Abstract

We have developed a light-sheet illumination microscope that can perform fast 3D imaging of transparent biological samples with inexpensive visible lasers and a single galvo mirror (GM). The light-sheet is created by raster scanning a Bessel beam with a GM, with this same GM also being used to rescan the fluorescence across a chip of a camera to construct an image in real time. A slit is used to reject out-of-focus fluorescence such that the image formed in real time has minimal contribution from the sidelobes of the Bessel beam. Compared with two-photon Bessel beam excitation or other confocal line-scanning approaches, our method is of lower cost, is simpler, and does not require calibration and synchronization of multiple GMs. We demonstrated the optical sectioning and out-of-focus background rejection capabilities of this microscope by imaging fluorescently labeled actin filaments in fixed 3T3 cells.

© 2014 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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  8. J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
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    [CrossRef]
  18. J. J. Han, A. P. Shreve, and J. H. Werner, in Characterization of Materials (Wiley, 2012), pp. 1026–1039.
  19. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
    [CrossRef]
  20. M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
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  21. C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
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  22. S. Quirin, S. R. P. Pavani, and R. Piestun, Proc. Natl. Acad. Sci. 109, 675 (2012).
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2014 (2)

P. Zhang, P. M. Goodwin, and J. H. Werner, Proc. SPIE 8947, 89471K (2014).
[CrossRef]

P. Zhang, P. M. Goodwin, and J. H. Werner, Opt. Express 22, 12398 (2014).
[CrossRef]

2013 (2)

F. O. Fahrbach, V. Gurchenkov, K. Alessandri, P. Nassoy, and A. Rohrbach, Opt. Express 21, 13824 (2013).
[CrossRef]

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

2012 (4)

F. O. Fahrbach and A. Rohrbach, Nat. Commun. 3, 632 (2012).
[CrossRef]

S. Quirin, S. R. P. Pavani, and R. Piestun, Proc. Natl. Acad. Sci. 109, 675 (2012).
[CrossRef]

L. Silvestri, A. Bria, L. Sacconi, G. Iannello, and F. S. Pavone, Opt. Express 20, 20582 (2012).
[CrossRef]

E. Baumgart and U. Kubitscheck, Opt. Express 20, 21805 (2012).
[CrossRef]

2011 (2)

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

2010 (3)

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

J. Mertz and J. Kim, J. Biomed. Opt. 15, 016027 (2010).
[CrossRef]

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[CrossRef]

2006 (2)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

2004 (2)

Y. Lill and B. Hecht, Appl. Phys. Lett. 84, 1665 (2004).
[CrossRef]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

2000 (1)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000).

1991 (1)

1990 (1)

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

Alessandri, K.

Basu, S.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Baumgart, E.

Berland, K. M.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000).

Betzig, E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Born, M.

M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).

Bria, A.

Cella Zanacchi, F.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Chapman, A. R.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Davidson, M. W.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

Del Bue, A.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Denk, W.

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

Diaspro, A.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Dong, C. Y.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000).

Fahrbach, F. O.

Faretta, M.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Furia, L.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Galbraith, C. G.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

Galbraith, J. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

Gao, L.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

Gebhardt, J. C. M.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Goodwin, P. M.

P. Zhang, P. M. Goodwin, and J. H. Werner, Proc. SPIE 8947, 89471K (2014).
[CrossRef]

P. Zhang, P. M. Goodwin, and J. H. Werner, Opt. Express 22, 12398 (2014).
[CrossRef]

Gurchenkov, V.

Han, J. J.

J. J. Han, A. P. Shreve, and J. H. Werner, in Characterization of Materials (Wiley, 2012), pp. 1026–1039.

Hecht, B.

Y. Lill and B. Hecht, Appl. Phys. Lett. 84, 1665 (2004).
[CrossRef]

Herman, R. M.

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

Iannello, G.

Joseph, N.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[CrossRef]

Kim, J.

J. Mertz and J. Kim, J. Biomed. Opt. 15, 016027 (2010).
[CrossRef]

Kubitscheck, U.

E. Baumgart and U. Kubitscheck, Opt. Express 20, 21805 (2012).
[CrossRef]

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

Lavagnino, Z.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Lidke, K. A.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[CrossRef]

Lill, Y.

Y. Lill and B. Hecht, Appl. Phys. Lett. 84, 1665 (2004).
[CrossRef]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Maniatis, T.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Masters, B. R.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000).

Mertz, J.

J. Mertz and J. Kim, J. Biomed. Opt. 15, 016027 (2010).
[CrossRef]

Milkie, D. E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

Nassoy, P.

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Pavani, S. R. P.

S. Quirin, S. R. P. Pavani, and R. Piestun, Proc. Natl. Acad. Sci. 109, 675 (2012).
[CrossRef]

Pavone, F. S.

Pawley, J.

J. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).

Perrone Donnorso, M.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

Piestun, R.

S. Quirin, S. R. P. Pavani, and R. Piestun, Proc. Natl. Acad. Sci. 109, 675 (2012).
[CrossRef]

Planchon, T. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

Quirin, S.

S. Quirin, S. R. P. Pavani, and R. Piestun, Proc. Natl. Acad. Sci. 109, 675 (2012).
[CrossRef]

Rieger, B.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[CrossRef]

Ritter, J. G.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

Rohrbach, A.

Roy, R.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Sacconi, L.

Shreve, A. P.

J. J. Han, A. P. Shreve, and J. H. Werner, in Characterization of Materials (Wiley, 2012), pp. 1026–1039.

Siebrasse, J. P.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

Silvestri, L.

Smith, C. S.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[CrossRef]

So, P. T. C.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000).

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Stelzer, E. H. K.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

Strickler, J.

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

Suter, D. M.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

Veenendaal, A.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

Veith, R.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

Webb, W.

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

Werner, J. H.

P. Zhang, P. M. Goodwin, and J. H. Werner, Opt. Express 22, 12398 (2014).
[CrossRef]

P. Zhang, P. M. Goodwin, and J. H. Werner, Proc. SPIE 8947, 89471K (2014).
[CrossRef]

J. J. Han, A. P. Shreve, and J. H. Werner, in Characterization of Materials (Wiley, 2012), pp. 1026–1039.

Wiggins, T. A.

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

Wolf, E.

M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).

Xie, X. S.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Zhang, P.

P. Zhang, P. M. Goodwin, and J. H. Werner, Proc. SPIE 8947, 89471K (2014).
[CrossRef]

P. Zhang, P. M. Goodwin, and J. H. Werner, Opt. Express 22, 12398 (2014).
[CrossRef]

Zhao, Z. W.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Ann. Rev. Biomed. Eng. (1)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, Ann. Rev. Biomed. Eng. 2, 399 (2000).

Appl. Phys. Lett. (1)

Y. Lill and B. Hecht, Appl. Phys. Lett. 84, 1665 (2004).
[CrossRef]

J. Biomed. Opt. (1)

J. Mertz and J. Kim, J. Biomed. Opt. 15, 016027 (2010).
[CrossRef]

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

Nat. Commun. (1)

F. O. Fahrbach and A. Rohrbach, Nat. Commun. 3, 632 (2012).
[CrossRef]

Nat. Methods (5)

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, Nat. Methods 8, 417 (2011).
[CrossRef]

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, Nat. Methods 10, 421 (2013).
[CrossRef]

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, Nat. Methods 8, 1047 (2011).
[CrossRef]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, Nat. Methods 7, 373 (2010).
[CrossRef]

Opt. Express (4)

PLoS ONE (1)

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, PLoS ONE 5, e11639 (2010).
[CrossRef]

Proc. Natl. Acad. Sci. (1)

S. Quirin, S. R. P. Pavani, and R. Piestun, Proc. Natl. Acad. Sci. 109, 675 (2012).
[CrossRef]

Proc. SPIE (1)

P. Zhang, P. M. Goodwin, and J. H. Werner, Proc. SPIE 8947, 89471K (2014).
[CrossRef]

Science (3)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef]

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

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef]

Other (3)

J. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).

M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).

J. J. Han, A. P. Shreve, and J. H. Werner, in Characterization of Materials (Wiley, 2012), pp. 1026–1039.

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Figures (4)

Fig. 1.
Fig. 1.

Confocal Bessel beam scanning microscope. (a) Beam path for the creation of a Bessel beam. GMP, galvo mirror plane; BFP, back focal plane; and EO, excitation objective. (b) Schematic diagram of the experimental setup. LD, laser diode; GM, galvo mirror; L, lens; DO, detection objective. The scanning frequency is 500 Hz.

Fig. 2.
Fig. 2.

Lateral locations of the scanned (P1), descanned (P2), and rescanned (P3) Bessel beam versus the galvo mirror angle.

Fig. 3.
Fig. 3.

Out-of-focus background rejection capability of the confocal Bessel beam microscope. (a) Typical image of a 3T3 cell with its actin labeled with Alexa Fluor 488 Phalloidin. (b) Image from the same area as in (a) when a 30 μm slit was used to reject the out-of-focus background. (c) Normalized fluorescence distributions along the dashed lines shown in (a) and (b).

Fig. 4.
Fig. 4.

3D imaging of fixed 3T3 cells with their actin labeled with Alexa Fluor 488 Phalloidin. (a) 3D volume rendering from 300 slices with z step size of 100 nm without (top) or with (bottom) spatial filter. (b) Two typical image slices taken without (upper row) and with (lower row) spatial filter. All images in (b) are in the same scale, and the scale bar is 10 μm.

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