Abstract

Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of in vivo three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength in vivo imaging.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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2018 (1)

2017 (1)

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

2016 (1)

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

2015 (3)

2014 (1)

K. Wang, N. G. Horton, K. Charan, and C. Xu, “Advanced Fiber Soliton Sources for Nonlinear Deep Tissue Imaging in Biophotonics,” IEEE J. Sel. Top. Quantum Electron. 20, 50–60 (2014).
[Crossref]

2013 (3)

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

S. L. Jacques, “Optical properties of biological tissues: a review,” Phys. Med. Biol.  58, R37 (2013).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

2012 (1)

2011 (1)

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt 16, 106014 (2011).
[Crossref] [PubMed]

2009 (2)

M. Balu, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt.  14, 010508 (2009).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17, 13354–13364 (2009).
[Crossref] [PubMed]

2006 (1)

M. Friebel, A. Roggan, G. J. Müller, and M. C. Meinke, “Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions,” J. Biomed. Opt 11, 034021 (2006).
[Crossref]

2005 (1)

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

2003 (1)

2002 (1)

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. 99, 11014–11019 (2002).
[Crossref]

2001 (1)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[Crossref] [PubMed]

2000 (2)

1999 (1)

A. Roggan, M. Friebel, K. Doerschel, A. Hahn, and G. J. Mueller, “Optical properties of circulating human blood in the wavelength range 400–2500 nm,” J. Biomed. Opt 4, 36–47 (1999).
[Crossref] [PubMed]

1998 (1)

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. 95, 15741–15746 (1998).
[Crossref] [PubMed]

1997 (2)

W. Denk and K. Svoboda, “Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

R. F. Reinoso, B. A. Telfer, and M. Rowland, “Tissue water content in rats measured by desiccation,” J. Pharmacol. Toxicol. Methods 38, 87–92 (1997).
[Crossref] [PubMed]

1993 (1)

1990 (2)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Ahrens, M. B.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Alfano, R. R.

Balu, M.

M. Balu, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt.  14, 010508 (2009).
[Crossref] [PubMed]

Baohan, A.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Bargmann, C. I.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Barry, S.

Beaurepaire, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[Crossref] [PubMed]

Berns, M. W.

Cable, A. E.

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[Crossref] [PubMed]

Charan, K.

K. Charan, B. Li, M. Wang, C. P. Lin, and C. Xu, “Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy,” Biomed. Opt. Express 9, 2304–2311 (2018).
[Crossref] [PubMed]

K. Wang, N. G. Horton, K. Charan, and C. Xu, “Advanced Fiber Soliton Sources for Nonlinear Deep Tissue Imaging in Biophotonics,” IEEE J. Sel. Top. Quantum Electron. 20, 50–60 (2014).
[Crossref]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[Crossref] [PubMed]

Chen, T.-W.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Cheng, Y.-T.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

Cheong, W. F.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Chong, S. P.

Clark, C. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Coleno, M.

Cooke, D. F.

Cruz-Hernández, J. C.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

Dana, H.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Denk, W.

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]

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. 95, 15741–15746 (1998).
[Crossref] [PubMed]

W. Denk and K. Svoboda, “Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

D. Kleinfeld and W. Denk, “Two-photon imaging of neocortical microcirculation,” in Imaging Neurons: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1999), pp. 1–23

Doerschel, K.

A. Roggan, M. Friebel, K. Doerschel, A. Hahn, and G. J. Mueller, “Optical properties of circulating human blood in the wavelength range 400–2500 nm,” J. Biomed. Opt 4, 36–47 (1999).
[Crossref] [PubMed]

Dunn, A. K.

Durst, M. E.

Feng, D. D.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

Friebel, M.

M. Friebel, A. Roggan, G. J. Müller, and M. C. Meinke, “Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions,” J. Biomed. Opt 11, 034021 (2006).
[Crossref]

A. Roggan, M. Friebel, K. Doerschel, A. Hahn, and G. J. Mueller, “Optical properties of circulating human blood in the wavelength range 400–2500 nm,” J. Biomed. Opt 4, 36–47 (1999).
[Crossref] [PubMed]

Gordus, A.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Hahn, A.

A. Roggan, M. Friebel, K. Doerschel, A. Hahn, and G. J. Mueller, “Optical properties of circulating human blood in the wavelength range 400–2500 nm,” J. Biomed. Opt 4, 36–47 (1999).
[Crossref] [PubMed]

Hasan, M. T.

Hasseman, J. P.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932–940 (2005).
[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. 95, 15741–15746 (1998).
[Crossref] [PubMed]

Holt, G. T.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Horton, N. G.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

N. G. Horton and C. Xu, “Dispersion compensation in three-photon fluorescence microscopy at 1,700 nm,” Biomed. Opt. Express 6, 1392–1397 (2015).
[Crossref] [PubMed]

K. Wang, N. G. Horton, K. Charan, and C. Xu, “Advanced Fiber Soliton Sources for Nonlinear Deep Tissue Imaging in Biophotonics,” IEEE J. Sel. Top. Quantum Electron. 20, 50–60 (2014).
[Crossref]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt 16, 106014 (2011).
[Crossref] [PubMed]

Hu, A.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Jacques, S. L.

S. L. Jacques, “Optical properties of biological tissues: a review,” Phys. Med. Biol.  58, R37 (2013).
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Jayaraman, V.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Jiang, J. Y.

Kerr, R. A.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Kim, D. S.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Kleinfeld, D.

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. 95, 15741–15746 (1998).
[Crossref] [PubMed]

D. Kleinfeld and W. Denk, “Two-photon imaging of neocortical microcirculation,” in Imaging Neurons: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1999), pp. 1–23

Kobat, D.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt 16, 106014 (2011).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17, 13354–13364 (2009).
[Crossref] [PubMed]

Kou, L.

Krubitzer, L.

Li, B.

Liang, R.

K. Wang, R. Liang, and P. Qiu, “Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy,” IEEE Photonics J..  7, 1–8 (2015).
[Crossref]

Lin, C. P.

Liu, F.

Looger, L. L.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Macklin, J. J.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Meinke, M. C.

M. Friebel, A. Roggan, G. J. Müller, and M. C. Meinke, “Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions,” J. Biomed. Opt 11, 034021 (2006).
[Crossref]

Merkle, C. W.

Mertz, J.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[Crossref] [PubMed]

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. 95, 15741–15746 (1998).
[Crossref] [PubMed]

Mohar, B.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Mueller, G. J.

A. Roggan, M. Friebel, K. Doerschel, A. Hahn, and G. J. Mueller, “Optical properties of circulating human blood in the wavelength range 400–2500 nm,” J. Biomed. Opt 4, 36–47 (1999).
[Crossref] [PubMed]

Müller, G. J.

M. Friebel, A. Roggan, G. J. Müller, and M. C. Meinke, “Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions,” J. Biomed. Opt 11, 034021 (2006).
[Crossref]

Narayan, S.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Nishimura, N.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17, 13354–13364 (2009).
[Crossref] [PubMed]

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, “Two-photon microscopy in brain tissue: parameters influencing the imaging depth,” J. Neurosci. Methods 111, 29–37 (2001).
[Crossref] [PubMed]

Orger, M. B.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Ouzounov, D.

T. Wang, D. Ouzounov, M. Wang, and C. Xu, “Quantitative Comparison of Two-photon and Three-photon Activity Imaging of GCaMP6s-labeled Neurons in vivo in the Mouse Brain,” in “Optics in the Life Sciences Congress (2017), paper BrM4B.4,” (Optical Society of America, 2017), p. BrM4B.4.

Ouzounov, D. G.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

Patel, R.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Prahl, S. A.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Pulver, S. R.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Qiu, P.

K. Wang, R. Liang, and P. Qiu, “Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy,” IEEE Photonics J..  7, 1–8 (2015).
[Crossref]

Radhakrishnan, H.

Reimer, J.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

Reinoso, R. F.

R. F. Reinoso, B. A. Telfer, and M. Rowland, “Tissue water content in rats measured by desiccation,” J. Pharmacol. Toxicol. Methods 38, 87–92 (1997).
[Crossref] [PubMed]

Renninger, S. L.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Roggan, A.

M. Friebel, A. Roggan, G. J. Müller, and M. C. Meinke, “Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions,” J. Biomed. Opt 11, 034021 (2006).
[Crossref]

A. Roggan, M. Friebel, K. Doerschel, A. Hahn, and G. J. Mueller, “Optical properties of circulating human blood in the wavelength range 400–2500 nm,” J. Biomed. Opt 4, 36–47 (1999).
[Crossref] [PubMed]

Rowland, M.

R. F. Reinoso, B. A. Telfer, and M. Rowland, “Tissue water content in rats measured by desiccation,” J. Pharmacol. Toxicol. Methods 38, 87–92 (1997).
[Crossref] [PubMed]

Schaffer, C. B.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17, 13354–13364 (2009).
[Crossref] [PubMed]

Schreiter, E. R.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Srinivasan, V. J.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

Sun, Y.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Svoboda, K.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

W. Denk and K. Svoboda, “Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

Telfer, B. A.

R. F. Reinoso, B. A. Telfer, and M. Rowland, “Tissue water content in rats measured by desiccation,” J. Pharmacol. Toxicol. Methods 38, 87–92 (1997).
[Crossref] [PubMed]

Theer, P.

Tolias, A. S.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. 99, 11014–11019 (2002).
[Crossref]

A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, and B. J. Tromberg, “Influence of optical properties on two-photon fluorescence imaging in turbid samples,” Appl. Opt. 39, 1194–1201 (2000).
[Crossref]

Tsegaye, G.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Wallace, V. P.

Walpita, D.

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

Wang, K.

K. Wang, R. Liang, and P. Qiu, “Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy,” IEEE Photonics J..  7, 1–8 (2015).
[Crossref]

K. Wang, N. G. Horton, K. Charan, and C. Xu, “Advanced Fiber Soliton Sources for Nonlinear Deep Tissue Imaging in Biophotonics,” IEEE J. Sel. Top. Quantum Electron. 20, 50–60 (2014).
[Crossref]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Wang, M.

K. Charan, B. Li, M. Wang, C. P. Lin, and C. Xu, “Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy,” Biomed. Opt. Express 9, 2304–2311 (2018).
[Crossref] [PubMed]

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

T. Wang, D. Ouzounov, M. Wang, and C. Xu, “Quantitative Comparison of Two-photon and Three-photon Activity Imaging of GCaMP6s-labeled Neurons in vivo in the Mouse Brain,” in “Optics in the Life Sciences Congress (2017), paper BrM4B.4,” (Optical Society of America, 2017), p. BrM4B.4.

Wang, T.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

T. Wang, D. Ouzounov, M. Wang, and C. Xu, “Quantitative Comparison of Two-photon and Three-photon Activity Imaging of GCaMP6s-labeled Neurons in vivo in the Mouse Brain,” in “Optics in the Life Sciences Congress (2017), paper BrM4B.4,” (Optical Society of America, 2017), p. BrM4B.4.

Wardill, T. J.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature. 499, 295–300 (2013).
[Crossref] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[Crossref] [PubMed]

C. Xu and W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” in Topics in Fluorescence Spectroscopy, vol. 5 (Springer, New York, 1997), pp. 471–540.

Welch, A. J.

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Wise, F. W.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Wong, A. W.

Xu, C.

K. Charan, B. Li, M. Wang, C. P. Lin, and C. Xu, “Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy,” Biomed. Opt. Express 9, 2304–2311 (2018).
[Crossref] [PubMed]

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14, 388–390 (2017).
[Crossref] [PubMed]

N. G. Horton and C. Xu, “Dispersion compensation in three-photon fluorescence microscopy at 1,700 nm,” Biomed. Opt. Express 6, 1392–1397 (2015).
[Crossref] [PubMed]

K. Wang, N. G. Horton, K. Charan, and C. Xu, “Advanced Fiber Soliton Sources for Nonlinear Deep Tissue Imaging in Biophotonics,” IEEE J. Sel. Top. Quantum Electron. 20, 50–60 (2014).
[Crossref]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt 16, 106014 (2011).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17, 13354–13364 (2009).
[Crossref] [PubMed]

C. Xu and W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” in Topics in Fluorescence Spectroscopy, vol. 5 (Springer, New York, 1997), pp. 471–540.

T. Wang, D. Ouzounov, M. Wang, and C. Xu, “Quantitative Comparison of Two-photon and Three-photon Activity Imaging of GCaMP6s-labeled Neurons in vivo in the Mouse Brain,” in “Optics in the Life Sciences Congress (2017), paper BrM4B.4,” (Optical Society of America, 2017), p. BrM4B.4.

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. 99, 11014–11019 (2002).
[Crossref]

Ying, J.

Zhang, T.

Zoumi, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. 99, 11014–11019 (2002).
[Crossref]

Appl. Opt. (3)

Biomed. Opt. Express (2)

eLife (1)

H. Dana, B. Mohar, Y. Sun, S. Narayan, A. Gordus, J. P. Hasseman, G. Tsegaye, G. T. Holt, A. Hu, D. Walpita, R. Patel, J. J. Macklin, C. I. Bargmann, M. B. Ahrens, E. R. Schreiter, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Sensitive red protein calcium indicators for imaging neural activity,” eLife 5, e12727 (2016).
[Crossref] [PubMed]

IEEE J. Quantum Electron. (1)

W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
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IEEE J. Sel. Top. Quantum Electron. (1)

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Figures (8)

Fig. 1
Fig. 1 Theoretical model of the effective attenuation lengths based on water absorption and Mie scattering. The black stars indicate the reported effective attenuation lengths in mouse brains in vivo, 131 µm at 775 nm [17], 152∼158 µm at 920 nm [18], 305 ~ 319 µm at 1300 nm [18] and 383 µm at 1680 nm [3].
Fig. 2
Fig. 2 (a) Measured spectra of the laser source operating at 1700 nm, 1550 nm, 1500 nm, 1450 nm and 1300 nm. Dependence of three-photon-excited fluorescence on excitation power for (b) 1700 nm and (c) 1450 nm in logarithmic scales. The blue diamonds are the measured data, and the red lines are linear fits to the experimental results. The slope is indicated in each figure.
Fig. 3
Fig. 3 Measured second-order interferometric autocorrelations of the laser pulse operating at (a) 1700 nm, (b) 1550 nm, (c) 1500 nm, and (d) 1450 nm. The intensity full-width-at-half-maximum (FWHM) of the pulse is indicated in each figure, assuming a deconvolution factor of 1.54 for sech2-pulse.
Fig. 4
Fig. 4 Beam size measurements at (a) 1700 nm, (b) 1550 nm, (c) 1500 nm, and (d) 1450 nm. The blue dots are the measured data, and the red lines are Gaussian fits to the measurements. There is some ellipticity in the excitation beam, therefore the measurements were taken along both the long axis (left) and the short axis (right). The FWHM is labeled in each figure.
Fig. 5
Fig. 5 (a) Comparison of the fluorescence signal as a function of depth at 1700 nm, 1550 nm, 1500 nm, 1450 nm, and 1700 nm (repeat experiment). The dots are the measured data, and the lines are linear fits to the measurements. With the same maximum average power, imaging at 1700 nm excitation includes both the neocortex (NC) and external capsule (EC), while imaging using the other wavelengths is limited to the NC. (b) 3D reconstruction of three-photon images of Texas Red-labeled brain vasculature, left, fluorescence, right, third harmonic generation (THG). Imaging depths are labeled in the middle. Scale bars, 100 µm.
Fig. 6
Fig. 6 Three-photon fluorescence images of the brain vasculature (upper) and THG images (lower) using 1700 nm excitation. The depths are indicated in the images. All the images are shown with the same contrast setting with the brightest 1% pixels saturated. Scale bars, 50 µm.
Fig. 7
Fig. 7 Beam size measurements at (a) 1300 nm and (b) 1450 nm. The blue dots are the measured data, and the red lines are Gaussian fits to the measurements. Measurements were taken along both the long axis (left) and the short axis (right). The FWHM is labeled in each figure. Measured second-order interferometric autocorrelations of the laser pulse operating at (c) 1300 nm and (d) 1450 nm. The FWHM of the pulse is indicated in the figure, assuming a deconvolution factor of 1.54 for sech2-pulse. (e) Comparison of the fluorescence signal as a function of depth at 1300 nm, 1450 nm, and 1300 nm (repeat experiment). The dots are the measured data, and the lines are linear fits to the measurements. With the same maximum average power, imaging by 1300 nm excitation includes both the NC and EC, while imaging by 1450 nm is limited to the NC.
Fig. 8
Fig. 8 Experimental data (averaged from all the measured EALs for each wavelength, black triangles) is shown on the same plot together with the theoretical model, indicating the accuracy of the model at predicting the experimental measurements at these excitation wavelengths.

Tables (1)

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Table 1 Summary of in vivo EAL measurements of the neocortex (NC) in three mice

Equations (3)

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P z = P 0 e z l e .
1 l e = 1 l a + 1 l s .
F 3 P P z 3 = P 0 3 e 3 z l e .

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