Abstract

There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio.

© 2016 Optical Society of America

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2015 (4)

2014 (3)

A. Suli, A. D. Guler, D. W. Raible, and D. Kimelman, “A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations,” Development 141(5), 1167–1174 (2014).
[Crossref] [PubMed]

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, twin-region two-photon imaging across extended cortical networks,” bioRxiv 10, 011320 (2014).

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

2013 (3)

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, “Whole-brain functional imaging at cellular resolution using light-sheet microscopy,” Nat. Methods 10(5), 413–420 (2013).
[Crossref] [PubMed]

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499(7458), 295–300 (2013).
[Crossref] [PubMed]

R. W. Friedrich, “Neuronal computations in the olfactory system of zebrafish,” Annu. Rev. Neurosci. 36(1), 383–402 (2013).
[Crossref] [PubMed]

2012 (4)

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

P. Zhu, O. Fajardo, J. Shum, Y. P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012).
[Crossref] [PubMed]

H. Dana and S. Shoham, “Remotely scanned multiphoton temporal focusing by axial grism scanning,” Opt. Lett. 37(14), 2913–2915 (2012).
[Crossref] [PubMed]

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

2011 (3)

2010 (1)

B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, “High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision,” Nat. Methods 7(5), 399–405 (2010).
[Crossref] [PubMed]

2009 (2)

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[Crossref] [PubMed]

S. Junek, T.-W. Chen, M. Alevra, and D. Schild, “Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations,” Biophys. J. 96(9), 3801–3809 (2009).
[Crossref] [PubMed]

2008 (2)

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

E. J. Botcherby, R. Juškaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
[Crossref]

2007 (3)

2006 (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]

2005 (1)

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

2004 (1)

R. Tabor, E. Yaksi, J.-M. Weislogel, and R. W. Friedrich, “Processing of odor mixtures in the zebrafish olfactory bulb,” J. Neurosci. 24(29), 6611–6620 (2004).
[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(1), 13 (2003).
[Crossref] [PubMed]

C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003).
[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]

1990 (1)

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

Ahrens, M. B.

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, “Whole-brain functional imaging at cellular resolution using light-sheet microscopy,” Nat. Methods 10(5), 413–420 (2013).
[Crossref] [PubMed]

Akerboom, J.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[Crossref] [PubMed]

Alevra, M.

S. Junek, T.-W. Chen, M. Alevra, and D. Schild, “Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations,” Biophys. J. 96(9), 3801–3809 (2009).
[Crossref] [PubMed]

Amir, W.

Baohan, A.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499(7458), 295–300 (2013).
[Crossref] [PubMed]

Bargmann, C. I.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[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]

Bifano, T.

Blumhagen, F.

F. Blumhagen, P. Zhu, J. Shum, Y.-P. Z. Schärer, E. Yaksi, K. Deisseroth, and R. W. Friedrich, “Neuronal filtering of multiplexed odour representations,” Nature 479(7374), 493–498 (2011).
[Crossref] [PubMed]

Booth, M. J.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

E. J. Botcherby, R. Juškaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
[Crossref]

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “Aberration-free optical refocusing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
[Crossref] [PubMed]

Botcherby, E. J.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

E. J. Botcherby, R. Juškaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
[Crossref]

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “Aberration-free optical refocusing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
[Crossref] [PubMed]

Boyden, E. S.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

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. Methods 111(1), 29–37 (2001).
[Crossref] [PubMed]

Chalasani, S. H.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[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]

Chen, T.-W.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499(7458), 295–300 (2013).
[Crossref] [PubMed]

S. Junek, T.-W. Chen, M. Alevra, and D. Schild, “Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations,” Biophys. J. 96(9), 3801–3809 (2009).
[Crossref] [PubMed]

Chiappe, M. E.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[Crossref] [PubMed]

Chiovini, B.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Colon, J.

Cui, M.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12(8), 759–762 (2015).
[Crossref] [PubMed]

Dana, H.

Débarre, D.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

Deisseroth, K.

F. Blumhagen, P. Zhu, J. Shum, Y.-P. Z. Schärer, E. Yaksi, K. Deisseroth, and R. W. Friedrich, “Neuronal filtering of multiplexed odour representations,” Nature 479(7374), 493–498 (2011).
[Crossref] [PubMed]

Denk, W.

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

Doris, E. A.

Duemani Reddy, G.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Durfee, C. G.

Fajardo, O.

P. Zhu, O. Fajardo, J. Shum, Y. P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012).
[Crossref] [PubMed]

Fink, R.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Friedrich, R. W.

R. W. Friedrich, “Neuronal computations in the olfactory system of zebrafish,” Annu. Rev. Neurosci. 36(1), 383–402 (2013).
[Crossref] [PubMed]

P. Zhu, O. Fajardo, J. Shum, Y. P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012).
[Crossref] [PubMed]

F. Blumhagen, P. Zhu, J. Shum, Y.-P. Z. Schärer, E. Yaksi, K. Deisseroth, and R. W. Friedrich, “Neuronal filtering of multiplexed odour representations,” Nature 479(7374), 493–498 (2011).
[Crossref] [PubMed]

R. Tabor, E. Yaksi, J.-M. Weislogel, and R. W. Friedrich, “Processing of odor mixtures in the zebrafish olfactory bulb,” J. Neurosci. 24(29), 6611–6620 (2004).
[Crossref] [PubMed]

Garaschuk, O.

C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003).
[Crossref] [PubMed]

Germain, R. N.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12(8), 759–762 (2015).
[Crossref] [PubMed]

Göbel, W.

W. Göbel, B. M. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
[Crossref] [PubMed]

Grewe, B. F.

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R. Tabor, E. Yaksi, J.-M. Weislogel, and R. W. Friedrich, “Processing of odor mixtures in the zebrafish olfactory bulb,” J. Neurosci. 24(29), 6611–6620 (2004).
[Crossref] [PubMed]

Tang, J.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12(8), 759–762 (2015).
[Crossref] [PubMed]

Taranto, J.

Tian, L.

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[Crossref] [PubMed]

van ’t Hoff, M.

Vaziri, A.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Veress, M.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Vizi, E. S.

G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

Voigt, F. F.

Wardill, T. J.

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499(7458), 295–300 (2013).
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Webb, W. W.

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

Weislogel, J.-M.

R. Tabor, E. Yaksi, J.-M. Weislogel, and R. W. Friedrich, “Processing of odor mixtures in the zebrafish olfactory bulb,” J. Neurosci. 24(29), 6611–6620 (2004).
[Crossref] [PubMed]

Wetzstein, G.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Wilson, T.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
[Crossref] [PubMed]

E. J. Botcherby, R. Juškaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
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E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, “Aberration-free optical refocusing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
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Yaksi, E.

F. Blumhagen, P. Zhu, J. Shum, Y.-P. Z. Schärer, E. Yaksi, K. Deisseroth, and R. W. Friedrich, “Neuronal filtering of multiplexed odour representations,” Nature 479(7374), 493–498 (2011).
[Crossref] [PubMed]

R. Tabor, E. Yaksi, J.-M. Weislogel, and R. W. Friedrich, “Processing of odor mixtures in the zebrafish olfactory bulb,” J. Neurosci. 24(29), 6611–6620 (2004).
[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]

Yoon, Y.-G.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Yu, Y.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12(8), 759–762 (2015).
[Crossref] [PubMed]

Zhang Schärer, Y. P.

P. Zhu, O. Fajardo, J. Shum, Y. P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012).
[Crossref] [PubMed]

Zhu, P.

P. Zhu, O. Fajardo, J. Shum, Y. P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012).
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F. Blumhagen, P. Zhu, J. Shum, Y.-P. Z. Schärer, E. Yaksi, K. Deisseroth, and R. W. Friedrich, “Neuronal filtering of multiplexed odour representations,” Nature 479(7374), 493–498 (2011).
[Crossref] [PubMed]

Zimmer, M.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Zinter, J. P.

Zipfel, W. R.

Annu. Rev. Neurosci. (1)

R. W. Friedrich, “Neuronal computations in the olfactory system of zebrafish,” Annu. Rev. Neurosci. 36(1), 383–402 (2013).
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Biomed. Eng. Online (1)

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S. Junek, T.-W. Chen, M. Alevra, and D. Schild, “Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations,” Biophys. J. 96(9), 3801–3809 (2009).
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bioRxiv (1)

J. N. Stirman, I. T. Smith, M. W. Kudenov, and S. L. Smith, “Wide field-of-view, twin-region two-photon imaging across extended cortical networks,” bioRxiv 10, 011320 (2014).

Development (1)

A. Suli, A. D. Guler, D. W. Raible, and D. Kimelman, “A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations,” Development 141(5), 1167–1174 (2014).
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J. Neurosci. (1)

R. Tabor, E. Yaksi, J.-M. Weislogel, and R. W. Friedrich, “Processing of odor mixtures in the zebrafish olfactory bulb,” J. Neurosci. 24(29), 6611–6620 (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).
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Nat. Methods (8)

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
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M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, “Whole-brain functional imaging at cellular resolution using light-sheet microscopy,” Nat. Methods 10(5), 413–420 (2013).
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F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
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B. F. Grewe, D. Langer, H. Kasper, B. M. Kampa, and F. Helmchen, “High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision,” Nat. Methods 7(5), 399–405 (2010).
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G. Katona, G. Szalay, P. Maák, A. Kaszás, M. Veress, D. Hillier, B. Chiovini, E. S. Vizi, B. Roska, and B. Rózsa, “Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes,” Nat. Methods 9(2), 201–208 (2012).
[Crossref] [PubMed]

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12(8), 759–762 (2015).
[Crossref] [PubMed]

L. Tian, S. A. Hires, T. Mao, D. Huber, M. E. Chiappe, S. H. Chalasani, L. Petreanu, J. Akerboom, S. A. McKinney, E. R. Schreiter, C. I. Bargmann, V. Jayaraman, K. Svoboda, and L. L. Looger, “Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators,” Nat. Methods 6(12), 875–881 (2009).
[Crossref] [PubMed]

W. Göbel, B. M. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
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Nat. Neurosci. (1)

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
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Nat. Protoc. (1)

P. Zhu, O. Fajardo, J. Shum, Y. P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012).
[Crossref] [PubMed]

Nature (2)

T.-W. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499(7458), 295–300 (2013).
[Crossref] [PubMed]

F. Blumhagen, P. Zhu, J. Shum, Y.-P. Z. Schärer, E. Yaksi, K. Deisseroth, and R. W. Friedrich, “Neuronal filtering of multiplexed odour representations,” Nature 479(7374), 493–498 (2011).
[Crossref] [PubMed]

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. Commun. (1)

E. J. Botcherby, R. Juškaitis, M. J. Booth, and T. Wilson, “An optical technique for remote focusing in microscopy,” Opt. Commun. 281(4), 880–887 (2008).
[Crossref]

Opt. Express (2)

Opt. Lett. (4)

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

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates,” Proc. Natl. Acad. Sci. U.S.A. 109(8), 2919–2924 (2012).
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C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003).
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Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
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Other (3)

B. F. Grewe, F. Helmchen, and B. M. Kampa, “Two-photon imaging of neuronal network dynamics in neocortex,” in Optical Imaging of Neocortical Dynamics (Springer, 2014), pp. 133–150.

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P. Rupprecht, “ABCD optics for beam propagation for remote z-scanning,” GitHub (2016). https://github.com/PTRRupprecht/remote-z-scanning-ABCD-optics

Supplementary Material (3)

NameDescription
» Code 1       ABCD optics for beam propagation for remote z-scanning using Mathematica.
» Visualization 1: MP4 (6085 KB)      Supplementary Movie 1. Data from Fig. 5. Reduced data quality due to compression.
» Visualization 2: MP4 (13949 KB)      Supplementary Movie 2. Data from Fig. 6. Reduced data quality due to compression.

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Figures (6)

Fig. 1
Fig. 1

Basic principle and design. (a) Basic principle. A remote z-scan mirror is inserted into a plane conjugate to the specimen plane. Moving the z-scan mirror away from this plane (D ≠ 0) changes the divergence of the beam and displaces the focus (d ≠ 0). (b) ZSU configuration 1 (single lens): the laser beam (1) is passed through the ZSU consisting of a single z-scan lens Lz with focal length fz and the z-scan mirror (2) before reaching the xy scanners and the xy-scan lens L2 (3). Directed passage of the beam is accomplished using a polarizing beam splitter (PBS) and a quarter-wave plate (QWP). The LSM consists of a scan lens (L2) and a tube lens (L1), which form a telescope with magnification M = f1/f2, and the objective (Lo). The minimum distance between the z-scan lens Lz and the xy-scanners is xZSU = 75 mm. (c) Experimental realization of configuration 1 (single-lens). The ZSU mounted onto the xy-scan unit is shown with correct relative scaling of the components. The discance xZSU between the z-scan lens and the xy-scanners is xZSU = fz + xz = 75 mm. This distance is required to insert the polarizing beam splitter, contained inside the cube, and the quarter-wave plate into the beam path. The voice coil motor is shown in a cross-sectional view, illustrating the working principle based on the coil inside a mostly homogeneous magnetic field. The z-scan mirror is attached to the coil by a rod and a scaffold. The Hall position sensor is glued to the top of the voice coil motor (not shown). (d) ZSU configuration 2 (with telescope). A second telescope with magnification M’ = f1’/f2’ is inserted into the beam path of the ZSU. This permits to maintain telecentric distances between the intermediate optics system and to increase the focal shift d by a factor M’2.

Fig. 2
Fig. 2

Focal shift, back aperture filling and beam path. (a) Focal shift d as a function of mirror displacement D for configuration 1 (single lens). Dots show experimental measurements, lines show theoretical predictions (Eqs. (1), (2)). Black: z-scan lens with fz = 30 mm; gray: z-scan lens with fz = 75 mm. In both cases, xZSU = 75 mm. xz = xZSU – fz gives the deviation from the telecentric arrangement. The dashed gray line shows theoretical prediction for fz = 30 mm and xz = 0, which was not realized experimentally. (b) Configuration 2 (telescope) with fz = 30 mm (xz = 0). The slope of the linear relationship d(D) is 56 μm/mm. (c) Schematic beam paths for single-lens (top) and telescope configuration (bottom). The diameter of the back aperture of the objective is approximately 18 mm. Rays indicate the outer shape of the beam; colors encode the z-scanning position. The focal length/free apertures of the xy-scan lens and the tube lens are 50 mm/30 mm and 200 mm/35 mm, respectively. The relay lenses have focal lengths/free apertures of 150 mm/50.8 mm and 75 mm/50.8 mm for the telescope configuration. Note that filling of the back focal aperture of the objective changes substantially during scanning in configuration 1 (single lens) but not in configuration 2 (telescope). (d) Modulation of FOV size in x and y direction as a function of focal shift d for configuration 2. Dots indicate measurements; solid lines show fits of Eq. (6) with Δw = 7 mm. Dashed lines indicate the theoretical FOV changes for Δw = 7 mm when the x- and y-scan mirrors are equidistant from the focal points of L’2 and L2. (e) Illustration of the separation of x- and y-scan mirrors by Δw = 7 mm in the beam path. All theoretical calculations for beam propagation were implemented in Mathematica, as we show in Code 1 (Ref [21]).

Fig. 3
Fig. 3

Dynamics of the voice coil scanning system. (a) Step current pulses for sawtooth scanning. (b) Displacement of the voice coil motor measured with the position sensor. Small deflections are measurement noise, not positional noise. (c) Overlay of voice coil trajectories (different colors) during four successive flyback periods. Blue values on the y-axis show the corresponding focal shifts for configuration 2. Grey shading indicates the time when no data are acquired (effective flyback time). Steps in the curves are due to digitization. (d) Effective flyback time as a function of scan amplitude (black) and the corresponding focal shift for configuration 2 (blue). Black line is a spline fit. (e) Stabilization of the voice coil (VC) movement. In open loop the position of the VC is not controlled directly, leading to slow positional drift (red). In the semi-closed loop circuit, the positional signal of the hall sensor (HS) is integrated over one z-scan cycle and compared to the setpoint. An error signal proportional to the difference is added to the z-scanning command signal, preventing positional drift (blue).

Fig. 4
Fig. 4

Dependence of PSF on focal shift d. (a) Axial extent (FWHM) of the PSF as a function of focal shift d in configuration 1 (single lens). Corresponding z-scan mirror displacements D are shown on top. Black dots show measurements; grey line is a spline fit. Dashed line shows FWHM considered acceptable for multiphoton calcium imaging of neuronal population activity (7 μm). (b) Examples of axial (left) and lateral (right) PSFs obtained in configuration 1 for different focal shifts d. Data are from the measurements marked by corresponding colors in (a). Individual data points show measurements; lines show Gaussian fits. (c) Black dots and continuous grey line: axial FWHM of the PSF as a function of focal shift d in configuration 2 (telescope; fz = 30 mm). Gray dots and dashed line: same for fz = 10 mm (20x objective as z-scan lens). (d) Examples of PSFs for configuration 2 (fz = 30 mm). Data are from the measurements marked by corresponding colors in (c). For both configurations, the FWHM at d = 0 (red curves) was ca. 2.6 μm axially and 0.4 μm laterally.

Fig. 5
Fig. 5

Calcium imaging in the olfactory bulb using configuration 1. (a) Calcium indicator fluorescence (512 x 512 pixels) in four optical sections through the OB of an adult Tg(NeuroD:GcaMP6F)icm05 fish (see Visualization 1). Values of d show the displacement of the center of each image from the reference plane (no mirror displacement; D = 0 and d = 0). Raw fluorescence images were averaged over the full 200 s. Two glomerular structures are outlined in black. Right: scan trajectory in yz; both scalebars are 50 μm. (b,c) Relative change in fluorescence intensity during application of each odor (40 s). (d) Time courses of first three principal components (PC 1-3) (e) Correlation maps showing the correlation coefficient between the time course of each pixel and each PC. For each optical section, the correlation maps for each PC are combined into a single RGB image (see color cube).

Fig. 6
Fig. 6

Calcium imaging in Dp using configuration 2. (a) Fluorescence in each plane, averaged over the full duration of 400 s (see Visualization 2). d indicates the focal shift d(D), ‘depth’ the axial distance of the plane from the brain surface. All scale bars are 50 μm. (b) ROIs depicting clearly identified somata of 1507 neurons. Colors encode the correlation between the fluorescence time courses of each ROI and the first three clusters (e) in RGB (see Fig. 5(e)). (c) Schematic depiction of scanned planes. (d) Scan trajectory in yz. Planes were tilted (angle, 9.5°) and lower planes were slightly smaller due to FOV compression. Dashed lines indicate flybacks of the y-scanner. (e) Time courses of fluorescence signals averaged over ROIs of the first three activity clusters. Color corresponds to the RGB code in (b) and (f). Shading depicts periods of odor application. (f) Fluorescence time courses of subsets of ROIs from each plane. RGB color code shows correlation to the time course of each cluster (e).

Equations (6)

Equations on this page are rendered with MathJax. Learn more.

d=2 f 0 2 f 2 2 f 1 2 f z 2 D
d=2 f 0 2 f 2 2 f 1 2 f 1 2 f z 2 f 2 2 D
d=2 f 0 2 f 2 2 f 1 2 ( f z 2 2D x z ) D.
R BA =R( f 1 f 2 2D f 0 f 2 f 1 f z 2 2D f 1 x z f 2 f z 2 ).
R BA =R( f 1 f 2 2D f 1 2 f 0 f 2 f 1 f 2 2 f z 2 ).
FOV f 0 f 2 f 1 R( 1 2 f 1 2 f 2 2 f z 2 Dw )β

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