Abstract

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

© 2012 OSA

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2012

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3, 632–639 (2012).
[CrossRef] [PubMed]

2011

J. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J Biophotonics4(1-2), 122–134 (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]

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]

2010

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

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

J. Palero, S. I. C. O. Santos, D. Artigas, and P. Loza-Alvarez, “A simple scanless two-photon fluorescence microscope using selective plane illumination,” Opt. Express18(8), 8491–8498 (2010).
[CrossRef] [PubMed]

2009

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

2008

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[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]

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[CrossRef] [PubMed]

S. Akturk, B. Zhou, B. Pasquiou, M. Franco, and A. Mysyrowicz, “Intensity distribution around the focal regions of real axicons,” Opt. Commun.281(17), 4240–4244 (2008).
[CrossRef]

O. Brzobohatý, T. Cizmár, and P. Zemánek, “High quality quasi-Bessel beam generated by round-tip axicon,” Opt. Express16(17), 12688–12700 (2008).
[PubMed]

2007

2006

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]

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2000

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

1997

1982

W. J. Alford, R. D. VanderNeut, and V. J. Zaleckas, “Laser scanning microscopy,” Proc. IEEE70(6), 641–651 (1982).
[CrossRef]

Akturk, S.

S. Akturk, B. Zhou, B. Pasquiou, M. Franco, and A. Mysyrowicz, “Intensity distribution around the focal regions of real axicons,” Opt. Commun.281(17), 4240–4244 (2008).
[CrossRef]

Alford, W. J.

W. J. Alford, R. D. VanderNeut, and V. J. Zaleckas, “Laser scanning microscopy,” Proc. IEEE70(6), 641–651 (1982).
[CrossRef]

Artigas, D.

Baird, G.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

Bao, Z.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[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]

Brzobohatý, O.

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]

Cizmár, T.

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]

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]

Engelbrecht, C. J.

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, 632–639 (2012).
[CrossRef] [PubMed]

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

Franco, M.

S. Akturk, B. Zhou, B. Pasquiou, M. Franco, and A. Mysyrowicz, “Intensity distribution around the focal regions of real axicons,” Opt. Commun.281(17), 4240–4244 (2008).
[CrossRef]

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]

Gratton, E.

Greger, K.

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

J. Swoger, P. Verveer, K. Greger, J. Huisken, and E. H. K. Stelzer, “Multi-view image fusion improves resolution in three-dimensional microscopy,” Opt. Express15(13), 8029–8042 (2007).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

J. Swoger, P. Verveer, K. Greger, J. Huisken, and E. H. K. Stelzer, “Multi-view image fusion improves resolution in three-dimensional microscopy,” Opt. Express15(13), 8029–8042 (2007).
[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]

Keller, P. J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[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]

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[CrossRef] [PubMed]

Kerr, R.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

Khairy, K.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

König, K.

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]

Kržic, U.

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

Lev-Ram, V.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

López-Schier, H.

J. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J Biophotonics4(1-2), 122–134 (2011).
[CrossRef] [PubMed]

Loza-Alvarez, P.

Mantulin, W. W.

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]

Muzzopappa, M.

J. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J Biophotonics4(1-2), 122–134 (2011).
[CrossRef] [PubMed]

Mysyrowicz, A.

S. Akturk, B. Zhou, B. Pasquiou, M. Franco, and A. Mysyrowicz, “Intensity distribution around the focal regions of real axicons,” Opt. Commun.281(17), 4240–4244 (2008).
[CrossRef]

Palero, J.

Pasquiou, B.

S. Akturk, B. Zhou, B. Pasquiou, M. Franco, and A. Mysyrowicz, “Intensity distribution around the focal regions of real axicons,” Opt. Commun.281(17), 4240–4244 (2008).
[CrossRef]

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]

Reynaud, E. G.

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

Rohrbach, A.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3, 632–639 (2012).
[CrossRef] [PubMed]

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

Santella, A.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Santos, S. I. C. O.

Schafer, W. R.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[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]

Sharpe, J.

J. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J Biophotonics4(1-2), 122–134 (2011).
[CrossRef] [PubMed]

Simon, P.

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

So, P. T. C.

Stainier, D. Y. R.

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009).
[CrossRef] [PubMed]

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

Stelzer, E. H.

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[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]

E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J2(5), 266–275 (2008).
[CrossRef] [PubMed]

J. Swoger, P. Verveer, K. Greger, J. Huisken, and E. H. K. Stelzer, “Multi-view image fusion improves resolution in three-dimensional microscopy,” Opt. Express15(13), 8029–8042 (2007).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[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]

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. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J Biophotonics4(1-2), 122–134 (2011).
[CrossRef] [PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78(2), 023705 (2007).
[CrossRef] [PubMed]

J. Swoger, P. Verveer, K. Greger, J. Huisken, and E. H. K. Stelzer, “Multi-view image fusion improves resolution in three-dimensional microscopy,” Opt. Express15(13), 8029–8042 (2007).
[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]

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]

Tsien, R. Y.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

VanderNeut, R. D.

W. J. Alford, R. D. VanderNeut, and V. J. Zaleckas, “Laser scanning microscopy,” Proc. IEEE70(6), 641–651 (1982).
[CrossRef]

Verveer, P.

Vincent, P.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron26(3), 583–594 (2000).
[CrossRef] [PubMed]

Webb, W. W.

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Supplementary Material (6)

» Media 1: AVI (171 KB)     
» Media 2: AVI (180 KB)     
» Media 3: AVI (188 KB)     
» Media 4: AVI (162 KB)     
» Media 5: AVI (257 KB)     
» Media 6: AVI (365 KB)     

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Figures (7)

Figure 1
Figure 1

Schematic diagram of our DLSM setup. The translation stage S1 allows switching between linear DSLM, where the beam pass through the SHG crystal and lenses L1 and L2, and 2p-DLSM where the femtosecond-laser beam pass unmodified. Gaussian or Bessel beams are selected by moving the translation stage S2 that contains an axicon and the Fourier transforming lens L3. A galvo mirror GM, a scan lens (SL), a tube lens (TLE) and an excitation objective lens (EO) are used to create the light sheet with the desired properties at the sample plane. The fluorescence generated in the sample is collected at 90 degrees by a collection objective lens (CO) and a regular tube lens (TLC) that forms image onto the CCD sensor with the designed magnification. Filters F1 and F2 are used to cut off the excitation light from the fluorescence images collected. The inset (top-right) shows the excitation-collection geometry that defines the notation of the axes used in this work.

Figure 2
Figure 2

Normalized fluorescence images and intensity profiles along x and y for the different excitation beams: a-c) DSLM with Gaussian beams, d-f) 2p-DSLM with Gaussian beams, g-i) DSLM with BB and j-l) 2p-DSLM with BB. The FWHM widths are indicated between arrows for each modality and their corresponding values Δx and Δy are included. These values are also summarized in Table 1. Profiles were taken along x and y directions with the point of maximum intensity located at the origin of coordinates, see the reference axes in Fig. 1(a). Scale bar: 50 μm, a pixel of the image corresponds to 0.44 μm in the sample.

Figure 3
Figure 3

Example of the PSFs obtained for the system using a sample of fluorescent beads in agar. The experimental intensity profiles (black dots) and Gaussian fits (red lines) for both transversal (δr) and axial (δz) dimensions are shown for the different modalities: a, b) DSLM with Gaussian beams, c, d) 2p-DSLM with Gaussian beams, e, f) DSLM with BB and g, h) 2p-DSLM with Bessel beams. The FWHM widths calculated from the Gaussian fit are shown over each graph. The average values of the PSF widths for 5 beads are reported in the Table 1

Figure 4
Figure 4

Images of a CFP-fluorescent pharynx of a C. elegans. Figures (a-c) are maximum intensity projections (MIP) of z stacks taken with a) the reference state-of-the-art SPIM system (Media 1), b) DSLM-Gauss (Media 2) and c) 2p-DSLM-Gauss (Media 3). Figures d) to f) show some individual sections of the z stacks obtained with SPIM (Media 4), DSLM (Media 5) and 2p-DSLM (Media 6) modalities using Gaussian beams, respectively. All z-stacks are composed of 54 images taken in steps of 2 μm. Scale bars: 20 μm.

Figure 5
Figure 5

Example of the contrast enhancement obtained by using 2p-DSLM. Regions of interest (ROI) taken from single optical sections (yellow squares on Fig. 4) for a) DSLM and b) 2p-DSLM. c) Plot of the intensity profiles along the selected lines for both modalities. Intensity values of the plots have been normalized to the maximum of each distribution.

Figure 6
Figure 6

Fluorescent images of a row of C. elegans aligned along the x direction. Figure a) shows the sample configuration as a reference (image taken using oblique illumination). Figures (b-e) are maximum intensity projections of z stacks taken with the four modalities available in our setup: b) DSLM-Gauss, c) 2p-DSLM-Gauss, d) DSLM-Bessel and e) 2p-DSLM-Bessel. The insets below each image indicate the position and extension of the excitation focal lines of Fig. 2. All z-stacks are composed of 50 images taken in steps of 2 μm. Scale bars: 50 μm.

Figure 7
Figure 7

Normalized profiles along a selected line taken from approximately the same optical section for all the DSLM modalities. Optical sections showing the same ROI and the selected line in yellow for a) DSLM with Gaussian beams, b) 2p-DSLM with Gaussian beams, c) DSLM with Bessel beams, and d) 2p-DSLM with Bessel beams. Plots of the intensity profiles along the selected lines are shown in e). Intensity of each plot has been normalized to its respective maximum value.

Tables (3)

Tables Icon

Table 1 Summary of the FWHM widths measured for the light lines (Fig. 2) and for the PSFs of the system (Fig. 3). An error of one pixel was assumed on the light lines. The mean value and statistical error in the PSFs was obtained over 30 measurements.

Tables Icon

Table 2 Summary of the experimental parameters employed to collect the data supporting Fig. 4

Tables Icon

Table 3 Summary of the experimental parameters employed to collect the data supporting Fig. 6

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