Abstract

Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF2P) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF2P at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF2P, thereby providing a basis for improved 2P microscopy.

© 2011 OSA

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2011 (3)

O. Katz, E. Small, Y. Bromberg, and Y. Silberberg, “Focusing and compression of ultrashort pulses through scattering media,” Nat. Photonics 5(6), 372–377 (2011).
[CrossRef]

N. Ji, D. E. Milkie, and E. Betzig, “Pupil-segmentation-based adaptive optics for microscopy,” Proc. SPIE 7931, 79310I (2011)

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

2010 (4)

I. M. Vellekoop and C. M. Aegerter, “Scattered light fluorescence microscopy: imaging through turbid layers,” Opt. Lett. 35(8), 1245–1247 (2010).
[CrossRef] [PubMed]

J. W. Cha, J. Ballesta, and P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15(4), 046022 (2010).
[CrossRef] [PubMed]

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

2009 (2)

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

D. Débarre, E. J. Botcherby, T. Watanabe, S. Srinivas, M. J. Booth, and T. Wilson, “Image-based adaptive optics for two-photon microscopy,” Opt. Lett. 34(16), 2495–2497 (2009).
[CrossRef] [PubMed]

2008 (2)

S. P. Poland, A. J. Wright, and J. M. Girkin, “Evaluation of fitness parameters used in an iterative approach to aberration correction in optical sectioning microscopy,” Appl. Opt. 47(6), 731–736 (2008).
[CrossRef] [PubMed]

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J. 94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

2007 (3)

Y. P. Zhou, T. Bifano, and C. Lin, “Adaptive optics two-photon fluorescence microscopy ” Proc. SPIE 6467, 646705 (2007).

A. J. Wright, S. P. Poland, J. M. Girkin, C. W. Freudiger, C. L. Evans, and X. S. Xie, “Adaptive optics for enhanced signal in CARS microscopy,” Opt. Express 15(26), 18209–18219 (2007).
[CrossRef] [PubMed]

M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. 365(1861), 2829–2843 (2007).
[CrossRef] [PubMed]

2006 (3)

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
[CrossRef] [PubMed]

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A. 103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

2005 (2)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

2004 (3)

M. Schwertner, M. J. Booth, and T. Wilson, “Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry,” J. Microsc. 215(3), 271–280 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12(26), 6540–6552 (2004).
[CrossRef] [PubMed]

2003 (2)

P. Marsh, D. Burns, and J. Girkin, “Practical implementation of adaptive optics in multiphoton microscopy,” Opt. Express 11(10), 1123–1130 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002 (3)

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, “Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,” J. Microsc. 206(1), 65–71 (2002).
[CrossRef] [PubMed]

H. U. Dodt, M. Eder, A. Schierloh, and W. Zieglgänsberger, “Infrared-guided laser stimulation of neurons in brain slices,” Sci. STKE 2002(120), 2pl (2002).
[CrossRef] [PubMed]

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(1), 29–37 (2001).
[CrossRef] [PubMed]

2000 (1)

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[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. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

1990 (2)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 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(12), 2166–2185 (1990).
[CrossRef]

1989 (1)

1959 (2)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 358–379 (1959).
[CrossRef]

E. Wolf, “Electromagnetic diffraction in optical systems. 1. An integral representation of the image field,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 349–357 (1959).
[CrossRef]

Aegerter, C. M.

Albert, O.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, “Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,” J. Microsc. 206(1), 65–71 (2002).
[CrossRef] [PubMed]

Ballesta, J.

J. W. Cha, J. Ballesta, and P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15(4), 046022 (2010).
[CrossRef] [PubMed]

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(1), 29–37 (2001).
[CrossRef] [PubMed]

Betzig, E.

N. Ji, D. E. Milkie, and E. Betzig, “Pupil-segmentation-based adaptive optics for microscopy,” Proc. SPIE 7931, 79310I (2011)

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Bifano, T.

Y. P. Zhou, T. Bifano, and C. Lin, “Adaptive optics two-photon fluorescence microscopy ” Proc. SPIE 6467, 646705 (2007).

Bolin, F. P.

Booth, M. J.

D. Débarre, E. J. Botcherby, T. Watanabe, S. Srinivas, M. J. Booth, and T. Wilson, “Image-based adaptive optics for two-photon microscopy,” Opt. Lett. 34(16), 2495–2497 (2009).
[CrossRef] [PubMed]

M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. 365(1861), 2829–2843 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12(26), 6540–6552 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, “Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry,” J. Microsc. 215(3), 271–280 (2004).
[CrossRef] [PubMed]

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[CrossRef] [PubMed]

Botcherby, E. J.

Brandt, A. U.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Bromberg, Y.

O. Katz, E. Small, Y. Bromberg, and Y. Silberberg, “Focusing and compression of ultrashort pulses through scattering media,” Nat. Photonics 5(6), 372–377 (2011).
[CrossRef]

Burns, D.

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

P. Marsh, D. Burns, and J. Girkin, “Practical implementation of adaptive optics in multiphoton microscopy,” Opt. Express 11(10), 1123–1130 (2003).
[CrossRef] [PubMed]

Cha, J. W.

J. W. Cha, J. Ballesta, and P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15(4), 046022 (2010).
[CrossRef] [PubMed]

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(1), 29–37 (2001).
[CrossRef] [PubMed]

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(1), 29–37 (2001).
[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(12), 2166–2185 (1990).
[CrossRef]

Cobb, S.

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

Débarre, D.

Denk, W.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A. 103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 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. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dodt, H. U.

H. U. Dodt, M. Eder, A. Schierloh, and W. Zieglgänsberger, “Infrared-guided laser stimulation of neurons in brain slices,” Sci. STKE 2002(120), 2pl (2002).
[CrossRef] [PubMed]

Eder, M.

H. U. Dodt, M. Eder, A. Schierloh, and W. Zieglgänsberger, “Infrared-guided laser stimulation of neurons in brain slices,” Sci. STKE 2002(120), 2pl (2002).
[CrossRef] [PubMed]

Evans, C. L.

Ference, R. J.

Freudiger, C. W.

Girkin, J.

Girkin, J. M.

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

S. P. Poland, A. J. Wright, and J. M. Girkin, “Evaluation of fitness parameters used in an iterative approach to aberration correction in optical sectioning microscopy,” Appl. Opt. 47(6), 731–736 (2008).
[CrossRef] [PubMed]

A. J. Wright, S. P. Poland, J. M. Girkin, C. W. Freudiger, C. L. Evans, and X. S. Xie, “Adaptive optics for enhanced signal in CARS microscopy,” Opt. Express 15(26), 18209–18219 (2007).
[CrossRef] [PubMed]

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

Hauser, A. E.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 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. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Herz, J.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, “Pupil-segmentation-based adaptive optics for microscopy,” Proc. SPIE 7931, 79310I (2011)

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Juskaitis, R.

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[CrossRef] [PubMed]

Katz, O.

O. Katz, E. Small, Y. Bromberg, and Y. Silberberg, “Focusing and compression of ultrashort pulses through scattering media,” Nat. Photonics 5(6), 372–377 (2011).
[CrossRef]

Kawata, S.

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[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. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Leray, A.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J. 94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

Leuenberger, T.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Lillis, K.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J. 94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

Lin, C.

Y. P. Zhou, T. Bifano, and C. Lin, “Adaptive optics two-photon fluorescence microscopy ” Proc. SPIE 6467, 646705 (2007).

Mack-Bucher, J. A.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A. 103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

Marsh, P.

Mertz, J.

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J. 94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

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(1), 29–37 (2001).
[CrossRef] [PubMed]

Milkie, D. E.

N. Ji, D. E. Milkie, and E. Betzig, “Pupil-segmentation-based adaptive optics for microscopy,” Proc. SPIE 7931, 79310I (2011)

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[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. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Neil, M. A.

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[CrossRef] [PubMed]

Neil, M. A. A.

M. Schwertner, M. J. Booth, M. A. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef] [PubMed]

Niesner, R. A.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Norris, T. B.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, “Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,” J. Microsc. 206(1), 65–71 (2002).
[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(1), 29–37 (2001).
[CrossRef] [PubMed]

Patterson, B. A.

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

Poland, S.

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

Poland, S. P.

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

S. P. Poland, A. J. Wright, and J. M. Girkin, “Evaluation of fitness parameters used in an iterative approach to aberration correction in optical sectioning microscopy,” Appl. Opt. 47(6), 731–736 (2008).
[CrossRef] [PubMed]

A. J. Wright, S. P. Poland, J. M. Girkin, C. W. Freudiger, C. L. Evans, and X. S. Xie, “Adaptive optics for enhanced signal in CARS microscopy,” Opt. Express 15(26), 18209–18219 (2007).
[CrossRef] [PubMed]

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[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(12), 2166–2185 (1990).
[CrossRef]

Preuss, L. E.

Radbruch, H.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 358–379 (1959).
[CrossRef]

Rueckel, M.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A. 103(46), 17137–17142 (2006).
[CrossRef] [PubMed]

Schierloh, A.

H. U. Dodt, M. Eder, A. Schierloh, and W. Zieglgänsberger, “Infrared-guided laser stimulation of neurons in brain slices,” Sci. STKE 2002(120), 2pl (2002).
[CrossRef] [PubMed]

Schober, R.

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Schulze, P. C.

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Schwarzmaier, H. J.

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Schwertner, M.

M. Schwertner, M. J. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12(26), 6540–6552 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, “Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry,” J. Microsc. 215(3), 271–280 (2004).
[CrossRef] [PubMed]

Sherman, L.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, “Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,” J. Microsc. 206(1), 65–71 (2002).
[CrossRef] [PubMed]

Siffrin, V.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Silberberg, Y.

O. Katz, E. Small, Y. Bromberg, and Y. Silberberg, “Focusing and compression of ultrashort pulses through scattering media,” Nat. Photonics 5(6), 372–377 (2011).
[CrossRef]

Small, E.

O. Katz, E. Small, Y. Bromberg, and Y. Silberberg, “Focusing and compression of ultrashort pulses through scattering media,” Nat. Photonics 5(6), 372–377 (2011).
[CrossRef]

So, P. T. C.

J. W. Cha, J. Ballesta, and P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15(4), 046022 (2010).
[CrossRef] [PubMed]

Srinivas, S.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Svoboda, K.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

Tanaka, T.

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[CrossRef] [PubMed]

Taylor, R. C.

Theer, P.

Ulrich, F.

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Valentine, G. J.

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

Vellekoop, I. M.

Vijverberg, J. C.

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

Watanabe, T.

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

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(12), 2166–2185 (1990).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Wilson, T.

D. Débarre, E. J. Botcherby, T. Watanabe, S. Srinivas, M. J. Booth, and T. Wilson, “Image-based adaptive optics for two-photon microscopy,” Opt. Lett. 34(16), 2495–2497 (2009).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12(26), 6540–6552 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, “Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry,” J. Microsc. 215(3), 271–280 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef] [PubMed]

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[CrossRef] [PubMed]

Wolf, E.

E. Wolf, “Electromagnetic diffraction in optical systems. 1. An integral representation of the image field,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 349–357 (1959).
[CrossRef]

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 358–379 (1959).
[CrossRef]

Wright, A. J.

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

S. P. Poland, A. J. Wright, and J. M. Girkin, “Evaluation of fitness parameters used in an iterative approach to aberration correction in optical sectioning microscopy,” Appl. Opt. 47(6), 731–736 (2008).
[CrossRef] [PubMed]

A. J. Wright, S. P. Poland, J. M. Girkin, C. W. Freudiger, C. L. Evans, and X. S. Xie, “Adaptive optics for enhanced signal in CARS microscopy,” Opt. Express 15(26), 18209–18219 (2007).
[CrossRef] [PubMed]

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

Xie, X. S.

Yaroslavsky, A. N.

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Yaroslavsky, I. V.

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Yasuda, R.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

Ye, J. Y.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, “Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,” J. Microsc. 206(1), 65–71 (2002).
[CrossRef] [PubMed]

Zhou, Y. P.

Y. P. Zhou, T. Bifano, and C. Lin, “Adaptive optics two-photon fluorescence microscopy ” Proc. SPIE 6467, 646705 (2007).

Zieglgänsberger, W.

H. U. Dodt, M. Eder, A. Schierloh, and W. Zieglgänsberger, “Infrared-guided laser stimulation of neurons in brain slices,” Sci. STKE 2002(120), 2pl (2002).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Zipp, F.

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Appl. Opt. (2)

Biophys. J. (2)

A. Leray, K. Lillis, and J. Mertz, “Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy,” Biophys. J. 94(4), 1449–1458 (2008).
[CrossRef] [PubMed]

J. Herz, V. Siffrin, A. E. Hauser, A. U. Brandt, T. Leuenberger, H. Radbruch, F. Zipp, and R. A. Niesner, “Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator,” Biophys. J. 98(4), 715–723 (2010).
[CrossRef] [PubMed]

Curr. Opin. Biotechnol. (1)

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[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(12), 2166–2185 (1990).
[CrossRef]

J. Biomed. Opt. (1)

J. W. Cha, J. Ballesta, and P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15(4), 046022 (2010).
[CrossRef] [PubMed]

J. Microsc. (4)

M. Schwertner, M. J. Booth, and T. Wilson, “Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry,” J. Microsc. 215(3), 271–280 (2004).
[CrossRef] [PubMed]

M. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microsc. 200(2), 105–108 (2000).
[CrossRef] [PubMed]

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, “Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,” J. Microsc. 206(1), 65–71 (2002).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef] [PubMed]

J. Neurosci. Methods (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(1), 29–37 (2001).
[CrossRef] [PubMed]

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

Micron (1)

S. P. Poland, A. J. Wright, S. Cobb, J. C. Vijverberg, and J. M. Girkin, “A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes,” Micron 42(4), 318–323 (2011).
[CrossRef]

Microsc. Res. Tech. (1)

A. J. Wright, D. Burns, B. A. Patterson, S. P. Poland, G. J. Valentine, and J. M. Girkin, “Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy,” Microsc. Res. Tech. 67(1), 36–44 (2005).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Methods (2)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Nat. Photonics (1)

O. Katz, E. Small, Y. Bromberg, and Y. Silberberg, “Focusing and compression of ultrashort pulses through scattering media,” Nat. Photonics 5(6), 372–377 (2011).
[CrossRef]

Neuron (1)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

Philos. Transact. A Math. Phys. Eng. Sci. (1)

M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. 365(1861), 2829–2843 (2007).
[CrossRef] [PubMed]

Phys. Med. Biol. (1)

A. N. Yaroslavsky, P. C. Schulze, I. V. Yaroslavsky, R. Schober, F. Ulrich, and H. J. 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(12), 2059–2073 (2002).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (2)

M. Rueckel, J. A. Mack-Bucher, and W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Natl. Acad. Sci. U.S.A. 103(46), 17137–17142 (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. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Proc. R. Soc. London A Math. Phys. Eng. Sci. (2)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 358–379 (1959).
[CrossRef]

E. Wolf, “Electromagnetic diffraction in optical systems. 1. An integral representation of the image field,” Proc. R. Soc. London A Math. Phys. Eng. Sci. 253(1274), 349–357 (1959).
[CrossRef]

Proc. SPIE (2)

Y. P. Zhou, T. Bifano, and C. Lin, “Adaptive optics two-photon fluorescence microscopy ” Proc. SPIE 6467, 646705 (2007).

N. Ji, D. E. Milkie, and E. Betzig, “Pupil-segmentation-based adaptive optics for microscopy,” Proc. SPIE 7931, 79310I (2011)

Sci. STKE (1)

H. U. Dodt, M. Eder, A. Schierloh, and W. Zieglgänsberger, “Infrared-guided laser stimulation of neurons in brain slices,” Sci. STKE 2002(120), 2pl (2002).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (5)

C. F. Bohren and D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, 1998).

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge, 2001).

R. K. Tyson, Principles of Adaptive Optics (Academic, 1998).

E. Chaigneau and R. A. Silver, University College London, London, UK, are preparing a manuscript entitled “Refractive index mismatch effects at edges: theory and application to two-photon imaging of biological samples.”

E. W. Weisstein, “Gaussian integral,” Wolfram MathWorld, (1999), http://mathworld.wolfram.com/GaussianIntegral.html.

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Figures (12)

Fig. 1
Fig. 1

Comparing the effects of static, statistically homogeneous scattering and wavefront distortion on excitation photons in 2P microscopy. Brain tissue is made of particles of a wide range of size and refractive index. Particles that are smaller than the wavelength of light create a statistically homogeneous effect. This decreases the power of ballistic photons while leaving the wavefront undistorted. Particles whose size is larger than the wavelength of light induce wavefront distortion.

Fig. 2
Fig. 2

Two-photon point spread function (PSF2P) characteristics in the cortex. (a) (Top left panel) experimental setup consisting of water immersion objective and beads used to measure the optical system PSF2P. Single images of beads acquired using the optical system in the focal plane (x-y) (Bottom left) and in a plane comprising the optical axis (z) (Bottom right). y axis indicated by a dashed line in the bottom left panel. Excitation wavelength (λ) = 725 nm. (b) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom left panel) 3D sketch of the cortex showing in blue a tangential slice in cortical layer II / III. (Top middle and right panels) x-y and y-z images of beads acquired by focusing through tangential slices at a depth of 150 μm. y axis indicated by a dashed line in the top middle panel. Same look up table as (a). (c) Gaussian fits of average PSF2P. (Top panel) x-y plane (Bottom panel) z axis. Error bars show the standard deviation.

Fig. 3
Fig. 3

Excitation mean free path in the cortex. (a) Maximum intensity projection along the z axis of a z-stack in layer II / III showing Alexa 488 filled pyramidal cell with dendrites spanning 200 μm in x-y and 84 μm in z. The scattering length or mean free path of excitation light (Lse) was estimated from the fluorescence of the dendrites at different depths. (b) Relationship between the fluorescence (F) divided by the square of the laser Power (P) and normalized by its value at the cortical slice surface (β) versus depth. Lse was calculated from an exponential fit (line) at a wavelength of 725 nm. (c) Dependence of Lse on wavelength (n = 4 −8 cells). Error bars give the standard error of the mean (sem), which is smaller than the symbols for λ ≤ 850 nm.

Fig. 4
Fig. 4

Effect of scattering on the PSF2P. (a) Conventions used when modeling the PSF2P. Ballistic photons form a cone that can be decomposed into beamlets of coordinates (r = sinθ / sinθΝΑ, ω). (b) Comparison of the xe and ye profiles of the measured cortical PSF2P with the theoretical, microscope and modeled PSF2P that accounts for the effect of Lse in the focal plane (Top panel) and along the optical axis (Bottom panel).

Fig. 5
Fig. 5

Conventional implementation of deformable membrane mirror (DMM). (a) Schematic diagram of conventional DMM implementation. (b) PSF2P of the microscope including the DMM in control conditions in the focal plane (Top) and in a plane comprising the optical axis (Bottom) indicated by the dashed line on the top panel.

Fig. 6
Fig. 6

Fluorescence and SNR enhancement of the PSF2P in the cortex with a DMM. (a) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom panel) 3D sketch of the cortex showing in blue a thalamocortical brain slice. (b) Single images of a bead acquired using the DMM in control conditions (CC) in the focal plane (top panel) and in a plane comprising the optical axis (z) (bottom panel, y axis indicated by dashed line in top panel) at a depth of 150 μm. (c) As for B but for the optimized mirror shape in the cortex (OMSc). Same laser power as (b). (d) Intensity projection (sum) of the z-stack of images of the bead shows that, in this example where there was no visible surrounding lobes, DMM optimization resulted in a decrease of the volume of the main lobe of the PSF2P and decrease in the background.

Fig. 7
Fig. 7

Spatial dependence of wavefront correction for the conventional DMM configuration. (a) Experimental protocol: an optimization was performed at the center of the field of view (1), at a particular cortical location. The cortical location was moved across the field of view at distances of 50 μm (position 2) or 100 μm (position 3) away from the optical axis and the fluorescence and SNR obtained using the optimized mirror shape (OMSc) and in control conditions (CC) were measured. The change in fluorescence (b) and SNR (c) across the field of view using the OMSc performed at position 1. There was no significant change in these parameters with distance to the optical axis (p > 0.08, paired t-test). Grey symbols: individual experiments, colored symbols: mean, black bars: sem.

Fig. 8
Fig. 8

Spatial dependence of wavefront distortions in the cortex. (a) (Left) A group of beads imaged through a 150 μm thalamocortical slice for the DMM set to control conditions (CC). The DMM shape was optimized on the central bead giving the optimized mirror shape in the cortex (OMSc). (Right) Using OMSc improved bead definition and fluorescence intensity in the lower half of the image but not in the upper half of the image, illustrating that wavefront distortions vary across a cortical slice. (b) Protocol used to examine the variability of wavefront distortions in the cortex: a first cortical column (C1) was positioned at the center of the field of view (indicated by the square box) and a first DMM shape optimization was performed there, giving OMSc (1). Then a neighboring cortical column (C2) was positioned at the center of the field of view, a second DMM shape optimization was performed, giving OMSc (2). Last a bead at the center of C2 was imaged using OMSc (1), OMSc (2) and CC. (c) Bead fluorescence using OMSc (1) was significantly smaller than using OMSc (2) (*) (p < 0.011, n = 8, paired t-test). Grey symbols: individual experiments, colored symbols: mean. The sem is smaller than the colored symbols.

Fig. 9
Fig. 9

Compensating for optical aberrations with light-efficient DMM implementation. (a) Light efficient configuration with the DMM implemented at 45°. (b) PSF2P of the microscope with the light-efficient DMM in control conditions in the focal plane (Left) and in a plane comprising the optical axis (Right). Y axis indicated by the dashed line on the top panel. (c) Single images of a bead under a cortical slice at a depth of 150 μm acquired using the DMM in control conditions (CC) in the focal plane. (d) As for (c) but for the optimized mirror shape in the cortex (OMSc). Same laser power as (c). (e) Z stacks of images of the previous bead were acquired and normalised to the maximal fluorescence for the DMM in CC. Focal plane (top panel) and plane comprising the optical axis (z) (bottom panel). Arrow indicates a surrounding lobe that disappeared after the DMM optimization. x axis indicated by dashed line in top panel. (f) Same as (e) but using the OMSc. (g) Maximum intensity projection (MIP) of data from (e). (h) MIP of data from (f).

Fig. 10
Fig. 10

Spatial dependence of wavefront correction for the light efficient DMM configuration. (a) Experimental protocol: an optimization was performed at the center of the field of view (position 1), at a particular cortical location. The cortical location was moved across the field of view at distances of 50 μm (position 2) or 100 μm (position 3) away from the optical axis, and the fluorescence and SNR obtained using the optimized mirror shape (OMSc) and in control conditions (CC) were measured. (b - c) The change in fluorescence (b) and SNR (c) across the field of view using the OMS performed at position 1. There was no significant change in these parameters with distance to the optical axis (p > 0.17, paired t-test). Grey symbols: individual experiments, colored symbols: mean, black bars: sem.

Fig. 11
Fig. 11

Optimization of the DMM shape on a cellular element. (a) Fine dendrite imaged with DMM in control conditions (CC). (b) Fluorescence change during the DMM shape optimization on a dendrite (arrow in panel A). (c) Same cellular element imaged using the optimized mirror shape (OMSc) and the same laser power as in CC.

Fig. 12
Fig. 12

Contributions of wavefront distortion and scattering to the cortical PSF2P and their effects on 2P microscopy. (a) Image of modeled microscope PSF2P assuming NA 0.7 in x-y plane (top) and y-z plane (bottom panel), y axis indicated by dashed line in top panel. (b) Example of a modeled PSF2P taking into account the optical aberrations corrected using the DMM in the cortex assuming NA 0.7. 2P excitation was normalized by its maximal value in (a) and (b). (c) Quantification of the average 2P excitation (integral of the distribution of the squared intensity of excitation light in the PSF2P) in the central 3D Gaussian lobe and in the surrounding lobes, for the modeled ideal PSF2P, the measured microscope PSF2P, the modeled PSF2P including measured cortical distortions and the experimentally measured cortical PSF2P, all at NA = 0.7. (*) p < 0.003, t-test, (**) p < 0.001, t-test. (d) Modeling the effects of wavefront distortions and scattering on fluorescence. (Top panel) The predicted fluorescence emitted by homogeneously labeled spherical objects using an ideal microscope, in the presence of scattering (Lse = 77 μm) and in the presence of optical aberrations, plotted versus the size of the object. Furthermore, to determine effects of the surrounding lobes, the fluorescence emitted by the main Gaussian core of the PSF2P was calculated in the presence of optical aberrations (dotted red line). (Bottom panel) Plots from top panel were normalized to the maximum value.

Tables (1)

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Table 1 PSF2P dimensions at wavelength (λ) = 725 nm

Equations (14)

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FWH M 2P = ( FWH M Image ) 2 ( FWH M Bead ) 2
F V 2P = e ( 4ln2 x e 2 (FWH M 2P xe ) 2 + 4ln2 y e 2 (FWH M 2P ye ) 2 + 4ln2 z e 2 (FWH M 2P ze ) 2 ) d x e d y e d z e
F V 2P =( e 4ln2 x e 2 (FWH M 2P xe ) 2 d x e )( e 4ln2 y e 2 (FWH M 2P ye ) 2 d y e )( e 4ln2 z e 2 (FWH M 2P ze ) 2 d z e )
e a x 2 dx= ( π a ) 0.5
F V 2P = ( π 4ln2 ) 1.5 FWH M 2P xe FWH M 2P ye FWH M 2P ze
F( z )=α P 0 2 e 2z L se
e x = if λ 0 θ NA 0 2π cos 0.5 θ sinθ(cosθ+(1cosθ) sin 2 ω) A(θ,ω) e ik(Φ(θ,ω)+ r p sinθcos(ω ω p )+ z p cosθ) dθdω
e y = if λ 0 θ NA 0 2π cos 0.5 θ sinθ(1cosθ)cosωsinω A(θ,ω) e ik(Φ(θ,ω)+ r p sinθcos(ω ω p )+ z p cosθ) dθdω
e z = if λ 0 θ NA 0 2π cos 0.5 θ sin 2 θcosω A(θ,ω) e ik(Φ(θ,ω)+ r p sinθcos(ω ω p )+ z p cosθ) dθdω
I 2 ( x,y,z )= ( | e x | 2 + | e y | 2 + | e z | 2 ) 2
A(θ,ω)= e ( ( sinθ sin θ NA ) 2 + z p 2 L se cosθ )
Φ(θ,ω)=0
Φ(θ,ω)= n,m>0 a n m R n m ( sinθ sin θ NA )cos(mω) + n,m<0 a n m R n m ( sinθ sin θ NA )sin(mω)
A(θ,ω)= e ( sinθ sin θ NA ) 2

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