascaded second-order contributions for the calibration of third-order nonlinearities," Phys. Rev. B 61, 10688-10701 (2000).
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U. Gubler and C. Bosshard, "Optical third-harmonic generation of fused silica in gas atmosphere: absolute value of the third-order nonlinear susceptibility chi(3)," Phys. Rev. B 61, 10702-10710 (2000).
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R. D. Schaller, J. C. Johnson, and R. J. Saykally, "Nonlinear chemical imaging microscopy: near-field third harmonic generation imaging of human blood cells," Anal. Chem. 72, 5361-5364 (2000).
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S. Y. Chen, A. Maksimichuk, E. Esarey, and D. Umstadter, "Observation of phase-matched relativistic harmonic generation," Phys. Rev. Lett. 84, 5528-5531 (2000).
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T. Lazaridis, "Solvent reorganization energy and entropy in hydrophobic hydration," J. Phys. Chem. B 104, 4964-4979 (2000).
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K. E. S. Tang and V. A. Bloomfield, "Excluded volume in solvation: sensitivity of scaled-particle theory to solvent size and density," Biophys. J. 79, 2222-2234 (2000).

H. Reis, M. G. Papadopoulos, and D. N. Theodorou, "Calculation of refractive indices and third-harmonic generation susceptibilities of liquid benzene and water: comparison of continuum and discrete local-field theories," J. Phys. Chem. 114, 876-881 (2000).

I. E. Borissevitch, N. Rakov, G. S. Maciel, and C. B. de Araujo, "Changes in porphyrin nonlinear absorption owing to interaction with bovine serum albumin," Appl. Opt. 39, 4431-4435 (2000).

A. Samokhvalov and R. Naaman, "Wavelength- and time-dependent two-photon photoemission spectroscopy of dye-coated silicon surface," J. Phys. Chem. B 104, 11248-11252 (2000).
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1999

A. H. Buist, M. Muller, R. I. Ghauharali, G. J. Brakenhoff, J. A. Squier, C. J. Bardeen, V. V. Yakeovlev, and K. R. Wilson, "Probing microscopic chemical environments with high-intensity chirped pulses," Opt. Lett. 24, 244-246 (1999).

C. E. Giacomelli, M. J. Esplandiu, P. I. Ortiz, M. J. Avena, and C. P. De Pauli, "Ellipsometric study of bovine serum albumin adsorped onto Ti/TiO2 electrodes," J. Colloid Interface Sci. 218, 404-411 (1999).
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A. C. Millard, P. W. Wiseman, D. N. Fittinghoff, K. Wilson, J. Squier, and M. Muller, "Third-harmonic generation microscopy by use of a compact, femtosecond fiber laser source," Appl. Opt. 38, 7393-7397 (1999).

D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999).

N. Sugimoto, H. Kanbara, S. Fujiwara, K. Tanaka, and Y. Shimizugawa, "Third-order optical nonlinearities and their ultrafast response in Bi2O3—B2O3—SiO2 glasses," J. Opt. Soc. Am. B 16, 1904-1908 (1999).

I. Ledoux, I. D. W. Samuel, J. Zyss, S. N. Yaliraki, F. J. Schattenmann, R. R. Schrock, and R. J. Silbey, "Third-order microscopic nonlinearities of very long chain polyenes: saturation phenomena and conformational effects," Chem. Phys. 245, 1-16 (1999).
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H. S. Nalwa, M. Hanack, G. Pawlowski, and M. K. Engel, "Third-order nonlinear optical properties of porphyrazine phthalocyanine and naphthalocyanine germanium derivities: demonstrating the effect of pi-conjugation length on third-order optical nonlinearity of two-dimensional molecules," Chem. Phys. 245, 17-26 (1999).
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1998

R. R. Tykwinski, U. Gubler, R. E. Martin, F. Diederich, C. Bosshard, and P. Gunter, "Structure-property relationships in third-order nonlinear optical chromophores," J. Phys. Chem. B 102, 4451-4465 (1998).
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M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, "3D-microscopy of transparent objects using third harmonic generation," J. Microsc. 191, 266-274 (1998).
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J. Squier, M. Muller, G. J. Brakenhoff, and K. Wilson, "Third harmonic generation microscopy," Opt. Express 3, 315-321 (1998).

W. P. de Boeij, M. S. Pshenichnikov, and D. A. Wiersma, "Ultrafast solvation dynamics explored by femtosecond photon echo spectroscopies," Annu. Rev. Phys. Chem. 49, 99-123 (1998).
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A. B. Myers, "Molecular electronic spectral broadening in liquids and glasses," Annu. Rev. Phys. Chem. 49, 267-295 (1998).
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D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, "Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex," Proc. Natl. Acad. Sci. U.S.A. 95, 15741-15746 (1998).
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J. Noack, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and A. Vogel, "Influence of pulse duration on mechanical effects after laser-induced breakdown in water," J. Appl. Phys. 83, 7488-7495 (1998).
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1997

P. D. T. Huibers, "Models for the wavelength dependence of the index of refraction of water," Appl. Opt. 36, 3785-3787 (1997).

P. R. Callis, "Two-photon induced fluorescence," Annu. Rev. Phys. Chem. 48, 271-297 (1997).
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S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, "Measuring serotonin distribution in live cells with three-photon excitation," Science 275, 530-532 (1997).
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Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
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W. Denk and K. Svoboda, "Photon upmanship: why multiphoton imaging is more than a gimmick," Neuron 18, 351-357 (1997).
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P. J. Campanola, D. W. Young, A. E. Cowan, C. Roychoudhuri, and L. M. Loew, "Applications in nonlinear optical microscopy," Commun. Dis. Public Health 4, 191-192 (1997).

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, "Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation," J. Phys. Chem. A 101, 7019-7023 (1997).
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1996

G. R. Fleming and M. Cho, "Chromophore-solvent dynamics," Annu. Rev. Phys. Chem. 47, 109-134 (1996).
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T. Tsang, "Third- and fifth-harmonic generation at the interfaces of glass and liquids," Phys. Rev. A 54, 5454-5457 (1996).
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D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, "Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser," Bioimaging 4, 1-7 (1996).

I. Graczynski, H. Malak, and J. R. Lakowicz, "Multi-photon excitation of DNA stains DAPI and Hoechst," Bioimaging 4, 138-148 (1996).

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Grycznyski, and J. R. Lakowicz, "Three-photon excitation in fluorescence microscopy," J. Biomed. Opt. 1, 71-74 (1996).

C. Xu and W. W. Webb, "Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050nm," J. Opt. Soc. Am. B 13, 481-491 (1996).

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, "Multiphoton excitation cross-sections of molecular fluorophores," Bioimaging 4, 198-207 (1996).
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C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proc. Natl. Acad. Sci. U.S.A. 93, 10763-10768 (1996).
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1995

X. Quan and E. Fry, "Empirical equation for the index of refraction of seawater," Appl. Opt. 34, 3477-3480 (1995).

L. V. Butov, A. Zrenner, G. Abstreiter, A. V. Petinova, and K. Eberl, "Direct and indirect magnetoexcitons in symmetrical lnxAg1−xAs/GaAs coupled quantum wells," Phys. Rev. B 52, 12153-12157 (1995).
[CrossRef]

T. Y. F. Tsang, "Optical third-harmonic generation at interfaces," Phys. Rev. A 52, 4116-4125 (1995).
[CrossRef]

M. A. Diaz-Garcia, F. Agullo-Lopez, W. E. Torruellas, and G. I. Stegeman, "Identification of two-photon states in phthalocyanines by third harmonic generation spectroscopy," Chem. Phys. Lett. 235, 535-540 (1995).
[CrossRef]

J. McElvain, M. Cha, H. Yu, N. Zhang, F. Wudl, and A. J. Heeger, "Third harmonic generation spectrum of a degenerate ground state conjugated polymer. Direct evidence of simultaneous two- and three-photon resonance," Chem. Phys. Lett. 247, 221-226 (1995).

H. Tajalli, J. P. Jiang, J. T. Murray, N. R. Armstrong, A. Schmidt, M. Chandross, S. Maxumdar, and N. Peyghambarian, "Spectra of third-order optical nonlinear susceptibilities of epitaxial chloro-indium-phthalocyanines," Appl. Phys. Lett. 67, 1639-1641 (1995).
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1994

J. Y. Huang and M. H. Wu, "Nonlinear optical studies of binary mixtures of hydrogen bonded liquids," Phys. Rev. E 50, 3737-3746 (1994).
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W. Denk, "Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions," Proc. Natl. Acad. Sci. U.S.A. 91, 6629-6633 (1994).

P. Vohringer, D. C. Arnett, R. A. Westervelt, M. J. Feldstein, and N. F. Scherer, "Optical dephasing on femtosecond time scales: direct measurement and calculation from solvent spectral densities," J. Chem. Phys. 102, 4027-4036 (1994).
[CrossRef]

H. Nasu, J. Matsuoka, and K. Kanichi, "Second- and third-order optical non-linearity of homogeneous glasses," J. Non-Cryst. Solids 178, 23-30 (1994).
[CrossRef]

1993

S. R. Marder, J. W. Perry, G. Bourhill, C. B. Gorman, B. G. Tiemann, and K. Mansour, "Relation between bond-length alternation and second electronic hyperpolarizability of conjugated molecules," Science 261, 186-189 (1993).

C. Halvorson, R. Wu, D. Moses, F. Wudl, and A. J. Heeger, "Third harmonic generation spectra of degenerate ground state derivatives of poly(1,6-heptadiyne)," Chem. Phys. Lett. 212, 85-89 (1993).
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1992

T. Hasegawa, Y. Iwasa, H. Kishida, T. Koda, Y. Tokura, H. Tachibana, and Y. Kawabata, "Two-photon resonant third-harmonic generation in polysilanes," Phys. Rev. B 45, 6317-6320 (1992).
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1991

1990

W. E. Torruellas, D. Neher, R. Zanoni, G. I. Stegeman, F. Kajzar, and M. Leclerc, "Dispersion measurements of the third-order nonlinear susceptiblity of polythiophene thin films," Chem. Phys. Lett. 175, 11-16 (1990).
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T. Hasegawa, K. Ishikawa, T. Kanetake, T. Koda, K. Takeda, H. Kobayashi, and K. Kubodera, "Excitonic resonant effect in the third-order nonlinear optical properties of blue- and red-form polydiacetylene films," Chem. Phys. Lett. 171, 239-244 (1990).

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

1989

D. W. Hall, M. A. Newhouse, N. F. Borrelli, W. H. Dumbaugh, and D. L. Weidman, "Nonlinear optical susceptibilities of high-index glasses," Appl. Phys. Lett. 54, 1293-1295 (1989).
[CrossRef]

E. M. Vogel, S. G. Kosinski, D. M. Krol, J. L. Jackel, S. R. Friberg, M. K. Oliver, and J. D. Powers, "Structural and optical study of silicate glasses for nonlinear optical devices," J. Non-Cryst. Solids 107, 244-250 (1989).
[CrossRef]

1988

M. Sinclair, D. Moses, K. Akagi, and A. J. Heeger, "Anisotropy of the third-order nonlinear-optical susceptibility in a degenerate-ground-state conjugated polymer: trans-(CH)x," Phys. Rev. B 38, 10724-10733 (1988).
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F. L. Arbelo, P. R. Ojeda, and I. L. Arbeloa, "On the aggregation of rhodamine B in ethanol," Chem. Phys. Lett. 148, 253-258 (1988).
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J. C. Baumert, G. C. Bjorklund, D. H. Jundt, M. C. Jurich, H. Looser, R. D. Miller, J. Rabolt, R. Sooriyakumaran, J. D. Swalen, and R. J. Twieg, "Temperature dependence of the third-order nonlinear optical susceptibilities in polysilanes and polygermanes," Appl. Phys. Lett. 53, 1147-1149 (1988).
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1987

F. Kajzar and J. Messier, "Original technique for third-harmonic-generation measurements in liquids," Rev. Sci. Instrum. 58, 2081-2085 (1987).
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1985

F. Kajzar and J. Messier, "Third-harmonic generation in liquids," Phys. Rev. A 32, 2352-2363 (1985).
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1983

G. R. Meredith, B. Buchalter, and C. Hanzlik, "Third-order optical susceptibility determination by third harmonic generation. 1," J. Chem. Phys. 78, 1533-1542 (1983).
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G. R. Meredith, B. Buchalter, and C. Hanzlik, "Third-order optical susceptibility determination by third harmonic generation. 2," J. Phys. Chem. 78, 1543-1551 (1983).

1978

N. L. Boling, A. J. Glass, and A. Owyoung, "Empirical relationships for predicting nonlinear refractive index changes in optical solids," IEEE J. Quantum Electron. QE-14, 601-608 (1978).
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R. L. Swofford and A. C. Albrecht, "Nonlinear spectroscopy," Annu. Rev. Phys. Chem. 29, 421-440 (1978).

1977

R. H. Hellwarth, "Third-order optical susceptibilities of liquids and solids," Prog. Quantum Electron. 5, 1-68 (1977).
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1975

H. J. Coles, B. R. Jennings, and V. J. Morris, "Refractive index increment measurement for bacterial suspensions," Phys. Med. Biol. 20, 310-313 (1975).
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1973

B. F. Levine and C. G. Bethea, "Molecular hyperpolarizabilities determined from conjugated and nonconjugated organic liquids," Appl. Phys. Lett. 24, 445-447 (1973).
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1971

J. E. Selwyn and J. I. Steinfeld, "Aggregation equilibria of xanthene dyes," J. Phys. Chem. 76, 762-774 (1971).
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1970

C. Wang, "Empirical relation between the linear and the third-order nonlinear optical susceptibilities," Phys. Rev. B 2, 2045-2048 (1970).
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H. T. Edward, "Molecular volumes and the Stokes-Einstein equation," J. Chem. Educ. 47, 261-270 (1970).

J. Jerphagnon and S. K. Kurtz, "Maker fringes: a detailed comparison of theory and experiment for isotropic and uniaxial crystals," J. Appl. Phys. 41, 1667-1681 (1970).
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1965

P. D. Maker and R. W. Terhune, "Study of optical effects due to an induced polarization third order in the electric field strength," Phys. Rev. 137, A801-A818 (1965).
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W. Orttung and J. Warner, "Refractive index dispersion in equine hemoglobin solutions," J. Phys. Chem. 69, 3188-3190 (1965).

I. H. Malitson, "Interspecimen comparison of refractive index of fused silica," J. Opt. Soc. Am. 55, 1205-1209 (1965).

1964

A. Bondi, "van der Waals volumes and radii," J. Phys. Chem. 68, 441-451 (1964).

1961

1951

M. Halwer, G. C. Nutting, and B. A. Brice, "Molecular weight of lactoglobulin, ovalbumin, lysozyme and serum albumin by light scattering," J. Am. Chem. Soc. 73, 2786-2790 (1951).

1948

G. E. Perlmann, L. G. Longsworth, "The specific refractive increment of some purified proteins," J. Am. Chem. Soc. 70, 2719-2724 (1948).

Aalders, A.

D. Faber, E. G. Mik, A. Aalders, and T. G. van Leeuwen, "Oxygen saturation dependent index of refraction of hemoglobin solutions assessed by OCT," in Coherence Domain Optical Methods and Optical Coherence Tomography in Biomedical VII, V. Tuchin, J. Izatt, and J. Fuijimoto, eds., Proc. SPIE4956, 271-281 (2003).

Abstreiter, G.

L. V. Butov, A. Zrenner, G. Abstreiter, A. V. Petinova, and K. Eberl, "Direct and indirect magnetoexcitons in symmetrical lnxAg1−xAs/GaAs coupled quantum wells," Phys. Rev. B 52, 12153-12157 (1995).
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Acuñ, A. U.

M. L. Ferrer, R. Duchowicz, B. Carrasco, J. G. de la Torre, and A. U. Acuñ, "The conformation of serum albumin in solution: a combined phosphorescence depolarization-hydrodymic modeling study," Biophys. J. 80, 2422-2430 (2001).

Agullo-Lopez, F.

M. A. Diaz-Garcia, F. Agullo-Lopez, W. E. Torruellas, and G. I. Stegeman, "Identification of two-photon states in phthalocyanines by third harmonic generation spectroscopy," Chem. Phys. Lett. 235, 535-540 (1995).
[CrossRef]

Akagi, K.

M. Sinclair, D. Moses, K. Akagi, and A. J. Heeger, "Anisotropy of the third-order nonlinear-optical susceptibility in a degenerate-ground-state conjugated polymer: trans-(CH)x," Phys. Rev. B 38, 10724-10733 (1988).
[CrossRef]

Akimov, D. A.

S. O. Konorov, D. A. Akimov, A. A. Ivanov, M. V. Alfimov, S. Botti, R. Ciardi, L. D. Deominicis, L. S. Asilyan, A. A. Podshivalov, D. A. Sidorov-Biryukov, R. Fantoni, and A. M. Zheltikov, "Femtosecond optical harmonic generation as a non-linear spectroscopic probe for carbon nanotubes," J. Raman Spectrosc. 34, 1018-1024 (2003).
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D. A. Akimov, A. A. Ivanov, M. V. Alfimov, E. P. Grabchak, A. A. Shtykova, A. N. Petrov, A. A. Podshivalov, and A. M. Zheltikov, "J-aggregation visualized with two-photon resonant third-harmonic generation," J. Raman Spectrosc. 34, 1007-1012 (2003).
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Albrecht, A. C.

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Figures (10)

Fig. 1
Fig. 1

Multiphoton imaging and THG spectroscopy apparatus. Images were collected using a 1.054 μ m , 100 fs Nd:glass pulsed laser. Spectroscopic measurements were made using a Ti:sapphire laser with 100 fs pulses and wavelengths between 760 and 1000 nm . PD, photodiode; PMT, photomultiplier tube.

Fig. 2
Fig. 2

Third-harmonic spectroscopic measurement procedure applied to deionized water. (A) Close-up of the sample and apparatus. Third-harmonic generated light is collected from both the sample/vessel interface and the vessel/air interface, and their ratio is used to infer sample properties. (B) A THG signal is generated as an interface is scanned through the focus of the beam. The peak of the THG intensity profile is measured for different incident laser powers. The half-width at half-maximum is proportional to the confocal parameter b. (C) The THG intensities from both the sample/vessel interface and the vessel/air interface demonstrate the anticipated cubic dependence on laser power.

Fig. 3
Fig. 3

(A) Theoretical curves of the confocal parameter times the wave-vector mismatch, b Δ κ . Curves are calculated from linear dispersion models for water, benzene, and Duran glass in the case of diffraction-limited geometry. The band representing benzene is bounded by the curves predicted by two different models of dispersion.[32, 72] (B) The phase-matching integral,[25] J ( b Δ κ ) .

Fig. 4
Fig. 4

Two-photon-excited fluorescence and THG imaging. (A) A comparison of two-photon-excited fluorescence and THG images. Unlike two-photon-excited fluorescence, THG can be collected from both fluorescent and nonfluorescent beads. THG can be seen to be primarily produced at interfaces perpendicular to the excitation beam, including the coverslip beneath the fluorescent samples. In this case a shadow of the beads is cast on the coverslip due to the distortion of the excitation beam. (B) Third-harmonic image showing the high contrast of red blood cells in solution without the use of dyes.

Fig. 5
Fig. 5

Third-harmonic spectra of water and benzene; each datum is the mean ± standard error ( ± SE ) . Linear absorption spectra between 250 to 1000 nm were obtained with a Cary 50 spectrophotometer. (A) The third-harmonic intensity ratio, R THG ( ω ) , of deionized water. The term R THG ( ω ) is the ratio of the cubic-fit coefficients of the sample/vessel interface to that of the vessel/air interface and is related to the ratio of the third-order susceptibility ratio by Eq. (18). ( A ) . We show the derived third-order susceptibility ratio of water and glass, χ H 2 O ( 3 ) ( 3 ω ) χ glass ( 3 ) (black arrow and left scale), and the linear absorption of water at the fundamental wavelength (gray arrow and right scale). The correspondence between the THG and the linear absorption spectrum indicates a one-photon resonant enhancement; see inset energy diagram (B) The third-harmonic intensity ratio, R THG ( ω ) , of neat benzene. ( B ) The calculated third-order susceptibility ratio of benzene and glass is relatively constant, suggesting that the dominant contribution to χ ( 3 ) ( 3 ω ) of benzene is nonresonant.

Fig. 6
Fig. 6

Third-harmonic spectrum of Rhodamine B, Fura-2, and BSA; each datum is the mean ± SE . (A) We show the third-harmonic intensity ratio, R THG ( ω ) , of an aqueous solution of 1 mM Rhodamine B. ( A ) We show the derived third-order susceptibility ratio of Rhodamine B, χ Rhodamine ( 3 ) ( 3 ω ) χ glass ( 3 ) , and the two-photon action cross section.[84] The correspondence between the THG and two-photon action indicates a strong two-photon resonant enhancement in Rhodamine B below 850 nm . (B) The third-harmonic intensity ratio, R THG ( ω ) , of an aqueous solution of 0.5 mM Fura-2. ( B ) The third-order susceptibility ratio of Fura-2, χ Fura - 2 ( 3 ) ( 3 ω ) χ glass ( 3 ) , and the two-photon action cross section[85, 86, 87] track each other, indicating a two-photon resonant enhancement. (C) The third-harmonic intensity ratio, R THG ( ω ) , of a 0.75 mM solution of BSA. ( C ) The calculated third-order susceptibility ratio of BSA and glass, χ BSA ( 3 ) ( 3 ω ) χ glass ( 3 ) suggests that χ ( 3 ) ( 3 ω ) for BSA has a three-photon resonant enhancement from 750 to 850 nm . We display the linear absorption of BSA over the region relevant to three-photon resonance.

Fig. 7
Fig. 7

(A) Linear absorption spectrum of oxy-, deoxy-, and carboxyhemoglobin.[56] The wavelength bands corresponding to potential one-photon, two-photon, and three-photon resonances are highlighted and labeled. (B) The estimated refractive index of oxyhemoglobin (Appendix A) and point index measurements taken by Orttung and Warner[88] (500 to 650 nm ), Arosio et al.[81] ( 750 nm ) and Faber et al.[89] ( 800 nm ) .

Fig. 8
Fig. 8

Third-harmonic spectrum of oxy-, carboxy-, and deoxyhemoglobin solutions (this work) together with linear absorption measurements;[56] each datum is the mean ± SE . (A) The third-harmonic intensity ratio, R THG ( ω ) , of a 2 mM oxyhemoglobin solution. ( A ) The correspondence between the derived third-order susceptibility ratio of oxyhemoglobin, χ HbO 2 ( 3 ) ( 3 ω ) χ glass ( 3 ) , and the linear absorption from 375 to 500 nm indicates a two-photon resonant enhancement. (B) We show the third-harmonic intensity ratio, R THG ( ω ) , of a 2 mM , 60% (v/v) carboxylated hemoglobin solution. ( B ) The derived third-order susceptibility ratio of 60% (v/v) carboxyhemoglobin, χ HbCO ( 3 ) ( 3 ω ) χ glass ( 3 ) , and the linear absorption in the two-photon resonance range suggest a two-photon resonant enhancement. (C) The third-harmonic intensity ratio, R THG ( ω ) , of a 2 mM , 95 % (v/v) deoxygenated hemoglobin solution. ( C ) The calculated third-order susceptibility ratio of deoxyhemoglobin and glass, χ Hb ( 3 ) ( 3 ω ) χ glass ( 3 ) , and the linear absorption of deoxyhemoglobin in the two-photon absorption regime.

Fig. 9
Fig. 9

Third-harmonic intensity ratio of hemoglobin in different oxidation states averaged over 20 nm bands. Error bars represent two SE; significance at the 95% confidence level is indicated by a bar.

Fig. 10
Fig. 10

Comparison of THG and linear absorption spectra with semiempirical rules and linear absorption spectra. The data from THG measurements made here have been shifted by the ratio of Miller’s rules for Duran glass and SiO 2 in order to compare them with values reported in the literature (this factor is 1.17 , Appendix B). (A) Benzene: the THG measurement at 1.06 μ m used 13 ns pulses and a Maker fringe technique,[66] and the 1.9 μ m measurement used 30 ns pulses and a triple-wedge technique.[53, 67] Linear absorption measurements in benzene are piecewise composed from measurements over wavelengths of 0.78 to 1.25 μ m [70] and 1.33 and 1.8 μ m .[71] Linear absorption values for the Duran glass are based on transmission measurements and are corrected to account for reflection using the Fresnel equations [Eq. (15)]. (B) Water: the THG measurement at 780 nm used 100 fs pulses and a variant of the Maker fringe approach,[16] those at 1.25 and 1.5 μ m used 40 to 130 fs pulses and an experimental protocol similar to that used here,[52, 54] and the measurement at 1.06 μ m used 13 ns pulses and a Maker fringe technique.[66] Linear absorption measurements are due to Segelstein.[69]

Tables (4)

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Table 1 Dispersion Estimates and Uncertainties at λ = 250 and 750 nm

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Table 2 Volume Fraction Estimates and Uncertainties of Solutes in Solution

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Table 3 Linear Dispersion Increment of Bovine Serum Albumin

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Table 4 Linear Dispersion Increment of Oxyhemoglobin

Equations (29)

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P ( 3 ) ( 3 ω ) = χ ( 3 ) ( 3 ω ) E 3 ( ω ) .
χ Total ( 3 ) ( 3 ω ) χ nonresonant ( 3 ) ( 3 ω ) + χ resonant ( 3 ) ( 3 ω ) .
χ resonant ( 3 ) ( 3 ω ) = l , k , j N μ 0 l μ l k μ k j μ j 0 3 ( ω l 0 3 ω i γ l 0 ) ( ω k 0 2 ω i γ k 0 ) ( ω j 0 ω i γ j 0 ) ,
χ nonresonant ( 3 ) ( 3 ω ) χ ( 1 ) ( 3 ω ) [ χ ( 1 ) ( ω ) ] 3 [ n 2 ( 3 ω ) 1 ] [ n 2 ( ω ) 1 ] 3 .
χ nonresonant ( 3 ) ( 3 ω ) [ χ ( 1 ) ( ω ) ] 2 [ n 2 ( ω ) 1 ] 2 .
χ nonresonant ( 3 ) ( 3 ω ) [ n 2 ( ω ) + 2 ] 2 [ n 2 ( ω ) 2 ] 2 .
P ( 3 ω ) = C ( ω ) b ( ω ) d ϕ exp ( i b Δ κ ϕ ) χ ( 3 ) ( 3 ω , ϕ ) ( 1 + i 2 ϕ ) 2 2 P 3 ( ω ) ,
b ( ω ) = 2 π n ( ω ) w o 2 λ = 2 n ( ω ) λ π [ n 2 ( ω ) NA 2 ] NA 2 ,
Δ κ = 6 π n ( ω ) n ( 3 ω ) λ .
P ( 3 ω ) Slab 1 Slab 2 = C ( ω ) χ Slab 1 ( 3 ) b Slab 1 J ( b Slab 1 Δ κ Slab 1 ) χ Slab 2 ( 3 ) b Slab 2 J ( b Slab 2 Δ κ Slab 2 ) 2 P 3 ( ω ) ,
J ( b Δ κ ) 0 d ϕ exp ( i b Δ κ ϕ ) ( 1 + i 2 ϕ ) 2 .
P ( 3 ω ) solution glass P ( 3 ω ) glass air = [ χ glass ( 3 ) ( 3 ω ) b glass ( ω ) J ( b glass Δ κ glass ) χ solution ( 3 ) ( 3 ω ) b solution ( ω ) J ( b solution Δ κ solution ) χ glass ( 3 ) ( 3 ω ) b glass ( ω ) J ( b glass Δ κ glass ) χ air ( 3 ) ( 3 ω ) b air ( ω ) J ( b air Δ κ air ) ] 2 .
P ( 3 ω ) solution glass P ( 3 ω ) glass air [ 1 χ solution ( 3 ) ( 3 ω ) b solution ( ω ) J ( b solution Δ κ solution ) χ glass ( 3 ) ( 3 ω ) b glass ( ω ) J ( b glass Δ κ glass ) ] 2 .
χ solution ( 3 ) ( 3 ω ) χ glass ( 3 ) ( 3 ω ) = J ( b glass Δ κ glass ) b glass ( ω ) J ( b solution Δ κ solution ) b solution ( ω ) { 1 ± [ P ( 3 ω ) solution glass P ( 3 ω ) glass air ] 1 2 } .
χ solute ( 3 ) ( 3 ω ) χ glass ( 3 ) ( 3 ω ) = J ( b glass Δ κ glass ) b glass ( ω ) J ( b solution Δ κ solution ) b solution ( ω ) ( 1 + 1 υ { ± [ P ( 3 ω ) solution glass P ( 3 ω ) glass air ] 1 2 ( 1 υ ) [ P ( 3 ω ) solvent glass P ( 3 ω ) glass air ] 1 2 } ) .
r ( ω ) SiO 2 solution = n glass ( ω ) n solution ( ω ) n solution ( ω ) + n glass ( ω ) ,
R THG ( ω ) α solution glass ( ω ) α glass air ( ω ) [ 1 r 2 ( ω ) glass solution ] 3 [ 1 a ( ω ) glass ] 3 [ 1 r 2 ( 3 ω ) glass solution ] [ 1 a ( 3 ω ) glass ] .
χ solution ( 3 ) ( ω ) χ glass ( 3 ) ( ω ) = J ( b glass Δ κ glass ) b glass ( ω ) J ( b solotion Δ κ solution ) b solution ( ω ) [ β ( ω ) ρ ( ω ) ± R THG ( ω ) ] ,
β ( ω ) J ( b glass solution Δ κ glass ) b glass solution ( ω ) J ( b glass air Δ κ glass ) b glass air ( ω )
ρ ( ω ) = { [ 1 r 2 ( ω ) glass solution ] 3 [ 1 r 2 ( 3 ω ) glass solution ] } 1 2
χ solute ( 3 ) ( ω ) χ glass ( 3 ) ( ω ) = J ( b glass Δ κ glass ) b glass ( ω ) J ( b solution Δ κ solution ) b solution ( ω ) { β ( ω ) ρ ( ω ) + 1 υ [ ± R THG solvent ( ω ) ( 1 υ ) R THG solvent ( ω ) ] } ,
n SiO 2 or Duran ( λ ) = [ 1 + n 1 1 ( λ 1 λ ) 2 + n 2 1 ( λ 2 λ ) 2 + n 3 1 ( λ 3 λ ) 2 ] 1 2 ,
n benzene ( λ ) = n 0 [ 1 + ( λ 0 λ ) 2 ] ,
n benzene ( λ ) = n 0 [ 1 + n 1 1 ( λ 1 λ ) 2 ] 1 2 ,
n water ( λ , S , T ) = n 0 + ( n 1 + n 2 T + n 3 T 2 ) S n 4 T 2 + n 5 + n 6 S + n 7 T λ ( n 8 λ ) 2 + ( n 9 λ ) 3 ,
d n BSA ( λ ) d c = λ 118 + 162 + 15 000 λ + ( 0.056 λ ) 2 ,
n ( ω ) = c ln ( 10 ) ϵ ( ω ) 2 ω ,
n ( ω ) = r A r exp [ ( Ω r ω ) 2 2 σ r 2 ] ,
Δ n ( ω ) = 2 r A r σ r ( Ω r ω ) exp [ ( Ω r ω ) 2 2 σ r 2 ] .

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