Abstract

The acquisition of high-resolution images in three dimensions is of utmost importance for the morphological and functional investigation of biological tissues. Here, we present a laser scanning two-photon microscope with remote and motionless control of the focus position. The movement of the excitation spot along the propagation direction is achieved by shaping the laser wavefront with a spatial light modulator. Depending on the optical properties of the objective in use, this approach allows z movements in a range of tens to hundreds of micrometers with small changes of the point spread function. We applied this technique for the three-dimensional (3D) imaging of fluorescent cells in the mouse neocortex in vivo. The presented system bypasses the limitations of microscopes based on moving objectives, enabling high-resolution inertia-free 3D imaging.

© 2011 Optical Society of America

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References

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  1. A. Diaspro and C. Sheppard, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, 2002).
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    [CrossRef] [PubMed]

2011 (2)

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

B. F. Grewe, F. F. Voigt, H. M. van’t Hoff, and F. Helmchen, Biomed. Opt. Express 2, 2035 (2011).
[CrossRef] [PubMed]

2010 (3)

M. Dal Maschio, F. Difato, R. Beltramo, A. Blau, F. Benfenati, and T. Fellin, Opt. Express 18, 18720 (2010).
[CrossRef] [PubMed]

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, Neuron 67, 858 (2010).
[CrossRef] [PubMed]

2009 (1)

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

2008 (3)

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

R. G. Duemani, K. Kelleher, R. Fink, and P. Saggau, Nat. Neurosci. 11, 713 (2008).
[CrossRef]

E. J. Botcherby, M. J. Booth, R. Juskaitis, and T. Wilson, Opt. Express 16, 21843 (2008).
[CrossRef] [PubMed]

2007 (1)

W. Gobel, B. M. Kampa, and F. Helmchen, Nat. Methods 4, 73 (2007).
[CrossRef]

2006 (1)

O. Garaschuk, R. I. Milos, and A. Konnerth, Nat. Protoc. 1, 380 (2006).
[CrossRef]

2005 (2)

2004 (2)

H. Oku, K. Hashimoto, and M. Ishikawa, Opt. Express 12, 2138 (2004).
[CrossRef] [PubMed]

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

2002 (1)

J. E. Curtis, B. A. Koss, and D. G. Grier, Opt. Commun. 207, 169 (2002).
[CrossRef]

1999 (1)

N. Callamaras and I. Parker, Cell Calcium 26, 271 (1999).
[CrossRef]

Andermann, M. L.

A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, Neuron 67, 858 (2010).
[CrossRef] [PubMed]

Araya, R.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Arisaka, K.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

Bachor, H. A.

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

Beltramo, R.

Benfenati, F.

Berezovskii, V. K.

A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, Neuron 67, 858 (2010).
[CrossRef] [PubMed]

Blau, A.

Booth, M. J.

Botcherby, E. J.

Bowman, R.

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

Callamaras, N.

N. Callamaras and I. Parker, Cell Calcium 26, 271 (1999).
[CrossRef]

Cheng, A.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

Cojoc, D.

Coppey-Moisan, M.

Curtis, J. E.

J. E. Curtis, B. A. Koss, and D. G. Grier, Opt. Commun. 207, 169 (2002).
[CrossRef]

Dal Maschio, M.

Daria, V. R.

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

Di, F. E.

Diaspro, A.

A. Diaspro and C. Sheppard, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, 2002).

Dickensheets, L. D.

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

Difato, F.

Duemani, R. G.

R. G. Duemani, K. Kelleher, R. Fink, and P. Saggau, Nat. Neurosci. 11, 713 (2008).
[CrossRef]

Durieux, C.

Emiliani, V.

Fellin, T.

Ferrari, E.

Fink, R.

R. G. Duemani, K. Kelleher, R. Fink, and P. Saggau, Nat. Neurosci. 11, 713 (2008).
[CrossRef]

Garaschuk, O.

O. Garaschuk, R. I. Milos, and A. Konnerth, Nat. Protoc. 1, 380 (2006).
[CrossRef]

Garbin, V.

Gobel, W.

W. Gobel, B. M. Kampa, and F. Helmchen, Nat. Methods 4, 73 (2007).
[CrossRef]

Golshani, P.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

Goncalves, J. T.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

Gordon, L. M.

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

Grewe, B. F.

B. F. Grewe, F. F. Voigt, H. M. van’t Hoff, and F. Helmchen, Biomed. Opt. Express 2, 2035 (2011).
[CrossRef] [PubMed]

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

Grier, D. G.

J. E. Curtis, B. A. Koss, and D. G. Grier, Opt. Commun. 207, 169 (2002).
[CrossRef]

Hashimoto, K.

Helmchen, F.

B. F. Grewe, F. F. Voigt, H. M. van’t Hoff, and F. Helmchen, Biomed. Opt. Express 2, 2035 (2011).
[CrossRef] [PubMed]

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

W. Gobel, B. M. Kampa, and F. Helmchen, Nat. Methods 4, 73 (2007).
[CrossRef]

Himmer, A. P.

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

Ishikawa, M.

Juskaitis, R.

Kampa, B. M.

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

W. Gobel, B. M. Kampa, and F. Helmchen, Nat. Methods 4, 73 (2007).
[CrossRef]

Kasper, H.

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

Kelleher, K.

R. G. Duemani, K. Kelleher, R. Fink, and P. Saggau, Nat. Neurosci. 11, 713 (2008).
[CrossRef]

Kerlin, A. M.

A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, Neuron 67, 858 (2010).
[CrossRef] [PubMed]

Konnerth, A.

O. Garaschuk, R. I. Milos, and A. Konnerth, Nat. Protoc. 1, 380 (2006).
[CrossRef]

Koss, B. A.

J. E. Curtis, B. A. Koss, and D. G. Grier, Opt. Commun. 207, 169 (2002).
[CrossRef]

Langer, D.

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

Milos, R. I.

O. Garaschuk, R. I. Milos, and A. Konnerth, Nat. Protoc. 1, 380 (2006).
[CrossRef]

Nikolenko, V.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Oku, H.

Parker, I.

N. Callamaras and I. Parker, Cell Calcium 26, 271 (1999).
[CrossRef]

Peterka, D. S.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Portera-Cailliau, C.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

Qi, B.

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

Reddy, G. D.

G. D. Reddy and P. Saggau, J. Biomed. Opt. 10, 064038(2005).
[CrossRef]

Redman, S.

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

Reid, R. C.

A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, Neuron 67, 858 (2010).
[CrossRef] [PubMed]

Saggau, P.

R. G. Duemani, K. Kelleher, R. Fink, and P. Saggau, Nat. Neurosci. 11, 713 (2008).
[CrossRef]

G. D. Reddy and P. Saggau, J. Biomed. Opt. 10, 064038(2005).
[CrossRef]

Sheppard, C.

A. Diaspro and C. Sheppard, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, 2002).

Stricker, C.

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

van’t Hoff, H. M.

Vitkin, I. A.

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

Voigt, F. F.

Watson, B. O.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Wilson, T.

Woodruff, A.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Yang, X. D. V.

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

Yuste, R.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

Appl. Phys. Lett. (1)

V. R. Daria, C. Stricker, R. Bowman, S. Redman, and H. A. Bachor, Appl. Phys. Lett. 95, 093701 (2009).
[CrossRef]

Biomed. Opt. Express (1)

Cell Calcium (1)

N. Callamaras and I. Parker, Cell Calcium 26, 271 (1999).
[CrossRef]

Front. Neural Circuits (1)

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, Front. Neural Circuits 2, 5 (2008).
[CrossRef]

J. Biomed. Opt. (1)

G. D. Reddy and P. Saggau, J. Biomed. Opt. 10, 064038(2005).
[CrossRef]

Nat. Methods (3)

W. Gobel, B. M. Kampa, and F. Helmchen, Nat. Methods 4, 73 (2007).
[CrossRef]

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, Nat. Methods 8, 139 (2011).
[CrossRef] [PubMed]

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, Nat. Methods 7, 399 (2010).
[CrossRef] [PubMed]

Nat. Neurosci. (1)

R. G. Duemani, K. Kelleher, R. Fink, and P. Saggau, Nat. Neurosci. 11, 713 (2008).
[CrossRef]

Nat. Protoc. (1)

O. Garaschuk, R. I. Milos, and A. Konnerth, Nat. Protoc. 1, 380 (2006).
[CrossRef]

Neuron (1)

A. M. Kerlin, M. L. Andermann, V. K. Berezovskii, and R. C. Reid, Neuron 67, 858 (2010).
[CrossRef] [PubMed]

Opt. Commun. (2)

B. Qi, A. P. Himmer, L. M. Gordon, X. D. V. Yang, L. D. Dickensheets, and I. A. Vitkin, Opt. Commun. 232, 123 (2004).
[CrossRef]

J. E. Curtis, B. A. Koss, and D. G. Grier, Opt. Commun. 207, 169 (2002).
[CrossRef]

Opt. Express (4)

Other (1)

A. Diaspro and C. Sheppard, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, 2002).

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Figures (3)

Fig. 1
Fig. 1

Schematic representation of the optical layout. A, the system consists of the Ti:sapphire laser source; P, Pockels cell; λ / 2 , half-wave plate; L 1 and L 2 , telescope #1; SLM, spatial light modulator; L 3 and L 4 , telescope #2; G, galvanometric mirrors; SL, scan lens; TL, tube lens; D 1 , 660 nm long-pass dichroic mirror; D 2 , 575 nm long-pass dichroic mirror; EF 1 , EF 2 , emission filters; PMT, photomultiplier tubes; and OBJ, objective. B, fluorescence scanning images ( λ ex = 800 nm ) of a pollen grain at different z positions obtained by moving the laser spot in the axial direction through phase modulation with the SLM. The numbers displayed on the images are the z positions with respect to the objective focal plane (negative values are given to positions below the focus plane). The objective used for these recordings was an Olympus 40 × , 0.8 NA. Scale bar: 30 μm .

Fig. 2
Fig. 2

Spatial resolution as a function of the axial position. A and A 1 , x z (A) and x y ( A 1 ) profiles for illumination of fluorescent beads 170 nm in diameter. B and B 1 and C and C 1 show the same as in A and A 1 at two distinct z positions. The values in micrometers on the image represent the z position obtained with SLM focus control with respect to the objective focal plane. The objective used is a 60 × , 0.9 NA. Scale bars: 3 μm . D, FWHM values of x y (gray circles) and z (black squares) profiles as a function of the axial position of the laser beam.

Fig. 3
Fig. 3

Inertia-free 3D imaging of fluorescent cells in vivo. A–D, two-photon fluorescence scanning images, at various z positions, of bulk loaded neocortical cells in an anesthetized mouse ( λ ex = 830 nm ). Images show merging of the green and red channels. All cells were loaded with Oregon Green BAPTA (green), while astrocytes are also loaded with the astrocyte- specific dye Sulforhodamine 101 (red). The number on the images refers to the z position obtained with SLM focus control with respect to the objective focal plane placed approximately 60 μm below the surface of the brain. Note that pictures A and B were taken in cortical layer I (where few cell bodies are present), while images C and D are taken in cortical layer II. Scale bar: 30 μm .

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