Abstract

We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 µm are performed.

© 2009 Optical Society of America

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2009 (4)

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

M. Friebel, J. Helfmann, U. Netz, and M. Meinke, "Influence of oxygen saturation on the optical scattering properties of human red blood cells in the spectral range 250 to 2000 nm," J. Biomed. Opt. 14, 034001-6 (2009).
[CrossRef] [PubMed]

T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, "Ex vivo and in vivo second-harmonic-generation imaging of dermal collagen fiber in skin: comparison of imaging characteristics between mode-locked Cr:forsterite and Ti:sapphire lasers," Appl. Opt. 48, D88-D95 (2009).
[CrossRef] [PubMed]

2008 (1)

2007 (3)

2006 (4)

T. Tsai, C. Lin, H. Tsai, S. Chen, S. Tai, K. Lin, and C. Sun, "Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section," Opt. Lett. 31, 930-932 (2006).
[CrossRef] [PubMed]

P. Theer, and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am. A 23, 3139-3149 (2006).
[CrossRef]

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Q. Nguyen, P.S. Tsai, and D. Kleinfeld, "Mpscope: a versatile software suite for multiphoton microscopy," J. Neurosci. Methods 156, 351-359 (2006).
[CrossRef] [PubMed]

2005 (1)

F. Helmchen, and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

2003 (3)

2002 (2)

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

I. Chen, S. Chu, C. Sun, P. Cheng, and B. Lin, "Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources," Opt. Quantum Electron. 34, 1251-1266 (2002).
[CrossRef]

2001 (1)

1999 (2)

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, "Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo," Nat. Neuroscience 2, 65-73 (1999).
[CrossRef]

F. Helmchen, K. Svoboda, W. Denk, and D. W. Tank, "In-vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons," Nat. Neuroscience 2, 989-996 (1999).
[CrossRef]

1998 (4)

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

M. Müller, J. Squier, K.R. Wilson, and G.J. Brakenhoff, "3d microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

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. USA 95, 15741-15746 (1998).
[CrossRef] [PubMed]

A. Schonle, and S.W. Hell, "Heating by absorption in the focus of an objective lens," Opt. Lett. 23, 325-327 (1998).
[CrossRef]

1996 (2)

1995 (1)

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

1994 (1)

J.M. Schmitt, A. Knuttel, M. Yadlowsky, and M.A. Eckhaus, "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys. Med. Biol. 39, 1705-1720 (1994).
[CrossRef] [PubMed]

1993 (1)

1990 (1)

W. Cheong, S. Prahl, and A. Welch, "A review of the optical properties of biological tissues," IEEE J. Quantum Electron. 26, 2166-2185 (1990).
[CrossRef]

Araki, T.

Baldacchini, T.

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

Balu, M.

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

Berns, M.W.

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Bilinsky, I.P.

Boas, D.A.

Bouma, B.E.

Brakenhoff, G.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

Brakenhoff, G.J.

M. Müller, J. Squier, K.R. Wilson, and G.J. Brakenhoff, "3d microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Carter, J.

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

Chapman, C.F.

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Chen, I.

I. Chen, S. Chu, C. Sun, P. Cheng, and B. Lin, "Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources," Opt. Quantum Electron. 34, 1251-1266 (2002).
[CrossRef]

S. Chu, I. Chen, T. Liu, P.C. Chen, C. Sun, and B. Lin, "Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser," Opt. Lett. 26, 1909-1911 (2001).
[CrossRef]

Chen, P.C.

Chen, S.

Cheng, D.K.

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Cheng, P.

I. Chen, S. Chu, C. Sun, P. Cheng, and B. Lin, "Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources," Opt. Quantum Electron. 34, 1251-1266 (2002).
[CrossRef]

Cheong, W.

W. Cheong, S. Prahl, and A. Welch, "A review of the optical properties of biological tissues," IEEE J. Quantum Electron. 26, 2166-2185 (1990).
[CrossRef]

Chong, A.

Christie, R.

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

Chu, S.

Chylek, P.

Demirbas, U.

Denk, W.

P. Theer, and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am. A 23, 3139-3149 (2006).
[CrossRef]

F. Helmchen, and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

P. Theer, M.T. Hasan, and W. Denk, "Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier," Opt. Lett. 28, 1022-1024 (2003).
[CrossRef] [PubMed]

F. Helmchen, K. Svoboda, W. Denk, and D. W. Tank, "In-vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons," Nat. Neuroscience 2, 989-996 (1999).
[CrossRef]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, "Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo," Nat. Neuroscience 2, 65-73 (1999).
[CrossRef]

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. USA 95, 15741-15746 (1998).
[CrossRef] [PubMed]

Drew, P.J.

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

Ebner, F.F.

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Eckhaus, M.A.

J.M. Schmitt, A. Knuttel, M. Yadlowsky, and M.A. Eckhaus, "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys. Med. Biol. 39, 1705-1720 (1994).
[CrossRef] [PubMed]

Friebel, M.

M. Friebel, J. Helfmann, U. Netz, and M. Meinke, "Influence of oxygen saturation on the optical scattering properties of human red blood cells in the spectral range 250 to 2000 nm," J. Biomed. Opt. 14, 034001-6 (2009).
[CrossRef] [PubMed]

Friedman, B.

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Fujimoto, J.G.

Fukushima, S.

Ghalmi, S.

Golubovic, B.

Hasan, M.T.

Helfmann, J.

M. Friebel, J. Helfmann, U. Netz, and M. Meinke, "Influence of oxygen saturation on the optical scattering properties of human red blood cells in the spectral range 250 to 2000 nm," J. Biomed. Opt. 14, 034001-6 (2009).
[CrossRef] [PubMed]

Hell, S.W.

Helmchen, F.

F. Helmchen, and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, "Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo," Nat. Neuroscience 2, 65-73 (1999).
[CrossRef]

F. Helmchen, K. Svoboda, W. Denk, and D. W. Tank, "In-vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons," Nat. Neuroscience 2, 989-996 (1999).
[CrossRef]

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. USA 95, 15741-15746 (1998).
[CrossRef] [PubMed]

Hyman, B.T.

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

Ito, M.

Kaertner, F.X.

Kleinfeld, D.

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Q. Nguyen, P.S. Tsai, and D. Kleinfeld, "Mpscope: a versatile software suite for multiphoton microscopy," J. Neurosci. Methods 156, 351-359 (2006).
[CrossRef] [PubMed]

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. USA 95, 15741-15746 (1998).
[CrossRef] [PubMed]

Knuttel, A.

J.M. Schmitt, A. Knuttel, M. Yadlowsky, and M.A. Eckhaus, "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys. Med. Biol. 39, 1705-1720 (1994).
[CrossRef] [PubMed]

Kou, L.

Krasieva, T.B.

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

Labrie, D.

Lee, J.H.

Lin, B.

I. Chen, S. Chu, C. Sun, P. Cheng, and B. Lin, "Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources," Opt. Quantum Electron. 34, 1251-1266 (2002).
[CrossRef]

S. Chu, I. Chen, T. Liu, P.C. Chen, C. Sun, and B. Lin, "Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser," Opt. Lett. 26, 1909-1911 (2001).
[CrossRef]

Lin, C.

Lin, K.

Liu, T.

Liu, Y.

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Lyden, P.D.

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Meinke, M.

M. Friebel, J. Helfmann, U. Netz, and M. Meinke, "Influence of oxygen saturation on the optical scattering properties of human red blood cells in the spectral range 250 to 2000 nm," J. Biomed. Opt. 14, 034001-6 (2009).
[CrossRef] [PubMed]

Mempel, T.R.

Mitra, P.P.

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. USA 95, 15741-15746 (1998).
[CrossRef] [PubMed]

Moore, A.

Müller, M.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

M. Müller, J. Squier, K.R. Wilson, and G.J. Brakenhoff, "3d microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Netz, U.

M. Friebel, J. Helfmann, U. Netz, and M. Meinke, "Influence of oxygen saturation on the optical scattering properties of human red blood cells in the spectral range 250 to 2000 nm," J. Biomed. Opt. 14, 034001-6 (2009).
[CrossRef] [PubMed]

Nguyen, Q.

Q. Nguyen, P.S. Tsai, and D. Kleinfeld, "Mpscope: a versatile software suite for multiphoton microscopy," J. Neurosci. Methods 156, 351-359 (2006).
[CrossRef] [PubMed]

Nikitin, A.Y.

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

Nishimura, N.

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Prahl, S.

W. Cheong, S. Prahl, and A. Welch, "A review of the optical properties of biological tissues," IEEE J. Quantum Electron. 26, 2166-2185 (1990).
[CrossRef]

Ramachandran, S.

Renninger, W. H.

Ruvinskaya, S.

Sakadzic, S.

Schaffer, C.B.

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Schmitt, J.M.

J.M. Schmitt, A. Knuttel, M. Yadlowsky, and M.A. Eckhaus, "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys. Med. Biol. 39, 1705-1720 (1994).
[CrossRef] [PubMed]

Schober, R.

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

Schonle, A.

Schroeder, L.F.

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Schulze, P.C.

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

Schwarzmaier, H.

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

Sennaroglu, A.

Shih, A.Y.

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

Simon, U.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

Sonek, G.J.

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Squier, J.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

M. Müller, J. Squier, K.R. Wilson, and G.J. Brakenhoff, "3d microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Sun, C.

Svoboda, K.

F. Helmchen, K. Svoboda, W. Denk, and D. W. Tank, "In-vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons," Nat. Neuroscience 2, 989-996 (1999).
[CrossRef]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, "Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo," Nat. Neuroscience 2, 65-73 (1999).
[CrossRef]

Tai, S.

Takahashi, Y.

Tank, D. W.

F. Helmchen, K. Svoboda, W. Denk, and D. W. Tank, "In-vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons," Nat. Neuroscience 2, 989-996 (1999).
[CrossRef]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, "Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo," Nat. Neuroscience 2, 65-73 (1999).
[CrossRef]

Tearney, G.J.

Theer, P.

Tromberg, B.J.

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Tsai, H.

Tsai, P.S.

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Q. Nguyen, P.S. Tsai, and D. Kleinfeld, "Mpscope: a versatile software suite for multiphoton microscopy," J. Neurosci. Methods 156, 351-359 (2006).
[CrossRef] [PubMed]

Tsai, T.

Ulrich, F.

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

van Howe, J.

Webb, W.W.

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

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

Welch, A.

W. Cheong, S. Prahl, and A. Welch, "A review of the optical properties of biological tissues," IEEE J. Quantum Electron. 26, 2166-2185 (1990).
[CrossRef]

Williams, R.M.

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

Wilson, K.R.

M. Müller, J. Squier, K.R. Wilson, and G.J. Brakenhoff, "3d microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

Wise, F.

Wise, F. W.

Wolleschensky, R.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

Xu, C.

Yadlowsky, M.

J.M. Schmitt, A. Knuttel, M. Yadlowsky, and M.A. Eckhaus, "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys. Med. Biol. 39, 1705-1720 (1994).
[CrossRef] [PubMed]

Yan, M.F.

Yaroslavsky, A.N.

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

Yaroslavsky, I.V.

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

Yasui, T.

Zadoyan, R.

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

Zhou, S.

Zipfel, W.R.

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

Appl. Opt. (2)

Biophys J. (1)

Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chapman, and B.J. Tromberg, "Evidence for localized cell heating induced by infrared optical tweezers.," Biophys J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

IEEE J. Quantum Electron. (1)

W. Cheong, S. Prahl, and A. Welch, "A review of the optical properties of biological tissues," IEEE J. Quantum Electron. 26, 2166-2185 (1990).
[CrossRef]

J. Biomed. Opt. (2)

M. Balu, T. Baldacchini, J. Carter, T.B. Krasieva, R. Zadoyan, and B.J. Tromberg, "Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media," J. Biomed. Opt. 14, 010508-3 (2009).
[CrossRef] [PubMed]

M. Friebel, J. Helfmann, U. Netz, and M. Meinke, "Influence of oxygen saturation on the optical scattering properties of human red blood cells in the spectral range 250 to 2000 nm," J. Biomed. Opt. 14, 034001-6 (2009).
[CrossRef] [PubMed]

J. Cereb. Blood. Flow. Metab. (1)

A.Y. Shih, B. Friedman, P.J. Drew, P.S. Tsai, P.D. Lyden, and D. Kleinfeld, "Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke," J. Cereb. Blood. Flow. Metab. 29, 738-751 (2009).
[CrossRef] [PubMed]

J. Microsc. (2)

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. Brakenhoff, "Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives," J. Microsc. 191, 141-150 (1998).
[CrossRef] [PubMed]

M. Müller, J. Squier, K.R. Wilson, and G.J. Brakenhoff, "3d microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

J. Neurosci. Methods (1)

Q. Nguyen, P.S. Tsai, and D. Kleinfeld, "Mpscope: a versatile software suite for multiphoton microscopy," J. Neurosci. Methods 156, 351-359 (2006).
[CrossRef] [PubMed]

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

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

Nat. Methods (1)

F. Helmchen, and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Nat. Neuroscience (2)

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, "Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo," Nat. Neuroscience 2, 65-73 (1999).
[CrossRef]

F. Helmchen, K. Svoboda, W. Denk, and D. W. Tank, "In-vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons," Nat. Neuroscience 2, 989-996 (1999).
[CrossRef]

Opt. Express (2)

Opt. Lett. (8)

T. Tsai, C. Lin, H. Tsai, S. Chen, S. Tai, K. Lin, and C. Sun, "Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section," Opt. Lett. 31, 930-932 (2006).
[CrossRef] [PubMed]

J. van Howe, J.H. Lee, S. Zhou, F. Wise, C. Xu, S. Ramachandran, S. Ghalmi, and M.F. Yan, "Demonstration of soliton self-frequency shift below 1300 nm in higher-order mode, solid silica-based fiber," Opt. Lett. 32, 340-342 (2007).
[CrossRef] [PubMed]

J.H. Lee, J. van Howe, C. Xu, S. Ramachandran, S. Ghalmi, and M.F. Yan, "Generation of femtosecond pulses at 1350 nm by Cerenkov radiation in higher-order-mode fiber," Opt. Lett. 32, 1053-1055 (2007).
[CrossRef] [PubMed]

A. Chong, W. H. Renninger, and F. W. Wise, "All-normal-dispersion femtosecond fiber laser with pulse energy above 20 nJ," Opt. Lett. 32, 2408-2410 (2007).
[CrossRef] [PubMed]

A. Schonle, and S.W. Hell, "Heating by absorption in the focus of an objective lens," Opt. Lett. 23, 325-327 (1998).
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B.E. Bouma, G.J. Tearney, I.P. Bilinsky, B. Golubovic, and J.G. Fujimoto, "Self-phase-modulated kerr-lens mode-locked Cr:forsterite laser source for optical coherence tomography," Opt. Lett. 21, 1839-1841 (1996).
[CrossRef] [PubMed]

S. Chu, I. Chen, T. Liu, P.C. Chen, C. Sun, and B. Lin, "Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser," Opt. Lett. 26, 1909-1911 (2001).
[CrossRef]

P. Theer, M.T. Hasan, and W. Denk, "Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier," Opt. Lett. 28, 1022-1024 (2003).
[CrossRef] [PubMed]

Opt. Quantum Electron. (1)

I. Chen, S. Chu, C. Sun, P. Cheng, and B. Lin, "Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources," Opt. Quantum Electron. 34, 1251-1266 (2002).
[CrossRef]

Phys. Med. Biol. (2)

J.M. Schmitt, A. Knuttel, M. Yadlowsky, and M.A. Eckhaus, "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys. Med. Biol. 39, 1705-1720 (1994).
[CrossRef] [PubMed]

A.N. Yaroslavsky, P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H. Schwarzmaier, "Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range," Phys. Med. Biol. 47, 2059-2073 (2002).
[CrossRef] [PubMed]

PLoS Biology (1)

C.B. Schaffer, B. Friedman, N. Nishimura, L.F. Schroeder, P.S. Tsai, F.F. Ebner, P.D. Lyden, and D. Kleinfeld, "Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion," PLoS Biology 4, e22 EP - (2006).
[CrossRef]

Proc. Natl. Acad. Sci. USA (2)

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

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. USA 95, 15741-15746 (1998).
[CrossRef] [PubMed]

Other (2)

D. Kleinfeld, and W. Denk, "Two-photon imaging of neocortical microcirculation," in Imaging Neurons: A Laboratory Manual, R. Yuste, F. Lanni, A. Konnerth, eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,2000) 23.1-23.15.

W. Denk, D.W. Piston, and W.W. Webb, "Multi-photon molecular excitation in laser-scanning microscopy," in Handbook of Biological Confocal Microscopy, 3.ed., J.B. Pawlay, ed. (Springer Science, New York, NY, 2006) 535-549.
[CrossRef]

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Figures (7)

Fig. 1.
Fig. 1.

TPM of fluorescent beads in a tissue phantom. (a) Maximum-intensity side projections of the image stacks for 1280-nm excitation and 775-nm excitation are shown on the left and right, respectively. Normalized single planar TPM images from several different depths are shown on each side. Each stack consists of 1200 x-y images (512×512 pixels, averaged over four scans, 4 fps) taken at a depth increment of 1 µm. (b) Fluorescence signal from single beads versus imaging depth. Points denote the average value of the brightest 0.1% of the pixels in a single x-y image on a logarithmic scale, and solid lines are exponential fits. (c) Absorption spectrum of water from 700 nm to 1900 nm [19].

Fig. 2.
Fig. 2.

Ex vivo TPM of cortical vasculature in a mouse brain. Blood vessels are filled with agarose gel that is stained by a mixture of fluorescein-dextran and Alexa680-dextran. (a) Maximum-intensity side projections of stacks for 1280-nm excitation (530 x-y images, 1-µm z increment) and for 775-nm excitation (330 x-y images, 1-µm z increment) are shown on the left and right, respectively. Each x-y image is 512×512 pixels, averaged over four scans and taken at a 3-fps rate. (b) Attenuation of the measured fluorescence intensity with depth. Fluorescence signal strength at a particular depth is represented by the average value of the brightest 10% of the pixels in the x-y image from that depth.

Fig. 3.
Fig. 3.

In vivo TPM of cortical vasculature in a mouse brain. Blood plasma is labeled by FITC-dextran and Alexa680-dextran. (a) Maximum intensity side projections of normalized image stacks for 1280-nm excitation and for 775-nm excitation are shown on the left and right, respectively. Images are normalized by stretching the histogram to allow 0.5% of the pixels to be saturated. Normalized x-y images from several different depths are shown on each side. x-y images (512×512 pixels, averaged over three scans, 3 fps) in the stacks are taken at a depth increment of 2 µm. An average-intensity z projection of the x-y images at depths from 980 µm to 1020 µm is at the bottom left corner of the figure. (b) Attenuation of the measured fluorescence intensity with depth. Fluorescence signal strength at a particular depth is represented by the average value of the brightest 10% of the pixels in the x-y image from that depth.

Fig. 4.
Fig. 4.

Blood flow speed measurement in a single cortical capillary at a depth of 900 µm. (a) A 3-D rendering of the stacks was created using the volume viewer plug-in in ImageJ software. (b) A single normalized planar TPM image from a depth of 900 µm. (c) Zoomed in planar view of a capillary. We recorded line scans along the central axis of the vessel at a rate of ~1 ms per line. Line scan trajectory is marked by the yellow line. (d) x (spatial dimension) - t (temporal dimension) data set from the line scans. Dark bands are due to the motion of the unstained red blood cells (RBCs) along the scan direction. The slope of the bands gives the inverse of the speed of the RBCs [23]. We calculated the speed of RBCs in this deep capillary to be 1.4 mm/s with a standard deviation of 0.5 mm/s.

Fig. 5.
Fig. 5.

Improved performance of in vivo two-photon imaging through blood vessels with longer wavelength excitation. Maximum intensity z-projections of TPM of the cortical vasculature with 1280-nm excitation (top row) and with 775-nm excitation (bottom row) are shown. The emission peak of Alexa680 (used with 1280-nm excitation) is at 700 nm, and the emission peak of FITC (used with 775-nm excitation) is at 518 nm. Left column is the projection of blood vessels between depths of 40 µm and 150 µm from the brain surface, showing relatively large blood vessels at the surface. Right column is the projection of the 60-µm slice beneath the large blood vessels. All images are normalized after the maximum intensity projections along the z-axis are performed. Reduction of the detected fluorescence intensity in the areas beneath the large blood vessels is significantly more pronounced with 775-nm excitation.

Fig. 6.
Fig. 6.

Maximum intensity z-projections of in vivo TPM images of cortical vasculature between 80-µm and 180-µm depth using (a) 775-nm excitation and 700-nm short pass emission filter, (b) 775-nm excitation and 525-nm band-pass filter with 50-nm spectral bandwidth, and (c) 1280-nm excitation and 700-nm emission band-pass filter with 75-nm bandwidth. Red arrows indicate the positions of two exemplary intrinsically fluorescent structures. All three images are normalized after maximum intensity projections along z-axis are performed.

Fig. 7.
Fig. 7.

Two-photon action cross-sections (quantum efficiency (η2) x absolute two-photon cross-section (σ2)) of 8 commercial dyes. Solid lines are guides for the eye.

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