Abstract

Two-photon microscopy (TPM) has been widely used for thick tissue imaging. However, its penetration depth is fundamentally limited by the loss of signal contrast. Differential aberration imaging (DAI) can reject out-of-focus fluorescence in TPM by subtracting an aberrated image from an unaberrated one. This technique is simple and effective but compromises imaging speed because two images must be taken sequentially. Here we report a new strategy for two-photon DAI based on near-instantaneous temporal multiplexing, enabling high-speed imaging with pixel rates limited only by fluorescence lifetime and laser repetition rate. Our technique can be implemented with standard two-photon microscopes since it does not require active optical elements and it is based on a synchronized sampling strategy that does not require specialized hardware. We demonstrate and characterize the resultant contrast improvement when imaging fluorescently-labeled mouse brain at video-rate.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Full Article  |  PDF Article
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References

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    [Crossref] [PubMed]
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    [Crossref]
  10. N. Chen, C.-H. Wong, and C. J. Sheppard, “Focal modulation microscopy,” Opt. Express 16, 18764–18769 (2008).
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]

2018 (2)

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

S. Piazza, P. Bianchini, C. Sheppard, A. Diaspro, and M. Duocastella, “Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping,” J. Biophotonics 11, e201700050 (2018).
[Crossref]

2016 (2)

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34, 857 (2016).
[Crossref] [PubMed]

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

2015 (1)

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE 9329, 93292c (2015).
[Crossref]

2013 (2)

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

H. Dehez, M. Piché, and Y. De Koninck, “Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging,” Opt. Express 21, 15912–15925 (2013).
[Crossref] [PubMed]

2011 (1)

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139 (2011).
[Crossref] [PubMed]

2009 (2)

K. K. Chu, A. Leray, T. G. Bifano, and J. Mertz, “Two-photon fluorescence microscopy with differential aberration imaging,” Proc. SPIE 7209, 720903 (2009).
[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]

2008 (2)

N. Chen, C.-H. Wong, and C. J. Sheppard, “Focal modulation microscopy,” Opt. Express 16, 18764–18769 (2008).
[Crossref]

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

2007 (1)

2006 (2)

2005 (1)

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

2003 (2)

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “Scanimage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online 2, 13 (2003).
[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:Al 2O 3 regenerative amplifier,” Opt. Lett. 28, 1022–1024 (2003).
[Crossref] [PubMed]

2001 (2)

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

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

Amir, W.

Antipov, A.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Arisaka, K.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139 (2011).
[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. Meth. 111, 29–37 (2001).
[Crossref]

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

Bianchini, P.

S. Piazza, P. Bianchini, C. Sheppard, A. Diaspro, and M. Duocastella, “Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping,” J. Biophotonics 11, e201700050 (2018).
[Crossref]

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Bifano, T. G.

K. K. Chu, A. Leray, T. G. Bifano, and J. Mertz, “Two-photon fluorescence microscopy with differential aberration imaging,” Proc. SPIE 7209, 720903 (2009).
[Crossref]

Carriles, R.

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. Meth. 111, 29–37 (2001).
[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. Meth. 111, 29–37 (2001).
[Crossref]

Chen, J.

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

Chen, J. L.

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE 9329, 93292c (2015).
[Crossref]

Chen, N.

Cheng, A.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139 (2011).
[Crossref] [PubMed]

Chu, K. K.

K. K. Chu, A. Leray, T. G. Bifano, and J. Mertz, “Two-photon fluorescence microscopy with differential aberration imaging,” Proc. SPIE 7209, 720903 (2009).
[Crossref]

De Koninck, Y.

Dehez, H.

Denk, W.

Diaspro, A.

S. Piazza, P. Bianchini, C. Sheppard, A. Diaspro, and M. Duocastella, “Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping,” J. Biophotonics 11, e201700050 (2018).
[Crossref]

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Duocastella, M.

S. Piazza, P. Bianchini, C. Sheppard, A. Diaspro, and M. Duocastella, “Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping,” J. Biophotonics 11, e201700050 (2018).
[Crossref]

Durfee, C. G.

Durst, M. E.

Ge, J.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Golshani, P.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139 (2011).
[Crossref] [PubMed]

Gonçalves, J. T.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139 (2011).
[Crossref] [PubMed]

Gong, W.

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

Griffin, F.

D. Tsyboulski, N. Orlova, F. Griffin, S. Seid, J. Lecoq, and P. Saggau, “Remote focusing system for simultaneous dual-plane mesoscopic multiphoton imaging,” bioRxiv p. 503052 (2018).

Gu, Z.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Hao, X.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Hasan, M. T.

Helmchen, F.

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE 9329, 93292c (2015).
[Crossref]

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

Hoover, E. E.

Huang, L.

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

Kobat, D.

Korobchevskaya, K.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Krueppel, R.

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE 9329, 93292c (2015).
[Crossref]

Kuang, C.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Kudenov, M. W.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34, 857 (2016).
[Crossref] [PubMed]

Lecoq, J.

D. Tsyboulski, N. Orlova, F. Griffin, S. Seid, J. Lecoq, and P. Saggau, “Remote focusing system for simultaneous dual-plane mesoscopic multiphoton imaging,” bioRxiv p. 503052 (2018).

Leray, A.

K. K. Chu, A. Leray, T. G. Bifano, and J. Mertz, “Two-photon fluorescence microscopy with differential aberration imaging,” Proc. SPIE 7209, 720903 (2009).
[Crossref]

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

A. Leray and J. Mertz, “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging,” Opt. Express 14, 10565–10573 (2006).
[Crossref] [PubMed]

Li, H.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Li, S.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Li, Z.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[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, 1449–1458 (2008).
[Crossref]

Liu, W.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Liu, X.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[Crossref] [PubMed]

Mertz, J.

K. K. Chu, A. Leray, T. G. Bifano, and J. Mertz, “Two-photon fluorescence microscopy with differential aberration imaging,” Proc. SPIE 7209, 720903 (2009).
[Crossref]

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

A. Leray and J. Mertz, “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging,” Opt. Express 14, 10565–10573 (2006).
[Crossref] [PubMed]

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

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

J. Mertz, Introduction to Optical Microscopy (Roberts and Company Publishers, 2010).

Nishimura, N.

Oheim, M.

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

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

Orlova, N.

D. Tsyboulski, N. Orlova, F. Griffin, S. Seid, J. Lecoq, and P. Saggau, “Remote focusing system for simultaneous dual-plane mesoscopic multiphoton imaging,” bioRxiv p. 503052 (2018).

Peres, C.

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

Piazza, S.

S. Piazza, P. Bianchini, C. Sheppard, A. Diaspro, and M. Duocastella, “Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping,” J. Biophotonics 11, e201700050 (2018).
[Crossref]

Piché, M.

Planchon, T. A.

Pologruto, T. A.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “Scanimage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online 2, 13 (2003).
[Crossref] [PubMed]

Portera-Cailliau, C.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139 (2011).
[Crossref] [PubMed]

Sabatini, B. L.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “Scanimage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online 2, 13 (2003).
[Crossref] [PubMed]

Saggau, P.

D. Tsyboulski, N. Orlova, F. Griffin, S. Seid, J. Lecoq, and P. Saggau, “Remote focusing system for simultaneous dual-plane mesoscopic multiphoton imaging,” bioRxiv p. 503052 (2018).

Schaffer, C. B.

Seid, S.

D. Tsyboulski, N. Orlova, F. Griffin, S. Seid, J. Lecoq, and P. Saggau, “Remote focusing system for simultaneous dual-plane mesoscopic multiphoton imaging,” bioRxiv p. 503052 (2018).

Sheppard, C.

S. Piazza, P. Bianchini, C. Sheppard, A. Diaspro, and M. Duocastella, “Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping,” J. Biophotonics 11, e201700050 (2018).
[Crossref]

Sheppard, C. J.

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

K. Korobchevskaya, C. Peres, Z. Li, A. Antipov, C. J. Sheppard, A. Diaspro, and P. Bianchini, “Intensity weighted subtraction microscopy approach for image contrast and resolution enhancement,” Sci. Rep. 6, 25816 (2016).
[Crossref] [PubMed]

N. Chen, C.-H. Wong, and C. J. Sheppard, “Focal modulation microscopy,” Opt. Express 16, 18764–18769 (2008).
[Crossref]

Shi, X.

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

Si, K.

Y. Zheng, J. Chen, X. Shi, X. Zhu, J. Wang, L. Huang, K. Si, C. J. Sheppard, and W. Gong, “Two photon focal modulation microscopy for high-resolution imaging in deep tissue,” J. Biophotonics 12, e201800247 (2018).
[Crossref]

Smith, I. T.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34, 857 (2016).
[Crossref] [PubMed]

Smith, S. L.

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain,” Nat. Biotechnol. 34, 857 (2016).
[Crossref] [PubMed]

Squier, J. A.

Stirman, J. N.

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Figures (5)

Fig. 1
Fig. 1 (a) Optical setup. f1 = -50 mm, f2 = 200 mm, f3 = 500 mm, f4 = 125 mm, f5 = 100 mm, f6 = 300 mm, f7 = 250 mm. (b) Demultiplexing scheme.
Fig. 2
Fig. 2 (a-d) Images from both channels when imaging a fixed GCaMP6-labeled mouse brain with the Gaussian beam (a,b) or the donut beam (c,d). For visualization, both (a,d) are normalized to their respective maxima. The upper right part of (b) is normalized to 1/8 the maximum of (a), and (c) to 1/4 the maximum of (d). Lower left parts of (b,c) are normalized to the maxima of (a,d) respectively. (e) Probability density function (PDF) of the measured crosstalk with and without the 90 MHz LPF. (f) Time-resolved fluorescence signals from both channels (red, blue curve) and system instrumentation response (purple curve), vertical gray dashed lines represent sampling events. (g) Same as (f) but without the LPF. Scale bars in (a-d) are 50 μm. Color bars in (a-d) represent intensity.
Fig. 3
Fig. 3 (a,b) Averaged unaberrated and aberrated images of a fluorescent bead sample with the 90 MHz LPF system. (c) DAI image taken with no-LPF system. (d) DAI image taken with 90 MHz LPF system. (e) DAI image with 50 MHz LPF system where unaberrated and aberrated images were taken separately. Inserts in (c-e) represent the SNR image over the red dashed square in (c-e). (f) Pixel SNR comparison of 90 MHz system (red dots) and no-LPF system (blue dots) versus the 50 MHz system. Only pixels with average intensity greater 14 are compared, as masked by the top left insert. (g) SNR profiles along the yellow dashed lines in the inserted SNR images of (c-e). (h,i) Time resolved fluorescence signals with the 90 MHz and no-LPF system, respectively. Scale bars in (a-e) are 50 μm. Color bars in (a-e) for the large images represent intensity (arbitrary unit). Color bars in (c-e) next to the small inserts represent SNR. All intensity and SNR images are normalized to maximum.
Fig. 4
Fig. 4 (a-d) Unaberrated (i.e. standard) two-photon images at depths 125 μm, 250 μm, 375 μm and 500 μm. (e-d) Corresponding DAI images at the same depths as in (a-d). (i) Intensity profiles along the red dashed lines in (a,e). (j) Intensity profiles along the red dashed lines in (d,h). The intermediate aberrated images corresponding to the orange traces in (i,j) are not shown. (k) Average contrast enhancement when using DAI as compared to standard two-photon imaging for depths ranging from 100 μm to 500 μm. All images are normalized to their maxima. Scale bars in (a-h) are 10 μm. A.U., arbitrary unit.
Fig. 5
Fig. 5 (a,b) Unaberrated and aberrated images acquired simultaneously at 30 Hz. (c) DAI image obtained by subtracting (a) from (b). (d) Image (b) after spatial lowpass Gaussian filtering. (e) DAI image obtained by subtracting (a) from (d). (f,g) Intensity profiles along the yellow dashed lines in (a,b,c) and (a,d,e). (h,i,j) Expanded views over the square areas in (a,c,e) respectively. All images are normalized to their maxima. Scale bars are 50 μm. A.U., arbitrary unit.

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