Abstract

“How thick is your light sheet?” is a question that has been asked frequently after talks showing impressive renderings of 3D data acquired by a light-sheet microscope. This question is motivated by the fact that most of the time the thickness of the light-sheet is uniquely associated to the axial resolution of the microscope. However, the link between light-sheet thickness and axial resolution has never been systematically assessed and it is still unclear how both are connected. The question is not trivial because commonly employed measures cannot readily be applied or do not lead to easily interpretable results for the many different types of light sheet. Here, we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution by using simulation data. Unexpectedly, our analysis revealed a trade-off between better axial resolution and thinner light-sheet thickness. Our results are surprising because thicker light-sheets that provide lower image contrast have previously not been associated with better axial resolution. We conclude that classical Gaussian illumination beams should be used when image contrast is most important, and more advanced types of illumination represent a way to optimize axial resolution at the expense of image contrast.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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2019 (3)

R. D. Vaadia, W. Li, V. Voleti, A. Singhania, E. M. C. Hillman, and W. B. Grueber, “Characterization of Proprioceptive System Dynamics in Behaving Drosophila Larvae Using High-Speed Volumetric Microscopy,” Curr. Biol. 29(6), 935–944.e4 (2019).
[Crossref]

B.-J. Chang, M. Kittisopikul, K. M. Dean, P. Roudot, E. S. Welf, and R. Fiolka, “Universal light-sheet generation with field synthesis,” Nat. Methods 16(3), 235–238 (2019).
[Crossref]

H. Jia, X. Yu, Y. Yang, X. Zhou, S. Yan, C. Liu, M. Lei, and B. Yao, “Axial resolution enhancement of light-sheet microscopy by double scanning of Bessel beam and its complementary beam,” J. Biophotonics 12(1), e201800094 (2019).
[Crossref]

2018 (4)

V. Le, X. Wang, C. Kuang, and X. Liu, “Axial resolution enhancement for light sheet fluorescence microscopy via using the subtraction method,” Opt. Eng. 57(10), 103107 (2018).
[Crossref]

M. Weigert, K. Subramanian, S. T. Bundschuh, E. W. Myers, and M. Kreysing, “Biobeam - Multiplexed wave-optical simulations of light sheet microscopy,” PLoS Comput. Biol. 14(4), e1006079 (2018).
[Crossref]

K. McDole, L. Guignard, F. Amat, A. Berger, G. Malandain, L. A. Royer, S. C. Turaga, K. Branson, and P. J. Keller, “In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level,” Cell 175(3), 859–876.e33 (2018).
[Crossref]

A.-K. Gustavsson, P. N. Petrov, and W. E. Moerner, “Light sheet approaches for improved precision in 3D localization-based super-resolution imaging in mammalian cells,” Opt. Express 26(10), 13122–13147 (2018).
[Crossref]

2017 (2)

C. Gohn-Kreuz and A. Rohrbach, “Light needles in scattering media using self-reconstructing beams and the STED principle,” Optica 4(9), 1134–1142 (2017).
[Crossref]

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref]

2016 (3)

P. Hoyer, G. de Medeiros, B. Balázs, N. Norlin, C. Besir, J. Hanne, H. G. Kräusslich, J. Engelhardt, S. J. Sahl, S. W. Hell, and L. Hufnagel, “Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT,” Proc. Natl. Acad. Sci. U. S. A. 113(13), 3442–3446 (2016).
[Crossref]

T. Zhao, S. C. Lau, Y. Wang, Y. Su, H. Wang, A. Cheng, K. Herrup, N. Y. Ip, S. Du, and M. Loy, “Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets,” Sci. Rep. 6(1), 26159 (2016).
[Crossref]

J. D. Manton and E. J. Rees, “triSPIM: light sheet microscopy with isotropic super-resolution,” Opt. Lett. 41(18), 4170–4173 (2016).
[Crossref]

2015 (5)

L. Gao, “Optimization of the excitation light sheet in selective plane illumination microscopy,” Biomed. Opt. Express 6(3), 881–890 (2015).
[Crossref]

L. Gao, “Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet,” Opt. Express 23(5), 6102–6111 (2015).
[Crossref]

E. G. Reynaud, J. Peychl, J. Huisken, and P. Tomancak, “Guide to light sheet microscopy for adventurous biologists,” Nat. Methods 12(1), 30–34 (2015).
[Crossref]

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref]

K. M. Dean, P. Roudot, E. S. Welf, G. Danuser, and R. Fiolka, “Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy,” Biophys. J. 108(12), 2807–2815 (2015).
[Crossref]

2014 (4)

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref]

K. Mohan, S. B. Purnapatra, and P. P. Mondal, “Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy,” PLoS One 9(6), e96551 (2014).
[Crossref]

Z. Yang, M. Prokopas, J. Nylk, C. Coll-Lladó, F. J. Gunn-Moore, D. E. Ferrier, T. Vettenburg, and K. Dholakia, “A compact Airy beam light sheet microscope with a tilted cylindrical lens,” Biomed. Opt. Express 5(10), 3434–3442 (2014).
[Crossref]

B. C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A. C. Reymann, R. Böhme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution,” Science 346(6208), 1257998 (2014).
[Crossref]

2013 (4)

2012 (1)

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]

2011 (2)

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. Methods 8(5), 417–423 (2011).
[Crossref]

T. V. Truong, Supatto, W., Koos, D. S., J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light sheet microscopy,” Nat. Methods 8(9), 757–760 (2011).
[Crossref]

2010 (2)

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

F. O. Fahrbach and A. Rohrbach, “A line scanned light sheet microscope with phase shaped self-reconstructing beams,” Opt. Express 18(23), 24229–24244 (2010).
[Crossref]

2009 (1)

2008 (1)

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

2007 (1)

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[Crossref]

2006 (1)

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]

2002 (1)

A. Rohrbach and E. H. K. Stelzer, “Three-dimensional position detection of optically trapped dielectric particles,” J. Appl. Phys. 91(8), 5474–5488 (2002).
[Crossref]

1997 (1)

C. J. R. Sheppard and K. G. Larkin, “Vectorial pupil functions and vectorial transfer functions,” Optik 107(2), 79–87 (1997).

1981 (1)

1964 (1)

Ahrens, M. B.

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

Alessandri, K.

Alghamdi, R. A.

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref]

Amat, F.

K. McDole, L. Guignard, F. Amat, A. Berger, G. Malandain, L. A. Royer, S. C. Turaga, K. Branson, and P. J. Keller, “In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level,” Cell 175(3), 859–876.e33 (2018).
[Crossref]

Andalman, A.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref]

Balázs, B.

P. Hoyer, G. de Medeiros, B. Balázs, N. Norlin, C. Besir, J. Hanne, H. G. Kräusslich, J. Engelhardt, S. J. Sahl, S. W. Hell, and L. Hufnagel, “Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT,” Proc. Natl. Acad. Sci. U. S. A. 113(13), 3442–3446 (2016).
[Crossref]

Bembenek, J. N.

B. C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A. C. Reymann, R. Böhme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution,” Science 346(6208), 1257998 (2014).
[Crossref]

Berger, A.

K. McDole, L. Guignard, F. Amat, A. Berger, G. Malandain, L. A. Royer, S. C. Turaga, K. Branson, and P. J. Keller, “In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level,” Cell 175(3), 859–876.e33 (2018).
[Crossref]

Besir, C.

P. Hoyer, G. de Medeiros, B. Balázs, N. Norlin, C. Besir, J. Hanne, H. G. Kräusslich, J. Engelhardt, S. J. Sahl, S. W. Hell, and L. Hufnagel, “Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT,” Proc. Natl. Acad. Sci. U. S. A. 113(13), 3442–3446 (2016).
[Crossref]

Betzig, E.

B. C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A. C. Reymann, R. Böhme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution,” Science 346(6208), 1257998 (2014).
[Crossref]

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. Methods 8(5), 417–423 (2011).
[Crossref]

Böhme, R.

B. C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A. C. Reymann, R. Böhme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution,” Science 346(6208), 1257998 (2014).
[Crossref]

Branson, K.

K. McDole, L. Guignard, F. Amat, A. Berger, G. Malandain, L. A. Royer, S. C. Turaga, K. Branson, and P. J. Keller, “In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level,” Cell 175(3), 859–876.e33 (2018).
[Crossref]

Broxton, M.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref]

Bundschuh, S. T.

M. Weigert, K. Subramanian, S. T. Bundschuh, E. W. Myers, and M. Kreysing, “Biobeam - Multiplexed wave-optical simulations of light sheet microscopy,” PLoS Comput. Biol. 14(4), e1006079 (2018).
[Crossref]

Burns, V. M.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref]

Buytaert, J. A. N.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[Crossref]

Chang, B.-J.

B.-J. Chang, M. Kittisopikul, K. M. Dean, P. Roudot, E. S. Welf, and R. Fiolka, “Universal light-sheet generation with field synthesis,” Nat. Methods 16(3), 235–238 (2019).
[Crossref]

Chen, B. C.

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Supplementary Material (1)

NameDescription
» Code 1       Code for simulation of beam propagation and assessment of light-sheet properties

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Figures (8)

Fig. 1.
Fig. 1. Illustration of the six light sheet types included in the comparison. The amplitude and phase of the electrical field in the back focal plane of the illumination objective is used to simulate the intensity distribution of the beams in the sample volume. Cross section along the illumination and detection axis (YZ) and along the scan axis and detection (XZ) of the sheets resulting from beam scanning or sheet dithering.
Fig. 2.
Fig. 2. Definition of Main Lobe Thickness and Optical Sectioning. yz-cross sections of the intensity of a single-lobe beam (a) and a multi-lobe beam (b) are shown. The respective profiles at the focus (purple line) and at a distance y = L, where the thickness has doubled (blue line) are shown in c-j. c, d and e, f illustrate the main-lobe thickness, defined as the range where the intensity is higher than 37% of the maximum intensity in the focus, in focus (c, e) and at distance y = L (d, f) for single-lobe beam (c, d) and multi-lobe beam, respectively. Sub-figures g-j correspondingly illustrate Optical Sectioning wOS, defined as the range that contains 63% of the beam’s power.
Fig. 3.
Fig. 3. Schematic illustration of the effect of different beam shapes on axial resolution dz and optical sectioning OS in a light sheet microscope. The illumination PSF (blue) indicates the intensity of the illumination light. The detection PSF (green) indicates the location-dependent detection probability for fluorescence photons. The dotted lines indicate iso-intensity surfaces, e.g. at 37% of the peak value. The dotted red line indicates the iso-surface of the combined illumination and detection PSF (product of illumination intensity and detection probability). Its extent along the detection axis is proportional to the axial resolution of the system. The green shaded area indicates the volume where fluorophores are illuminated, and fluorescence is collected. The optical sectioning measures the extent of this volume along the detection axis. In a), where a single lobe illumination beam is shown, resolution is anisotropic, i.e. lateral resolution is better than axial resolution, but optical sectioning is close to axial resolution. In b), where a multi-lobe illumination beam is sketched, resolution is isotropic, but additional fluorescence signal is collected from the areas illuminated by the side lobes. Optical sectioning is inferior to axial resolution. The side lobes illuminate out-of-focus planes that blur the resulting image.
Fig. 4.
Fig. 4. Measurements of main lobe thickness and optical sectioning from simulated data. Main lobe thickness (a, c) and optical sectioning (c, d) are shown as a function of length for linear (a, b) and two-photon excitation (c, d). Parameters used in the simulations are NA = 0.14 to 0.26 for Gaussian beams; NA = 0.16 to 0.40 Focused flat-top beams; NA = 0.05 and separation NA = 0.14 to 0.3 for Double beams; NA = 0.5 and phase parameter a = 4 to 12 radians for Airy beams; NA = 0.32 to 0.6 with ring thickness parameter ɛ = 0.7 for the Bessel beam, NA = 0.25 with quartic phase amplitude a = 0.04 to 0.09 radians for the Spherically aberrated beam; and NA = 0.3 to 0.55 for ɛ = 0.7 for the dithered Square Bessel beam lattice.
Fig. 5.
Fig. 5. Ratio of Optical Sectioning (blue) and Main Lobe Thickness (orange) for each beam type with respect to the Gaussian beam. All values are computed for beams with length L∼30 µm. The insert shows a scheme to interpret the graph. Values larger than 100% mean that the main lobe or optical section is thicker than for the Gaussian beam.
Fig. 6.
Fig. 6. The Main Lobe Thickness (orange) and Light Sheet Thickness / Optical Sectioning (blue) is shown for the double beam. The figure illustrates how the two parameters of multi lobe beams can be used to control beam length at the expense of a thicker light sheet or a thicker main lobe (a) To change the length of the light-sheet the NA of the two sub-beams is varied (NA = 0.2 to NA = 0.05) while the angle of interference is held constant (NAsep = 0.3) (b) the sub-beams NA is held constant while the angle of interference in varied (NAsep = 0.45 to NAsep = 0.15 and NA = 0.05).
Fig. 7.
Fig. 7. The main lobe thickness and optical sectioning for different values of parameter b that determines the period of the lattice.
Fig. 8.
Fig. 8. User interface of the beam simulator.

Equations (9)

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h ill ( x , y , z ) = | E ( x , y , z ) | 2 .
E ( x , y 0 + d y , z ) = FT { E ~ ( k x , y 0 , k z ) exp { i ( ( k 0 n ) 2 k r 2 ) 1 / 2 d y } }
E ( x , y 0 , z ) = FT { E ~ BFP ( k x , k z ) } .
E ~ BFP ( k r ) = exp { ( k r / ( k 0 NA ) ) 2 }
E ~ BFP ( k r ) = circ { k r / ( k 0 NA ) }
E ~ BFP ( k x , k z ) = comb ( k x / δ k x ) exp { ( ( k r k c ) / k t ) 2 }
E ~ BFP ( k r ) = circ { k r / ( k 0 NA ) } exp { i a ( k y 3 + k z 3 ) / ( k 0 3 N A 3 ) }
E ~ BFP ( k r ) = circ { k r / ( k 0 NA ) } exp { i a k r 4 }
E ~ BFP ( k x , k z ) = exp { ( k r 1 / ( k 0 NA ) ) 2 } + exp { ( k r 2 / ( k 0 NA ) ) 2 }