Abstract

Line-temporal focusing has been recognized as an elegant strategy that provides two-photon microscopy with an effective means for fast imaging through parallelization, together with an improved resilience to scattering for deep imaging. However, the axial resolution remains sub-optimal, except when using high NA objectives and a small field-of-view. With the introduction of an intracavity control of the spectral width of the femtosecond laser to adaptively fill the back aperture of the objective lens, line-temporal focusing two-photon microscopy is demonstrated to reach near-diffraction-limited axial resolution with a large back-aperture objective lens, and improved immunity to sample scattering. In addition, a new incoherent flattop beam shaping method is proposed which provides a uniform contrast with little degradation of the axial resolution along the focus line, even deep in the sample. This is demonstrated in large volumetric imaging of mouse lung samples.

© 2018 Optical Society of America

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References

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2016 (1)

2015 (1)

J. H. Park, W. Sun, and M. Cui, Proc. Natl. Acad. Sci. USA 112, 9236 (2015).
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2013 (2)

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T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, Nat. Methods 10, 1013 (2013).
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2012 (3)

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C. G. Durfee, M. Greco, E. Block, D. Vitek, and J. A. Squier, Opt. Express 20, 14244 (2012).
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2011 (1)

2010 (1)

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2008 (1)

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T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, Nat. Methods 10, 1013 (2013).
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[Crossref]

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E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Debarre, M. J. Booth, R. Juskaitis, O. Paulsen, and T. Wilson, Proc. Natl. Acad. Sci. USA 109, 2919 (2012).
[Crossref]

Brosh, I.

H. Dana, A. Marom, S. Paluch, R. Dvorkin, I. Brosh, and S. Shoham, Nat. Commun. 5, 3997 (2014).
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Chang, C. Y.

Chang, H. Y.

Chen, S. J.

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G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
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Choi, H. J.

H. J. Choi and P. T. C. So, Sci. Rep. 4, 6626 (2014).
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J. H. Park, W. Sun, and M. Cui, Proc. Natl. Acad. Sci. USA 112, 9236 (2015).
[Crossref]

Dana, H.

de Sars, V.

Debarre, D.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Debarre, M. J. Booth, R. Juskaitis, O. Paulsen, and T. Wilson, Proc. Natl. Acad. Sci. USA 109, 2919 (2012).
[Crossref]

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P. Theer and W. Denk, J. Opt. Soc. Am. A 23, 3139 (2006).
[Crossref]

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[Crossref]

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[Crossref]

Ding, J. P.

Durfee, C. G.

Durst, M. E.

Dvorkin, R.

H. Dana, A. Marom, S. Paluch, R. Dvorkin, I. Brosh, and S. Shoham, Nat. Commun. 5, 3997 (2014).
[Crossref]

Ellman, A.

Emiliani, V.

Granick, S.

K. Lou, S. Granick, and F. Amblard, Proc. Natl. Acad. Sci. USA 115, 6554 (2018).
[Crossref]

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Guo, C. S.

Hell, S. W.

Helmchen, F.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[Crossref]

Hillier, D.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
[Crossref]

Hu, Y. Y.

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2010).
[Crossref]

Juskaitis, R.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Debarre, M. J. Booth, R. Juskaitis, O. Paulsen, and T. Wilson, Proc. Natl. Acad. Sci. USA 109, 2919 (2012).
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Kaszás, A.

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Kohl, M. M.

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Lin, C. H.

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K. Lou, S. Granick, and F. Amblard, Proc. Natl. Acad. Sci. USA 115, 6554 (2018).
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Maák, P.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
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N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2010).
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Paluch, S.

H. Dana, A. Marom, S. Paluch, R. Dvorkin, I. Brosh, and S. Shoham, Nat. Commun. 5, 3997 (2014).
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Park, J. H.

J. H. Park, W. Sun, and M. Cui, Proc. Natl. Acad. Sci. USA 112, 9236 (2015).
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Paulsen, O.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Debarre, M. J. Booth, R. Juskaitis, O. Paulsen, and T. Wilson, Proc. Natl. Acad. Sci. USA 109, 2919 (2012).
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Roska, B.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
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G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
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T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, Nat. Methods 10, 1013 (2013).
[Crossref]

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Silberberg, Y.

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H. J. Choi and P. T. C. So, Sci. Rep. 4, 6626 (2014).
[Crossref]

Squier, J. A.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[Crossref]

Sun, W.

J. H. Park, W. Sun, and M. Cui, Proc. Natl. Acad. Sci. USA 112, 9236 (2015).
[Crossref]

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K. Svoboda and R. Yasuda, Neuron 50, 823 (2006).
[Crossref]

Szalay, G.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
[Crossref]

Tal, E.

Theer, P.

Tsai, S. F.

Vaziri, A.

T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, Nat. Methods 10, 1013 (2013).
[Crossref]

Veress, M.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
[Crossref]

Vitek, D.

Vizi, E.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
[Crossref]

Wang, H. T.

Wang, X. L.

Webb, W. W.

C. Xu and W. W. Webb, J. Opt. Soc. Am. B 13, 481 (1996).
[Crossref]

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[Crossref]

Weiner, A. M.

A. M. Weiner, Ultrafast Optics (Wiley, 2009).

Wilson, T.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Debarre, M. J. Booth, R. Juskaitis, O. Paulsen, and T. Wilson, Proc. Natl. Acad. Sci. USA 109, 2919 (2012).
[Crossref]

Xu, C.

Yasuda, R.

K. Svoboda and R. Yasuda, Neuron 50, 823 (2006).
[Crossref]

Zhu, G. H.

Zimmer, M.

T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, Nat. Methods 10, 1013 (2013).
[Crossref]

Appl. Opt. (1)

Biomed. Opt. Express (1)

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

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

Nat. Commun. (1)

H. Dana, A. Marom, S. Paluch, R. Dvorkin, I. Brosh, and S. Shoham, Nat. Commun. 5, 3997 (2014).
[Crossref]

Nat. Methods (4)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[Crossref]

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141 (2010).
[Crossref]

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rózsa, Nat. Methods 9, 201 (2012).
[Crossref]

T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, Nat. Methods 10, 1013 (2013).
[Crossref]

Neuron (1)

K. Svoboda and R. Yasuda, Neuron 50, 823 (2006).
[Crossref]

Opt. Express (6)

Opt. Lett. (3)

Proc. Natl. Acad. Sci. USA (3)

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Debarre, M. J. Booth, R. Juskaitis, O. Paulsen, and T. Wilson, Proc. Natl. Acad. Sci. USA 109, 2919 (2012).
[Crossref]

J. H. Park, W. Sun, and M. Cui, Proc. Natl. Acad. Sci. USA 112, 9236 (2015).
[Crossref]

K. Lou, S. Granick, and F. Amblard, Proc. Natl. Acad. Sci. USA 115, 6554 (2018).
[Crossref]

Sci. Rep. (1)

H. J. Choi and P. T. C. So, Sci. Rep. 4, 6626 (2014).
[Crossref]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[Crossref]

Other (1)

A. M. Weiner, Ultrafast Optics (Wiley, 2009).

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Figures (3)

Fig. 1.
Fig. 1. Schematic diagram of the system implemented. (a) Output from a femtosecond laser. The laser power was controlled using a half-wave plate (HWP) and a Faraday isolator (FI). The beam was expanded by a telescope (T), impinged on a SLM, passed through an afocal cylindrical lens pair (CL1 and CL2), and then reflected from a 1D GM and focused through another cylindrical lens (CL3, focal length f L ) onto a 1200 line/mm blazed grating (G). The spectral components dispersed by the grating were collected by a spherical collimating lens (L, focal length f L ) and reflected by a dichroic mirror (DM) into a water-immersion objective lens ( 10 × , NA0.45). The fluorescence emission passed through the objective, a tube lens (TL), and an emission filter before being detected by an electron-multiplcation CCD camera (EMCCD). (b) Schematic representation of the spatially dispersed spectral components at the objective back aperture (radius R ). Each diffraction order is similarly dispersed by G, but with a slightly different incidence angle. The spatial and temporal fill factors are b / R and a T / R respectively, in the x and y directions. (c) Schematic diagram of the dispersed spectral components passing through the objective, where τ B A and τ R are the pulse durations at the back aperture and focal plane of the objective. (d) Effect of the intracavity control on the spectral width of the laser. The initially narrow spectrum ( Ω = 6.9    nm , dotted curve) broadens up to a maximum one ( Ω = 21.4    nm , solid curve) used for comparison experiments.
Fig. 2.
Fig. 2. Axial sectioning for two-photon point scanning and LTFM implemented here with the same laser, objective, and samples. (a) Axial FWHM, plotted against the laser spectral width Ω with f L = 500    mm , using a 10 × objective lens with NA = 0.45 . The sample is made of 0.5 μm diameter fluorescent beads immobilized at 0.1 mg/mL in agarose. Blue, line temporal focusing. Star, point scanning. Diamond, classical line scanning. (b), (c) Axial FWHM is shown as a function of imaging depth using LTFM (blue) and TP-LSM (red) modalities implemented in our laboratory with the same laser, objective ( 10 × , NA = 0.45 ), and same samples (4 μm fluorescent beads dispersed in diluted intralipid emulsions). (b) and (c) correspond to a 1:1000 and 1:100 intralipid dilution respectively. Insets show x z sections of the point spread functions for different depths.
Fig. 3.
Fig. 3. Uniform photoexcitation. (a)  x z -section diagram of the beam path between SLM and G. The three diffracted beam orders ( 0 , ± 1 ) generated by an SLM focus through CL3 as three slightly offset adjacent line beams on G, that are conjugated to three adjacent lines in the sample focal plane. (b) Duration of laser pulses on the grating plotted against x -positions with active flattop beam generation. (c) Fluorescence response profiles along x for line temporal focusing without (top image and blue line) or with flattop photoexcitation (bottom image and red line) using a 10 × , NA = 0.45 objective. (d) Axial fluorescence profiles of Gaussian (blue squares) and flattop (red circles) measured from 0.5 μm diameter fluorescence beads. The x z -section of a 780 × 780 × 900    μm 3 volumetric image taken from a mouse lung slice respectively by (e) LTFM and (f) TP-LSM using the same 10 × objective with NA = 0.45 . The corresponding Fourier transform images are compared on the right of each image.

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