Abstract

Light-sheet-based fluorescence imaging techniques rely on simultaneous excitation of a single optical plane and thus permit high-contrast optically sectioned imaging of extended tissue samples. Here, we introduce a miniaturized fiber-optic implementation of a selective plane-illumination microscope (miniSPIM). The excitation light was delivered through a single-mode optical fiber, and a light-sheet was created with a cylindrical gradient-index lens and a right-angle microprism. Fluorescence emission was collected orthogonally to the light-sheet through a gradient-index lens assembly and a coherent fiber bundle. The end face of the fiber bundle was imaged onto a charge-coupled device camera. The spatial resolutions of the miniSPIM were 3.2μm laterally and 5.1μm axially. Images of fluorescent beads and neurons in mouse neocortex exhibited superior axial resolution and contrast in the miniSPIM-mode compared to images recorded in epi-illumination mode. The miniSPIM may thus enable novel in vivo imaging approaches.

© 2010 Optical Society of America

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  1. B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
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J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, Development 136, 1963 (2009).
[CrossRef] [PubMed]

T. H. Chia and M. J. Levene, J. Neurophysiol. 102, 1310 (2009).
[CrossRef] [PubMed]

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

2008 (3)

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, Opt. Express 16, 5556 (2008).
[CrossRef] [PubMed]

D. Turaga and T. E. Holy, Opt. Lett. 33, 2302 (2008).
[CrossRef] [PubMed]

2006 (1)

2005 (2)

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

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

2003 (1)

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

2001 (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Aguilera, T. A.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Barretto, R. P. J.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

Burns, L. D.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

Cheung, E. L. M.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Chia, T. H.

T. H. Chia and M. J. Levene, J. Neurophysiol. 102, 1310 (2009).
[CrossRef] [PubMed]

Cocker, E. D.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Denk, W.

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

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

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Engelbrecht, C. J.

Fee, M. S.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Flusberg, B. A.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Greenberg, D. S.

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

Grossmann, S.

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

Helmchen, F.

C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, Opt. Express 16, 5556 (2008).
[CrossRef] [PubMed]

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

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Holy, T. E.

Huisken, J.

J. Huisken and D. Y. R. Stainier, Development 136, 1963 (2009).
[CrossRef] [PubMed]

Johnston, R. S.

Jung, J. C.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Kaneko, T.

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

Kerr, J. N. D.

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

Ko, T. H.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

Levene, M. J.

T. H. Chia and M. J. Levene, J. Neurophysiol. 102, 1310 (2009).
[CrossRef] [PubMed]

Lev-Ram, V.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Lin, M. Z.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Miyazaki, J. -I.

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

Mukamel, E. A.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

Nimmerjahn, A.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

Obata, K.

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

Piyawattanametha, W.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Royant, A.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Sawinski, J.

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

Schnitzer, M. J.

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

Seibel, E. J.

Shu, X.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Stainier, D. Y. R.

J. Huisken and D. Y. R. Stainier, Development 136, 1963 (2009).
[CrossRef] [PubMed]

Steinbach, P. A.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Stelzer, E. H.

Tamamaki, N.

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

Tank, D. W.

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Tomioka, R.

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

Tsien, R. Y.

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

Turaga, D.

Wallace, D. J.

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

Yanagawa, Y.

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

Development (1)

J. Huisken and D. Y. R. Stainier, Development 136, 1963 (2009).
[CrossRef] [PubMed]

J. Comp. Neurol. (1)

N. Tamamaki, Y. Yanagawa, R. Tomioka, J.-I. Miyazaki, K. Obata, and T. Kaneko, J. Comp. Neurol. 467, 60 (2003).
[CrossRef] [PubMed]

J. Neurophysiol. (1)

T. H. Chia and M. J. Levene, J. Neurophysiol. 102, 1310 (2009).
[CrossRef] [PubMed]

Nat. Methods (3)

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

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, Nat. Methods 2, 941 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A. Mukamel, R. P. J. Barretto, T. H. Ko, L. D. Burns, J. C. Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
[CrossRef] [PubMed]

Neuron (1)

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. USA (1)

J. Sawinski, D. J. Wallace, D. S. Greenberg, S. Grossmann, W. Denk, and J. N. D. Kerr, Proc. Natl. Acad. Sci. USA 106, 19557 (2009).
[CrossRef] [PubMed]

Science (1)

X. Shu, A. Royant, M. Z. Lin, T. A. Aguilera, V. Lev-Ram, P. A. Steinbach, and R. Y. Tsien, Science 324, 804 (2009).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

miniSPIM. (a) Proximal optical system consisting of L, laser; M, mirror; λ / 2 , half-wave plate; PBS, polarizing beam splitter cube; CL, fiber coupling lens; L1 and L2, Galileo telescope; L3, projection lens; DCM, dichroic mirror; OL, objective lens; EF, emission filter; TL, tube lens; and camera. SMF and CFB connect the proximal optical system (a) to the miniaturized miniSPIM front piece (b). The front piece consists of two GRIN-assemblies, GRIN 1 and GRIN 2. (c) and (d) Photographs of the front part of the front piece taken in fluorescein solution to visualize (c) epi-excitation and (d) miniSPIM-excitation modes.

Fig. 2
Fig. 2

Characterization of miniSPIM. (a) 1 μ m fluorescent microspheres were used to quantify lateral and axial miniSPIM-resolutions. Left column, raw pixelated x y image. Center column, depixelated x y image using a Gaussian blur filter. Right column, x z projection after isotropic rescaling. (b) 1 μ m fluorescent microsphere imaged in epi-mode. Same columns as above. All images are maximum intensity projections and were brightness/contrast adjusted. (c) Line profiles through depixelated x y images of beads and Gaussian fits for miniSPIM- (red) and epi- (black) modes. Maximum and minimum intensity values are indicated. (d) Axial profiles through beads with Gaussian fits for miniSPIM-(red) and epi- (black) modes.

Fig. 3
Fig. 3

Application of miniSPIM to image GFP-expressing interneurons in fixed tissue (top row) and in vivo (bottom row). (a) Experimental setup for imaging in fixed mouse neocortex. Arrow (with asterisk) indicates the distance between microprism face and tissue edge. (b) Images acquired at a depth of z 230 μ m below the brain surface. Left: epi-mode; right: miniSPIM-mode. (c) In vivo imaging setup with spacer. (d) In vivo images acquired at a depth of z 125 μ m in epi- (left) and miniSPIM- (right) modes. All images are single planes from z-stacks and were brightness/contrast adjusted. Incoming light-sheet direction for miniSPIM-mode is indicated by an arrow.

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