Abstract

We present a beam splitter mask that can be easily added to a multiphoton raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.

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

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  1. R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
    [Crossref]
  2. R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
    [Crossref]
  3. J. Xing, J. Kim, and H. Yoo, “Design and fabrication of an optical probe with a phase filter for extended depth of focus,” Opt. Express 24(2), 1037–1044 (2016).
    [Crossref]
  4. L. Liu, F. Diaz, L. Wang, B. Loiseaux, J.-P. Huignard, C. J. R. Sheppard, and N. Chen, “Superresolution along extended depth of focus with binary-phase filters for the Gaussian beam,” J. Opt. Soc. Am. A 25(8), 2095–2101 (2008).
    [Crossref]
  5. S. Ryu and C. Joo, “Design of binary phase filters for depth-of-focus extension via binarization of axisymmetric aberrations,” Opt. Express 25(24), 30312–30326 (2017).
    [Crossref]
  6. P. Dufour, M. Piché, Y. D. Koninck, and N. McCarthy, “Two-photon excitation fluorescence microscopy with a high depth of field using an axicon,” Appl. Opt. 45(36), 9246–9252 (2006).
    [Crossref]
  7. G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
    [Crossref]
  8. S. Abrahamsson, S. Usawa, and M. Gustafsson, “A new approach to extended focus for high-speed high-resolution biological microscopy,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII (International Society for Optics and Photonics, 2006), 6090, p. 60900N.
  9. C. W. McCutchen, “Generalized Aperture and the Three-Dimensional Diffraction Image,” J. Opt. Soc. Am. 54(2), 240–244 (1964).
    [Crossref]
  10. J. D. Manton and E. J. Rees, “triSPIM: light sheet microscopy with isotropic super-resolution,” Opt. Lett. 41(18), 4170–4173 (2016).
    [Crossref]
  11. J. D. Manton, “Matlab simulations for extended depth of focus point spread functions,” figshare (2020), https://doi.org/10.6084/m9.figshare.12049020
  12. G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163(1-3), 95–102 (1999).
    [Crossref]
  13. T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
    [Crossref]
  14. N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
    [Crossref]

2020 (1)

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

2019 (1)

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

2017 (2)

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

S. Ryu and C. Joo, “Design of binary phase filters for depth-of-focus extension via binarization of axisymmetric aberrations,” Opt. Express 25(24), 30312–30326 (2017).
[Crossref]

2016 (2)

2014 (1)

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

2008 (2)

2006 (1)

1999 (1)

G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163(1-3), 95–102 (1999).
[Crossref]

1964 (1)

Abrahamsson, S.

S. Abrahamsson, S. Usawa, and M. Gustafsson, “A new approach to extended focus for high-speed high-resolution biological microscopy,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII (International Society for Optics and Photonics, 2006), 6090, p. 60900N.

Betzig, E.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[Crossref]

Bezprozvanny, I.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Bierfeld, J.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Castonguay, A.

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

Chakraborty, T.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Chang, B.-J.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Chen, N.

Cottet, M.

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

Danuser, G.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

De Koninck, Y.

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

De La Cruz, E. D.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Dean, K. M.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Diaz, F.

Driscoll, M. K.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Dufour, P.

Fiolka, R.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Fitzpatrick, D.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Ghosh, G.

G. Ghosh, “Dispersion-equation coefficients for the refractive index and birefringence of calcite and quartz crystals,” Opt. Commun. 163(1-3), 95–102 (1999).
[Crossref]

Gustafsson, M.

S. Abrahamsson, S. Usawa, and M. Gustafsson, “A new approach to extended focus for high-speed high-resolution biological microscopy,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII (International Society for Optics and Photonics, 2006), 6090, p. 60900N.

Heintzmann, R.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Hiremath, C.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Huignard, J.-P.

Jeffery, E.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Ji, N.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[Crossref]

Joo, C.

Kerlin, A.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Kim, J.

Koninck, Y. D.

Koyama, M.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Liang, Y.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Liu, L.

Loiseaux, B.

Lu, R.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Magee, J. C.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[Crossref]

Manton, J. D.

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

J. D. Manton, “Matlab simulations for extended depth of focus point spread functions,” figshare (2020), https://doi.org/10.6084/m9.figshare.12049020

Marciano, D. K.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

McCarthy, N.

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

P. Dufour, M. Piché, Y. D. Koninck, and N. McCarthy, “Two-photon excitation fluorescence microscopy with a high depth of field using an axicon,” Appl. Opt. 45(36), 9246–9252 (2006).
[Crossref]

McCutchen, C. W.

Meeks, J. P.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Meng, G.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

Mohar, B.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Morrison, S. J.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Murphy, M. M.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Nielson, C. D.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Orger, M. B.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Paninski, L.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

Piché, M.

Rees, E. J.

Roudot, P.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Ryu, S.

Scholl, B.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Seelig, J. D.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Sheppard, C. J. R.

Sun, W.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Svoboda, K.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

Tanimoto, M.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Thériault, G.

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

Tomer, R.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Usawa, S.

S. Abrahamsson, S. Usawa, and M. Gustafsson, “A new approach to extended focus for high-speed high-resolution biological microscopy,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII (International Society for Optics and Photonics, 2006), 6090, p. 60900N.

Vora, S.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Wang, L.

Wilson, D. E.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

Wong, W. M.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Xing, J.

Yi, Y.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Yoo, H.

Zhang, H.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Zhao, H.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Zhemkov, V.

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

Zhou, P.

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

Appl. Opt. (1)

Front. Cell. Neurosci. (1)

G. Thériault, M. Cottet, A. Castonguay, N. McCarthy, and Y. De Koninck, “Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging,” Front. Cell. Neurosci. 8, 44 (2014).
[Crossref]

J. Opt. Soc. Am. (1)

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

Nat. Methods (3)

T. Chakraborty, M. K. Driscoll, E. Jeffery, M. M. Murphy, P. Roudot, B.-J. Chang, S. Vora, W. M. Wong, C. D. Nielson, H. Zhang, V. Zhemkov, C. Hiremath, E. D. De La Cruz, Y. Yi, I. Bezprozvanny, H. Zhao, R. Tomer, R. Heintzmann, J. P. Meeks, D. K. Marciano, S. J. Morrison, G. Danuser, K. M. Dean, and R. Fiolka, “Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution,” Nat. Methods 16(11), 1109–1113 (2019).
[Crossref]

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[Crossref]

R. Lu, Y. Liang, G. Meng, P. Zhou, K. Svoboda, L. Paninski, and N. Ji, “Rapid mesoscale volumetric imaging of neural activity with synaptic resolution,” Nat. Methods 17(3), 291–294 (2020).
[Crossref]

Nat. Neurosci. (1)

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, M. Koyama, D. Fitzpatrick, M. B. Orger, and N. Ji, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20(4), 620–628 (2017).
[Crossref]

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[Crossref]

Opt. Express (2)

Opt. Lett. (1)

Other (2)

S. Abrahamsson, S. Usawa, and M. Gustafsson, “A new approach to extended focus for high-speed high-resolution biological microscopy,” in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII (International Society for Optics and Photonics, 2006), 6090, p. 60900N.

J. D. Manton, “Matlab simulations for extended depth of focus point spread functions,” figshare (2020), https://doi.org/10.6084/m9.figshare.12049020

Supplementary Material (1)

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Figures (9)

Fig. 1.
Fig. 1. Principle of extended depth of focus for ultrafast laser pulses. (a) Schematic of the beam splitter mask consisting of multiple glass disks of varying diameter. (b) Illustration of the working principle. An ultrafast laser pulse (red disk) is incident on the beam splitter mask. Portions of the pulse travelling through the different annular zones are time delayed into separate beamlets (red annuli). Each beamlet forms a focus at the front focal plane of the objective at a different time. (c) Numerical simulations of the squared intensity of the laser foci produced by the different beamlets. Numbers label annular zones, with 1 the innermost zone and 5 the outermost one. The effective EDF point spread function is the incoherent sum of all five foci. Scale bar: 10 um.
Fig. 2.
Fig. 2. Geometric considerations for calculating the depth-of-focus. (a) The Ewald sphere wavevector construction for a high-NA objective lens (NA1), with a subset of wavevectors highlighted in cyan. α1, α2 angles defining the selected subset, k wavenumber, λ0 wavelength, kX component of wavevector in x direction, kZ component of wavevector in z direction (b) Mapping the pupil mask zones onto the Ewald sphere via the sine condition.
Fig. 3.
Fig. 3. Numerical simulation of PSFs. (a), from top to bottom: high NA PSF using an NA of 0.67 and 900 nm wavelength. Middle: EDF PSF. Bottom: low NA PSF to match axial extent of the EDF PSF. The low NA corresponds to 0.3. (b-c) Axial and lateral cross-sections through the PSFs shown above.
Fig. 4.
Fig. 4. Schematic diagram of the experimental setup. 1, Pockel cell, 350-80-2, Conoptics; 2, Lens, AC254-50-B, Thorlabs; 3, Pinhole, PH-100, Newport; 4, Lens, AC254-300-B, Thorlabs; 5, Iris; 6, Beam splitter mask, A12802-35-040, Hamamatsu; 7, Lens, AC254-125-B, Thorlabs; 8, Lens, AC254-75-B, Thorlabs; 9 and 12, Galvo mirror, 6215H, Cambridge technology; 10 and 11, Plössl lens, F-75 mm; 13, Scan Lens, SL50-2P2, Thorlabs; 14, Tube Lens, TL200-2P2, Thorlabs; 15, Dichroic Mirror, FF735-Di02-50.8-D, Semrock; 16, Objective, XLPLN25XWMP2, Olympus; 17, Filters, FF02-694/sp-25, FF01-527/70-25, Semrock; 18, Lens, AC254-45-A, Thorlabs; 19, PMT, H7422-40, Hamamatsu.
Fig. 5.
Fig. 5. Point spread functions measured with 200 nm green fluorescent beads. (a) Axial cross-sectional views of (top) high NA PSF for a laser focus using an NA of 0.67 and no beam splitter mask; (middle) PSF using an EDF beam splitter mask; (bottom) Low NA PSF corresponding to the innermost zone of the beam splitter mask. The low NA corresponded to 0.3 (b-c) Axial and lateral profiles through the point spread functions of high NA PSF without beam splitter mask (black), EDF PSF using the beam splitter mask (red) and low NA laser focus (blue).
Fig. 6.
Fig. 6. Volumetric two-photon imaging of Thy1-GFP labelled neurons in fixed brain tissue using either the beam splitter mask or conventional two-photon laser scanning with z-stepping with an excitation NA of 0.67. (a) Single frame acquired with the beam splitter mask. (b) Projection of a 3D volume acquired with conventional laser scanning over 16 z-planes spanning a range of 22.5 microns. Color encodes z-position. (c) Magnified view of the boxed region of panel (a). (d) Magnified region of the boxed region of panel (b). (e-g) Individual z-planes from the region shown in panel (d) at 22.5 um, 10.5 um and 0 um depth. Arrows mark selected cell bodies that appear at these distinct depths.
Fig. 7.
Fig. 7. Cross-sectional profiles of the PSFs shown in Fig. 1(c), centered at the waist of each beam. (a-e) Lateral profiles of PSFs arising from zone 1 through 5 of the beam splitter mask. (f) Corresponding profile of the EDF PSF. The intensities in (a-e) were normalized to the highest peak intensity of these five beams (profile shown in c). The EDF PSF was normalized separately to its own peak intensity.
Fig. 8.
Fig. 8. Point spread functions measured with 200nm green fluorescent beads. (a) Axial cross-sectional views from top to bottom: PSF for laser focus using an NA of 1.05 and no beam splitter mask; EDF PSF using the beam splitter mask placed directly in front of the galvo mirror. (b-c) Axial and lateral profiles through the point spread functions. Black curves correspond to the high NA PSF without EDF mask and the blue curves correspond to the PSF using the beam splitter mask with no relay telescope (No-RL).
Fig. 9.
Fig. 9. Point spread functions measured with 200nm green fluorescent beads. (a) Axial cross-sectional views; (top) a laser focus using an NA of 0.9 and no beam splitter mask; (middle) EDF PSF using the beam splitter mask; (bottom) Low NA PSF corresponding to the innermost zone of the beam splitter mask. The NA corresponded to 0.4. (b-c) Axial and lateral profiles through the point spread functions of the high NA PSF (black), the EDF PSF using the beam splitter mask (red) and the low NA PSF (blue).

Equations (10)

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d a x i a l = λ o n × ( 1 cos α 2 )
d a x i a l = λ o n × { cos [ sin 1 ( N A 1 n ) ] cos [ sin 1 ( N A 2 n ) ] }
d = λ n ( cos α inner cos α outer )
d = λ n ( cos α n 1 cos α n )
cos α n = cos α n 1 λ n d
Z N = Z 0 + N δ
cos α N = cos α 0 N λ n d
cos α N = 1 N λ n d
d = N λ n ( 1 cos α N )
d = λ n ( 1 cos α N )

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