Abstract

In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving the quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstrate that this pivoting system is compatible with confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) by using a pivoted elliptical-Gaussian beam. We tested its performance by acquiring signals emitted by specific fluorophores in several mouse brain areas, comparing the pivoting beam illumination and a traditional static one, measuring the point spread function response and quantifying the striping reduction. We observed real-time shadow suppression, while preserving the advantages of confocal detection for image contrast.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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    [Crossref]
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    [Crossref]
  34. A. K. Glaser, Y. Chen, C. Yin, L. Wei, L. Barner, N. Reder, and J. Liu, “Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging,” Sci. Rep. 8(1), 13878 (2018).
    [Crossref]
  35. Q. Fu, B. L. Martin, D. Q. Matus, and L. Gao, “Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy,” Nat. Commun. 7(1), 11088 (2016).
    [Crossref]
  36. M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
    [Crossref]
  37. C. Guenthner, K. Miyamichi, H. Yang, H. Heller, and L. Luo, “Permanent genetic access to transiently active neurons via trap: Targeted recombination in active populations,” Neuron 78(5), 773–784 (2013).
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  38. I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
    [Crossref]
  39. D. Storace, M. S. Rad, B. Kang, L. B. Cohen, T. Hughes, and B. J. Baker, “Toward better genetically encoded sensors of membrane potential,” Trends Neurosci. 39(5), 277–289 (2016).
    [Crossref]
  40. Y. Bando, M. Sakamoto, S. Kim, I. Ayzenshtat, and R. Yuste, “Comparative evaluation of genetically encoded voltage indicators,” Cell Rep. 26(3), 802–813.e4 (2019).
    [Crossref]

2019 (5)

G. Sancataldo, L. Silvestri, A. L. A. Mascaro, L. Sacconi, and F. S. Pavone, “Advanced fluorescence microscopy for in vivo imaging of neuronal activity,” Optica 6(6), 758–765 (2019).
[Crossref]

Y. Liu, J. D. Lauderdale, and P. Kner, “Stripe artifact reduction for digital scanned structured illumination light sheet microscopy,” Opt. Lett. 44(10), 2510–2513 (2019).
[Crossref]

V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
[Crossref]

G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
[Crossref]

Y. Bando, M. Sakamoto, S. Kim, I. Ayzenshtat, and R. Yuste, “Comparative evaluation of genetically encoded voltage indicators,” Cell Rep. 26(3), 802–813.e4 (2019).
[Crossref]

2018 (6)

A. K. Glaser, Y. Chen, C. Yin, L. Wei, L. Barner, N. Reder, and J. Liu, “Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging,” Sci. Rep. 8(1), 13878 (2018).
[Crossref]

M. A. Taylor, G. C. Vanwalleghem, I. A. Favre-Bulle, and E. K. Scott, “Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations,” J. Biophotonics 11(12), e201800088 (2018).
[Crossref]

M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
[Crossref]

M. C. Müllenbroich, L. Silvestri, A. P. Di Giovanna, G. Mazzamuto, I. Costantini, L. Sacconi, and F. S. Pavone, “High-fidelity imaging in brain-wide structural studies using light-sheet microscopy,” eNeuro 5(6), ENEURO.0124-18.2018 (2018).
[Crossref]

S. M. Salili, M. Harrington, and D. J. Durian, “Note: Eliminating stripe artifacts in light-sheet fluorescence imaging,” Rev. Sci. Instrum. 89(3), 036107 (2018).
[Crossref]

J. Mayer, A. Robert-Moreno, J. Sharpe, and J. Swoger, “Attenuation artifacts in light sheet fluorescence microscopy corrected by optispim,” Light: Sci. Appl. 7(1), 70 (2018).
[Crossref]

2017 (2)

M. Duocastella, G. Sancataldo, P. Saggau, P. Ramoino, P. Bianchini, and A. Diaspro, “Fast inertia-free volumetric light-sheet microscope,” ACS Photonics 4(7), 1797–1804 (2017).
[Crossref]

R. M. Power and J. Huisken, “A guide to light-sheet fluorescence microscopy for multiscale imaging,” Nat. Methods 14(4), 360–373 (2017).
[Crossref]

2016 (4)

L. A. Royer, W. C. Lemon, R. K. Chhetri, Y. Wan, M. Coleman, E. W. Myers, and P. J. Keller, “Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms,” Nat. Biotechnol. 34(12), 1267–1278 (2016).
[Crossref]

L. Silvestri, I. Costantini, L. Sacconi, and F. S. Pavone, “Clearing of fixed tissue: a review from a microscopist’s perspective,” J. Biomed. Opt. 21(8), 081205 (2016).
[Crossref]

Q. Fu, B. L. Martin, D. Q. Matus, and L. Gao, “Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy,” Nat. Commun. 7(1), 11088 (2016).
[Crossref]

D. Storace, M. S. Rad, B. Kang, L. B. Cohen, T. Hughes, and B. J. Baker, “Toward better genetically encoded sensors of membrane potential,” Trends Neurosci. 39(5), 277–289 (2016).
[Crossref]

2015 (4)

M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
[Crossref]

I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
[Crossref]

G. d. Medeiros, N. Norlin, S. Gunther, M. Albert, L. Panavaite, U.-M. Fiuza, F. Peri, T. Hiiragi, U. Krzic, and L. Hufnagel, “Confocal multiview light-sheet microscopy,” Nat. Commun. 6(1), 8881 (2015).
[Crossref]

P. Keller and M. Ahrens, “Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy,” Neuron 85(3), 462–483 (2015).
[Crossref]

2014 (2)

M. Mickoleit, B. Schmid, M. Weber, F. O. Fahrbach, S. Hombach, S. Reischauer, and J. Huisken, “High-resolution reconstruction of the beating zebrafish heart,” Nat. Methods 11(9), 919–922 (2014).
[Crossref]

N. Vladimirov, Y. Mu, T. Kawashima, D. V. Bennett, C.-T. Yang, L. L. Looger, P. J. Keller, J. Freeman, and M. B. Ahrens, “Light-sheet functional imaging in fictively behaving zebrafish,” Nat. Methods 11(9), 883–884 (2014).
[Crossref]

2013 (4)

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]

T. Panier, S. Romano, R. Olive, T. Pietri, G. Sumbre, R. Candelier, and G. Debrégeas, “Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy,” Front. Neural Circuits 7, 65 (2013).
[Crossref]

F. O. Fahrbach, V. Gurchenkov, K. Alessandri, P. Nassoy, and A. Rohrbach, “Self-reconstructing sectioned bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy,” Opt. Express 21(9), 11425–11440 (2013).
[Crossref]

C. Guenthner, K. Miyamichi, H. Yang, H. Heller, and L. Luo, “Permanent genetic access to transiently active neurons via trap: Targeted recombination in active populations,” Neuron 78(5), 773–784 (2013).
[Crossref]

2012 (4)

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing bessel beams enables contrast-enhanced imaging in thick media,” Nat. Commun. 3(1), 632 (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. Express 20(18), 20582–20598 (2012).
[Crossref]

E. Baumgart and U. Kubitscheck, “Scanned light sheet microscopy with confocal slit detection,” Opt. Express 20(19), 21805–21814 (2012).
[Crossref]

P. J. Keller and H.-U. Dodt, “Light sheet microscopy of living or cleared specimens. Neurotechnology,” Curr. Opin. Neurobiol. 22(1), 138–143 (2012).
[Crossref]

2010 (2)

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS One 5(7), e11639 (2010).
[Crossref]

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics 4(11), 780–785 (2010).
[Crossref]

2009 (1)

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development 136(12), 1963–1975 (2009).
[Crossref]

2008 (1)

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref]

2007 (1)

2005 (1)

D. McGloin and K. Dholakia, “Bessel beams: diffraction in a new light,” Contemp. Phys. 46(1), 15–28 (2005).
[Crossref]

2004 (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,” Science 305(5686), 1007–1009 (2004).
[Crossref]

1993 (1)

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(3), 229–236 (1993).
[Crossref]

Ahrens, M.

P. Keller and M. Ahrens, “Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy,” Neuron 85(3), 462–483 (2015).
[Crossref]

Ahrens, M. B.

N. Vladimirov, Y. Mu, T. Kawashima, D. V. Bennett, C.-T. Yang, L. L. Looger, P. J. Keller, J. Freeman, and M. B. Ahrens, “Light-sheet functional imaging in fictively behaving zebrafish,” Nat. Methods 11(9), 883–884 (2014).
[Crossref]

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]

Albert, M.

G. d. Medeiros, N. Norlin, S. Gunther, M. Albert, L. Panavaite, U.-M. Fiuza, F. Peri, T. Hiiragi, U. Krzic, and L. Hufnagel, “Confocal multiview light-sheet microscopy,” Nat. Commun. 6(1), 8881 (2015).
[Crossref]

Alessandri, K.

Alterini, T.

M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
[Crossref]

Ayzenshtat, I.

Y. Bando, M. Sakamoto, S. Kim, I. Ayzenshtat, and R. Yuste, “Comparative evaluation of genetically encoded voltage indicators,” Cell Rep. 26(3), 802–813.e4 (2019).
[Crossref]

Baker, B. J.

D. Storace, M. S. Rad, B. Kang, L. B. Cohen, T. Hughes, and B. J. Baker, “Toward better genetically encoded sensors of membrane potential,” Trends Neurosci. 39(5), 277–289 (2016).
[Crossref]

Bando, Y.

Y. Bando, M. Sakamoto, S. Kim, I. Ayzenshtat, and R. Yuste, “Comparative evaluation of genetically encoded voltage indicators,” Cell Rep. 26(3), 802–813.e4 (2019).
[Crossref]

Barner, L.

A. K. Glaser, Y. Chen, C. Yin, L. Wei, L. Barner, N. Reder, and J. Liu, “Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging,” Sci. Rep. 8(1), 13878 (2018).
[Crossref]

Baumgart, E.

Bennett, D. V.

N. Vladimirov, Y. Mu, T. Kawashima, D. V. Bennett, C.-T. Yang, L. L. Looger, P. J. Keller, J. Freeman, and M. B. Ahrens, “Light-sheet functional imaging in fictively behaving zebrafish,” Nat. Methods 11(9), 883–884 (2014).
[Crossref]

Bianchini, P.

M. Duocastella, G. Sancataldo, P. Saggau, P. Ramoino, P. Bianchini, and A. Diaspro, “Fast inertia-free volumetric light-sheet microscope,” ACS Photonics 4(7), 1797–1804 (2017).
[Crossref]

Bria, A.

Burns, D. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(3), 229–236 (1993).
[Crossref]

Candelier, R.

T. Panier, S. Romano, R. Olive, T. Pietri, G. Sumbre, R. Candelier, and G. Debrégeas, “Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy,” Front. Neural Circuits 7, 65 (2013).
[Crossref]

Chédotal, A.

H. R. Ueda, A. Ertürk, K. Chung, V. Gradinaru, A. Chédotal, P. Tomancak, and P. J. Keller, “Tissue clearing and its applications in neuroscience,” Nat. Rev. Neurosci. (2020).

Chen, Y.

A. K. Glaser, Y. Chen, C. Yin, L. Wei, L. Barner, N. Reder, and J. Liu, “Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging,” Sci. Rep. 8(1), 13878 (2018).
[Crossref]

Chhetri, R. K.

L. A. Royer, W. C. Lemon, R. K. Chhetri, Y. Wan, M. Coleman, E. W. Myers, and P. J. Keller, “Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms,” Nat. Biotechnol. 34(12), 1267–1278 (2016).
[Crossref]

Chung, K.

H. R. Ueda, A. Ertürk, K. Chung, V. Gradinaru, A. Chédotal, P. Tomancak, and P. J. Keller, “Tissue clearing and its applications in neuroscience,” Nat. Rev. Neurosci. (2020).

Cohen, L. B.

D. Storace, M. S. Rad, B. Kang, L. B. Cohen, T. Hughes, and B. J. Baker, “Toward better genetically encoded sensors of membrane potential,” Trends Neurosci. 39(5), 277–289 (2016).
[Crossref]

Coleman, M.

L. A. Royer, W. C. Lemon, R. K. Chhetri, Y. Wan, M. Coleman, E. W. Myers, and P. J. Keller, “Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms,” Nat. Biotechnol. 34(12), 1267–1278 (2016).
[Crossref]

Conti, V.

I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
[Crossref]

Costantini, I.

M. C. Müllenbroich, L. Silvestri, A. P. Di Giovanna, G. Mazzamuto, I. Costantini, L. Sacconi, and F. S. Pavone, “High-fidelity imaging in brain-wide structural studies using light-sheet microscopy,” eNeuro 5(6), ENEURO.0124-18.2018 (2018).
[Crossref]

L. Silvestri, I. Costantini, L. Sacconi, and F. S. Pavone, “Clearing of fixed tissue: a review from a microscopist’s perspective,” J. Biomed. Opt. 21(8), 081205 (2016).
[Crossref]

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M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
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Orger, 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).
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Panier, T.

T. Panier, S. Romano, R. Olive, T. Pietri, G. Sumbre, R. Candelier, and G. Debrégeas, “Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy,” Front. Neural Circuits 7, 65 (2013).
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Pavone, F. S.

G. Sancataldo, L. Silvestri, A. L. A. Mascaro, L. Sacconi, and F. S. Pavone, “Advanced fluorescence microscopy for in vivo imaging of neuronal activity,” Optica 6(6), 758–765 (2019).
[Crossref]

V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
[Crossref]

G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
[Crossref]

M. C. Müllenbroich, L. Silvestri, A. P. Di Giovanna, G. Mazzamuto, I. Costantini, L. Sacconi, and F. S. Pavone, “High-fidelity imaging in brain-wide structural studies using light-sheet microscopy,” eNeuro 5(6), ENEURO.0124-18.2018 (2018).
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M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
[Crossref]

L. Silvestri, I. Costantini, L. Sacconi, and F. S. Pavone, “Clearing of fixed tissue: a review from a microscopist’s perspective,” J. Biomed. Opt. 21(8), 081205 (2016).
[Crossref]

M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
[Crossref]

I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
[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. Express 20(18), 20582–20598 (2012).
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G. d. Medeiros, N. Norlin, S. Gunther, M. Albert, L. Panavaite, U.-M. Fiuza, F. Peri, T. Hiiragi, U. Krzic, and L. Hufnagel, “Confocal multiview light-sheet microscopy,” Nat. Commun. 6(1), 8881 (2015).
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V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
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T. Panier, S. Romano, R. Olive, T. Pietri, G. Sumbre, R. Candelier, and G. Debrégeas, “Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy,” Front. Neural Circuits 7, 65 (2013).
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R. M. Power and J. Huisken, “A guide to light-sheet fluorescence microscopy for multiscale imaging,” Nat. Methods 14(4), 360–373 (2017).
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D. Storace, M. S. Rad, B. Kang, L. B. Cohen, T. Hughes, and B. J. Baker, “Toward better genetically encoded sensors of membrane potential,” Trends Neurosci. 39(5), 277–289 (2016).
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M. Duocastella, G. Sancataldo, P. Saggau, P. Ramoino, P. Bianchini, and A. Diaspro, “Fast inertia-free volumetric light-sheet microscope,” ACS Photonics 4(7), 1797–1804 (2017).
[Crossref]

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A. K. Glaser, Y. Chen, C. Yin, L. Wei, L. Barner, N. Reder, and J. Liu, “Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging,” Sci. Rep. 8(1), 13878 (2018).
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M. Mickoleit, B. Schmid, M. Weber, F. O. Fahrbach, S. Hombach, S. Reischauer, and J. Huisken, “High-resolution reconstruction of the beating zebrafish heart,” Nat. Methods 11(9), 919–922 (2014).
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V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
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J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS One 5(7), e11639 (2010).
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J. Mayer, A. Robert-Moreno, J. Sharpe, and J. Swoger, “Attenuation artifacts in light sheet fluorescence microscopy corrected by optispim,” Light: Sci. Appl. 7(1), 70 (2018).
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Robson, D. N.

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. O. Fahrbach, V. Gurchenkov, K. Alessandri, P. Nassoy, and A. Rohrbach, “Self-reconstructing sectioned bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy,” Opt. Express 21(9), 11425–11440 (2013).
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F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing bessel beams enables contrast-enhanced imaging in thick media,” Nat. Commun. 3(1), 632 (2012).
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F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics 4(11), 780–785 (2010).
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T. Panier, S. Romano, R. Olive, T. Pietri, G. Sumbre, R. Candelier, and G. Debrégeas, “Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy,” Front. Neural Circuits 7, 65 (2013).
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L. A. Royer, W. C. Lemon, R. K. Chhetri, Y. Wan, M. Coleman, E. W. Myers, and P. J. Keller, “Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms,” Nat. Biotechnol. 34(12), 1267–1278 (2016).
[Crossref]

Sacconi, L.

G. Sancataldo, L. Silvestri, A. L. A. Mascaro, L. Sacconi, and F. S. Pavone, “Advanced fluorescence microscopy for in vivo imaging of neuronal activity,” Optica 6(6), 758–765 (2019).
[Crossref]

M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
[Crossref]

M. C. Müllenbroich, L. Silvestri, A. P. Di Giovanna, G. Mazzamuto, I. Costantini, L. Sacconi, and F. S. Pavone, “High-fidelity imaging in brain-wide structural studies using light-sheet microscopy,” eNeuro 5(6), ENEURO.0124-18.2018 (2018).
[Crossref]

L. Silvestri, I. Costantini, L. Sacconi, and F. S. Pavone, “Clearing of fixed tissue: a review from a microscopist’s perspective,” J. Biomed. Opt. 21(8), 081205 (2016).
[Crossref]

M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
[Crossref]

I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
[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. Express 20(18), 20582–20598 (2012).
[Crossref]

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M. Duocastella, G. Sancataldo, P. Saggau, P. Ramoino, P. Bianchini, and A. Diaspro, “Fast inertia-free volumetric light-sheet microscope,” ACS Photonics 4(7), 1797–1804 (2017).
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G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
[Crossref]

G. Sancataldo, L. Silvestri, A. L. A. Mascaro, L. Sacconi, and F. S. Pavone, “Advanced fluorescence microscopy for in vivo imaging of neuronal activity,” Optica 6(6), 758–765 (2019).
[Crossref]

V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
[Crossref]

M. Duocastella, G. Sancataldo, P. Saggau, P. Ramoino, P. Bianchini, and A. Diaspro, “Fast inertia-free volumetric light-sheet microscope,” ACS Photonics 4(7), 1797–1804 (2017).
[Crossref]

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M. Mickoleit, B. Schmid, M. Weber, F. O. Fahrbach, S. Hombach, S. Reischauer, and J. Huisken, “High-resolution reconstruction of the beating zebrafish heart,” Nat. Methods 11(9), 919–922 (2014).
[Crossref]

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P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
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M. A. Taylor, G. C. Vanwalleghem, I. A. Favre-Bulle, and E. K. Scott, “Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations,” J. Biophotonics 11(12), e201800088 (2018).
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Sharpe, J.

J. Mayer, A. Robert-Moreno, J. Sharpe, and J. Swoger, “Attenuation artifacts in light sheet fluorescence microscopy corrected by optispim,” Light: Sci. Appl. 7(1), 70 (2018).
[Crossref]

Siebrasse, J. P.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS One 5(7), e11639 (2010).
[Crossref]

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G. Sancataldo, L. Silvestri, A. L. A. Mascaro, L. Sacconi, and F. S. Pavone, “Advanced fluorescence microscopy for in vivo imaging of neuronal activity,” Optica 6(6), 758–765 (2019).
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G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
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V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
[Crossref]

M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
[Crossref]

M. C. Müllenbroich, L. Silvestri, A. P. Di Giovanna, G. Mazzamuto, I. Costantini, L. Sacconi, and F. S. Pavone, “High-fidelity imaging in brain-wide structural studies using light-sheet microscopy,” eNeuro 5(6), ENEURO.0124-18.2018 (2018).
[Crossref]

L. Silvestri, I. Costantini, L. Sacconi, and F. S. Pavone, “Clearing of fixed tissue: a review from a microscopist’s perspective,” J. Biomed. Opt. 21(8), 081205 (2016).
[Crossref]

M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
[Crossref]

I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
[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. Express 20(18), 20582–20598 (2012).
[Crossref]

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F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics 4(11), 780–785 (2010).
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A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(3), 229–236 (1993).
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P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
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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,” Science 305(5686), 1007–1009 (2004).
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D. Storace, M. S. Rad, B. Kang, L. B. Cohen, T. Hughes, and B. J. Baker, “Toward better genetically encoded sensors of membrane potential,” Trends Neurosci. 39(5), 277–289 (2016).
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T. Panier, S. Romano, R. Olive, T. Pietri, G. Sumbre, R. Candelier, and G. Debrégeas, “Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy,” Front. Neural Circuits 7, 65 (2013).
[Crossref]

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J. Mayer, A. Robert-Moreno, J. Sharpe, and J. Swoger, “Attenuation artifacts in light sheet fluorescence microscopy corrected by optispim,” Light: Sci. Appl. 7(1), 70 (2018).
[Crossref]

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,” Science 305(5686), 1007–1009 (2004).
[Crossref]

Taylor, M. A.

M. A. Taylor, G. C. Vanwalleghem, I. A. Favre-Bulle, and E. K. Scott, “Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations,” J. Biophotonics 11(12), e201800088 (2018).
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V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
[Crossref]

G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
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G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
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V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
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M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
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H. R. Ueda, A. Ertürk, K. Chung, V. Gradinaru, A. Chédotal, P. Tomancak, and P. J. Keller, “Tissue clearing and its applications in neuroscience,” Nat. Rev. Neurosci. (2020).

van’t Hoff, M.

M. C. Müllenbroich, L. Silvestri, L. Onofri, I. Costantini, M. van’t Hoff, L. Sacconi, G. Iannello, and F. S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (2015).
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M. A. Taylor, G. C. Vanwalleghem, I. A. Favre-Bulle, and E. K. Scott, “Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations,” J. Biophotonics 11(12), e201800088 (2018).
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G. Sancataldo, V. Gavryusev, G. de Vito, L. Turrini, M. Locatelli, C. Fornetto, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts,” Front. Neuroanat. 13, 7 (2019).
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V. Gavryusev, G. Sancataldo, P. Ricci, A. Montalbano, C. Fornetto, L. Turrini, A. Laurino, L. Pesce, G. de Vito, N. Tiso, F. Vanzi, L. Silvestri, and F. S. Pavone, “Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector,” J. Biomed. Opt. 24(10), 1–6 (2019).
[Crossref]

M. C. Müllenbroich, L. Turrini, L. Silvestri, T. Alterini, A. Gheisari, F. Vanzi, L. Sacconi, and F. S. Pavone, “Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy,” Front. Cell. Neurosci. 12, 315 (2018).
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I. Costantini, J.-P. Ghobril, A. P. Di Giovanna, A. L. A. Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5(1), 9808 (2015).
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Veenendaal, A.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS One 5(7), e11639 (2010).
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Veith, R.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS One 5(7), e11639 (2010).
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A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(3), 229–236 (1993).
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L. A. Royer, W. C. Lemon, R. K. Chhetri, Y. Wan, M. Coleman, E. W. Myers, and P. J. Keller, “Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms,” Nat. Biotechnol. 34(12), 1267–1278 (2016).
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M. Mickoleit, B. Schmid, M. Weber, F. O. Fahrbach, S. Hombach, S. Reischauer, and J. Huisken, “High-resolution reconstruction of the beating zebrafish heart,” Nat. Methods 11(9), 919–922 (2014).
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Wei, L.

A. K. Glaser, Y. Chen, C. Yin, L. Wei, L. Barner, N. Reder, and J. Liu, “Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging,” Sci. Rep. 8(1), 13878 (2018).
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Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref]

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,” Science 305(5686), 1007–1009 (2004).
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Figures (6)

Fig. 1.
Fig. 1. Schematic of the multi-directional DSLM setup, presenting the excitation and imaging paths. The red dashed rectangle denotes an alternative light path, selectable via flip-flop mirrors, where two cylindrical lenses CL shape the circular Gaussian beam into an elliptical profile that is then pivoted by an AOD.
Fig. 2.
Fig. 2. Setup characterization: (A) shows the AOD’s angular response as a function of the input radio frequency shift relative to the referenced central value. (B) and (C) show the simulated dependence, respectively, of the beam width $w$ and of the detection efficiency $Eff$ from the pivoting angle $\alpha$, up to the maximum angle admitted by the excitation objective.
Fig. 3.
Fig. 3. Schematic of the beam pivoting arrangement at the sample: $w$ is the beam width; $D$ is the virtual slit width; $L$ is the length of the digital slit on the camera sensor (sepia rectangle); $\alpha$ is the pivoting angle which defines also the complementary angles $\beta + \delta = \pi /2 - \alpha$.
Fig. 4.
Fig. 4. Panels (a)-(c) and (d)-(f)) show the raw data points (encoded by a different color for each of the 6 measurements) and the average Gaussian fit (black line), obtained respectively in radial and axial direction. From left to right, the results obtained respectively for the NO AOD, 3LS and SB configurations. Panels (g) and (h) show the average FWHM and the standard deviation extracted from these measurements. Panel (i) shows a representative frame containing the fluorescence signal detected from a Tetraspeck fluorescent microsphere ($r={50}\,{\rm{nm}}$) embedded in agarose gel. Scale bar size: 10 µm.
Fig. 5.
Fig. 5. On the right LSFM images of a mouse brain, expressing fluorescent protein tdTomato, obtained by pivoting the beam with different scanning configurations, respectively from up to down, NO AOD, 1LS, 3LS, 5Ls and SB. On the left, the normalized gray-scale intensity profiles taken along the vertical directions. Scale bar size: 100 µm.
Fig. 6.
Fig. 6. Shadowing suppression quantification. (A1), (B1) and (C1) show the normalized intensity profiles, together with their corresponding difference, obtained by a longitudinally averaged intensity projection of the images; (A2), (B2) and (C2) show single frames acquired in three different mouse brains expressing fluorescent protein tdTomato, taken in NO AOD configuration; correspondingly, A(3), B(3) and C(3) represent the same view taken with SB scanning configuration. Areas of larger striping attenuation are indicated by red arrows. Scale bar size: 100 µm.

Tables (3)

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Table 1. Detection parameters used for PSF measurements with fluorescent beads and during mouse brain imaging.

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Table 2. Shadowing suppression ratios, obtained by a longitudinally averaged intensity projection of a substack of ten consecutive frames.

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Table 3. Contrast enhancement by confocal detection, calculated over stacks acquired with SB and NO AOD configurations.

Equations (4)

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

$$\overline{OH}=\frac{1}{2}\sqrt{L^2+D^2}$$
$$\left\{ \begin{array}{lcl} w/2= \overline{OH} \cos{(\delta)} = \overline{OH} \cos{(\frac{\pi}{2}-\beta -\alpha)}\\ w/2= \overline{OH} \sin{(\beta+\alpha)} \end{array}\right.$$
$$\beta = \arcsin{ {\biggl(}\frac{D}{2 \cdot \overline{OH}}{\biggr)}}.$$
$$Eff=\frac{D}{w}$$