Abstract

One of main challenges in light-sheet microscopy is to design the light-sheet as extended and thin as possible - extended to cover a large field of view, thin to optimize resolution and contrast. However, a decrease of the beam’s waist also decreases the illumination beam’s depth of field. Here, we introduce a new kind of beam that we call sectioned Bessel beam. These beams can be generated by blocking opposite sections of the beam’s angular spectrum. In combination with confocal-line detection the optical sectioning performance of the light-sheet can be decoupled from the depth of field of the illumination beam. By simulations and experiments we demonstrate that these beams exhibit self-reconstruction capabilities and penetration depths into thick scattering media equal to those of conventional Bessel beams. We applied sectioned Bessel beams to illuminate tumor multicellular spheroids and prove the increase in contrast. Sectioned Bessel beams turn out to be highly advantageous for the investigation of large strongly scattering samples in a light-sheet microscope.

© 2013 OSA

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  1. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by Selective Plane Illumination Microscopy,” Science305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  2. H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
    [CrossRef] [PubMed]
  3. P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
    [CrossRef] [PubMed]
  4. F. O. Fahrbach and A. Rohrbach, “A line scanned light-sheet microscope with phase shaped self-reconstructing beams,” Opt. Express18(23), 24229–24244 (2010).
    [CrossRef] [PubMed]
  5. F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
    [CrossRef]
  6. T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
    [CrossRef] [PubMed]
  7. F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3, 632 (2012).
    [CrossRef] [PubMed]
  8. L. Silvestri, A. Bria, L. Sacconi, G. Iannello, and F. S. Pavone, “Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain,” Opt. Express20(18), 20582–20598 (2012).
    [CrossRef] [PubMed]
  9. E. Baumgart and U. Kubitscheck, “Scanned light sheet microscopy with confocal slit detection,” Opt. Express20(19), 21805–21814 (2012).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  11. J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
  16. M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc.191(2), 135–140 (1998).
    [CrossRef] [PubMed]
  17. G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
    [CrossRef] [PubMed]
  18. T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
    [CrossRef] [PubMed]

2012

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3, 632 (2012).
[CrossRef] [PubMed]

T. Ersoy, B. Yalizay, and S. Akturk, “Self-reconstruction of diffraction-free and accelerating laser beams in scattering media,” J. Quantum Spec. Rad. Trans.113(18), 2470–2475 (2012).
[CrossRef]

L. Silvestri, A. Bria, L. Sacconi, G. Iannello, and F. S. Pavone, “Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain,” Opt. Express20(18), 20582–20598 (2012).
[CrossRef] [PubMed]

E. Baumgart and U. Kubitscheck, “Scanned light sheet microscopy with confocal slit detection,” Opt. Express20(19), 21805–21814 (2012).
[CrossRef] [PubMed]

2011

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

2010

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
[CrossRef]

F. O. Fahrbach and A. Rohrbach, “A line scanned light-sheet microscope with phase shaped self-reconstructing beams,” Opt. Express18(23), 24229–24244 (2010).
[CrossRef] [PubMed]

2009

2008

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

2007

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

2004

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by Selective Plane Illumination Microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2001

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

2000

1999

1998

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc.191(2), 135–140 (1998).
[CrossRef] [PubMed]

1978

Akturk, S.

T. Ersoy, B. Yalizay, and S. Akturk, “Self-reconstruction of diffraction-free and accelerating laser beams in scattering media,” J. Quantum Spec. Rad. Trans.113(18), 2470–2475 (2012).
[CrossRef]

Baumgart, E.

Becker, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Betzig, E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Bianchini, P.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

Bria, A.

Chávez-Cerda, S.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, and S. Chávez-Cerda, “Alternative formulation for invariant optical fields: Mathieu beams,” Opt. Lett.25(20), 1493–1495 (2000).
[CrossRef] [PubMed]

Choi, J. M.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Davidson, M. W.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Deininger, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by Selective Plane Illumination Microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Deussing, J. M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Diaspro, A.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

Dodt, H.-U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Eder, M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Ersoy, T.

T. Ersoy, B. Yalizay, and S. Akturk, “Self-reconstruction of diffraction-free and accelerating laser beams in scattering media,” J. Quantum Spec. Rad. Trans.113(18), 2470–2475 (2012).
[CrossRef]

Fahrbach, F. O.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3, 632 (2012).
[CrossRef] [PubMed]

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
[CrossRef]

F. O. Fahrbach and A. Rohrbach, “A line scanned light-sheet microscope with phase shaped self-reconstructing beams,” Opt. Express18(23), 24229–24244 (2010).
[CrossRef] [PubMed]

Feit, M. D.

Fleck, J. A.

Fraser, S. E.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Galbraith, C. G.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Galbraith, J. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Gao, L.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Grill, S.

Gutiérrez-Vega, J. C.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, and S. Chávez-Cerda, “Alternative formulation for invariant optical fields: Mathieu beams,” Opt. Lett.25(20), 1493–1495 (2000).
[CrossRef] [PubMed]

Hell, S. W.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc.191(2), 135–140 (1998).
[CrossRef] [PubMed]

Hofmann, U. G.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc.191(2), 135–140 (1998).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by Selective Plane Illumination Microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Iannello, G.

Iturbe-Castillo, M. D.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, and S. Chávez-Cerda, “Alternative formulation for invariant optical fields: Mathieu beams,” Opt. Lett.25(20), 1493–1495 (2000).
[CrossRef] [PubMed]

Jährling, N.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Keller, P. J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Koos, D. S.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Krol, S.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

Kubitscheck, U.

Leischner, U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Mauch, C. P.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Milkie, D. E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

New, G. H. C.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

Pavone, F. S.

Planchon, T. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Ramirez, G. A.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

Rodríguez-Dagnino, R. M.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

Rohrbach, A.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3, 632 (2012).
[CrossRef] [PubMed]

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
[CrossRef]

F. O. Fahrbach and A. Rohrbach, “A line scanned light-sheet microscope with phase shaped self-reconstructing beams,” Opt. Express18(23), 24229–24244 (2010).
[CrossRef] [PubMed]

A. Rohrbach, “Artifacts resulting from imaging in scattering media: a theoretical prediction,” Opt. Lett.34(19), 3041–3043 (2009).
[CrossRef] [PubMed]

Sacconi, L.

Schierloh, A.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Schneider, M.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

Schrader, M.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc.191(2), 135–140 (1998).
[CrossRef] [PubMed]

Silvestri, L.

Simon, P.

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
[CrossRef]

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by Selective Plane Illumination Microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

S. Grill and E. H. K. Stelzer, “Method to calculate lateral and axial gain factors of optical setups with a large solid angle,” J. Opt. Soc. Am. A16(11), 2658–2665 (1999).
[CrossRef]

Supatto, W.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by Selective Plane Illumination Microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Szellas, T.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

Tepichín, E.

J. C. Gutiérrez-Vega, M. D. Iturbe-Castillo, G. A. Ramirez, E. Tepichín, R. M. Rodríguez-Dagnino, S. Chávez-Cerda, and G. H. C. New, “Experimental demonstration of optical Mathieu beams,” Opt. Commun.195(1-4), 35–40 (2001).
[CrossRef]

Truong, T. V.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Vicidomini, G.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc.225(1), 88–95 (2007).
[CrossRef] [PubMed]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

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H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
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[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish early embryonic development by Scanned Light Sheet Microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

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Figures (9)

Fig. 1
Fig. 1

A sectioned Bessel beam in k-space and in real space. (a) The extents dkx, dky, dkz, of the sectioned angular spectrum (red areas) as a projection of the Ewald spherical cap define the dimensions of the beam. (b) The intensity cross-sections I(x,y,z = 0), I(x,y = 0,z) and I(x = 0,y,z) illustrate the section angle β, but also show the propagation invariance.

Fig. 2
Fig. 2

Change of the effective beam widths. (a) The lateral beams widths of a sectioned Bessel beam change slowly along the detection axis y, but fall off strongly in beam scanning x-direction for angles β > 50°. (b) The depth of field dz can be controlled by the ring thickness ε, which has only a minor effect on the lateral width dx and no effect on dy. The curves shown are for β = 0.6π~110°. (c) The width dy of the Gaussian beam increases with increasing depth of field dz, which is not the case for both types of Bessel beams according to the angular spectrum estimation.

Fig. 3
Fig. 3

Simulated intensity cross-sections of a Gaussian and a Bessel beam propagating through a scattering medium. Transverse intensity cross-sections I(x,y,z = zi) are shown for three different penetration depths z0 = 12µm, z1 = 24µm, z2 = 36µm into a scattering medium for a Gaussian (a), a Bessel beam (b) and a sectioned Bessel beam (c). The medium consists of spheres with d = 2µm, n = 1.41 at a volume concentration of ρ = 6\%. Integration over the area around the beam’s propagation axis yields the beam’s on axis power P(z,R = 2µm). The region is marked by a dashed circle.

Fig. 4
Fig. 4

On-axis power for different illumination beams propagating through scattering media. The graphs show the on-axis power Q ¯ (z;R=2µm) for different beams. The shown data was averaged over 9 beam positions in 15 different scattering media and normalized to equal values at z = 0µm. The scattering medium consists of randomly distributed spheres with a difference in refractive index of Δn = 0.08 with respect to the surrounding volume. The diameter of the spheres is d = 2.0µm in (a), (b) and d = 4.0µm in (c), (d). The volume concentration is ρV = 6% in (a), (c) and ρV = 12% in (b), (d).

Fig. 5
Fig. 5

Average normalized on-axis power for beams propagating through distributions of scattering spheres at different beam positions. The average on-axis power Q ¯ i,j for beams that propagate through a scattering medium consisting of spheres (d = 2µm, Δn = 0.08, ρV = 6%) is shown for different beam positions i and different random position configurations of the spheres j. The color codes the on-axis power average along the propagation distance through the scattering spheres for: (a) a Gaussian beam with NA = 0.08, (b) a Bessel beam with a low NA = 0.2, (c) a Bessel beam with a high NA = 0.4, and (d) a sectioned Bessel beam with the same NA and β = 88°. The variation in the values Q ¯ i,j is strongest for the Gaussian beam and weakest for the high-NA Bessel beam and sectioned Bessel beam.

Fig. 6
Fig. 6

Transfer functions of fields and intensities for (sectioned) Bessel beams and a conventional (flat-top) beam. The table shows the coherent transfer functions Ẽ(kx,ky) (angular spectrum) in the upper rows, the intensity transfer functions AC() in the second row, and the system transfer function Hsys(kx,ky) in the bottom row. The red and yellow areas in Hsys(kx,ky) give an estimate for the ratio of high-frequency and medium frequency information of the beam. Vertical line scans Hsys(ky) along the detection axis are plotted on the right and reveal the ratios of high- and low-frequency contributions. The detection MTFdet(kx,ky) is shown as an inset (bottom right).

Fig. 7
Fig. 7

Optical sectioning performance in light-sheet microscope with sectioned Bessel beam illumination for various section angles. (a-c) The simulated cross-section of three sectioned Bessel beams for β = 180°, β = 100°, and β = 20°, respectively. Small insets in the upper left corner illustrate the beams’ angular spectrum (kx,ky). (d) The z-projection of the detection PSF for NA = 0.8. (e-g) The system-PSFs are shown below the corresponding illumination beams. (h) The fluorescence contribution to the total detected signal from each layer F(y) is shown in (i) The normalized primitive of F(y), G(y), where the signal (S) and background (BG) according to Eq. (13) are marked by arrows. (j) The optical sectioning dyOS as a function of the section angle β for wide-field detection (green) with different depths of field dz1/e = 40µm and dz1/e = 80µm for ε = 0.8 and ε = 0.9, respectively and for confocal-line detection (red).

Fig. 8
Fig. 8

Optical Sectioning dependency on the depth of field of the illumination beam with confocal-line detection (simulation). The depth of field dz1/e is steered by the NA for the Gaussian beam. For the Bessel beam and the sectioned Bessel beam (β = 110°) with NA = 0.4 the ring parameter ε is adapted.

Fig. 9
Fig. 9

Images of Tumor multicellular spheroids. Images of spheroidal cell clusters were imaged with Gaussian beam illumination using wide-field detection (a), and using confocal-line detection (b) For Bessel beams (c) and sectioned Bessel beams (d) images are shown only with confocal line-detection. The size of the scalebar is 20µm. The illumination beams propagate from left to right. (e) The average image intensity along the propagation axis for b,c,d. (f) The image contrast measured by the ratio of and low spatial frequencies for the whole image stack. Layers deeper within the spheroid (larger i) show less contrast.

Equations (14)

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E ˜ SeB ( k x , k y )= E ˜ 0 ( Θ( k 0 NA B k r )Θ( ε k 0 NA B k r ) )rect( k y 2 k 0 NA B sin(β/2) )
E SeB (x,y,z)= A r ( J 1 ( k 0 NA B r ) ε J 1 ( ε k 0 NA B r ) )sinc( k 0 NA B sin(β/2)y )
dx= λ NA B ( 1 ε cos(β/2) )
dy= λ NA B sin(β/2)
dz= 4λn NA B 2 ( 1ε )
P( z;R )= x 2 + y 2 < R 2 I( x,y,z )dxdy .
Q( z;R )= P scat (z;R) P ideal (z;R)
Q ¯ (R)= 1 Δz Δz Q(z;R)dz
h sys (x,y)= h ill (x,y) h det (x,y)
p CL (r)=[ h ill (r) h det (r) ] c F (r)
H sys (k)= H ill (k) H det (k)=AC{ E ˜ ill (k) }AC{ E ˜ det (k) }
G( y )= 1 F tot y F(y')dy' ,
SBG= δ δ F(y)dy / ( δ F(y)dy + δ F(y)dy ) = G(δ)G(δ) G(δ)+(1G(δ)) .
C= HSF LSF = k r k F | p ˜ ( k x , k z ) | d k x d k z k r < k F | p ˜ ( k x , k z ) | d k x d k z

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