Abstract

We demonstrate a simple scanless two-photon (2p) excited fluorescence microscope based on selective plane illumination microscopy (SPIM). Optical sectioning capability is presented and depth-resolved imaging of cameleon protein in C. elegans pharyngeal muscle is implemented.

© 2010 OSA

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  1. Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001).
    [CrossRef] [PubMed]
  2. M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
    [CrossRef] [PubMed]
  3. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
    [CrossRef] [PubMed]
  4. A. Egner and S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000).
    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  15. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  16. P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
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  22. H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77(4), 2226–2236 (1999).
    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
  26. Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
    [CrossRef] [PubMed]
  27. J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999).
    [CrossRef] [PubMed]
  28. M. Takenaka, T. Horiuchi, and R. Yanagimachi, “Effects of light on development of mammalian zygotes,” Proc. Natl. Acad. Sci. U.S.A. 104(36), 14289–14293 (2007).
    [CrossRef] [PubMed]

2009

B. O. Watson, V. Nikolenko, and R. Yuste, “Two-photon imaging with diffractive optical elements,” Front Neural Circuits 3, 6 (2009).
[PubMed]

E. Papagiakoumou, V. de Sars, V. Emiliani, and D. Oron, “Temporal focusing with spatially modulated excitation,” Opt. Express 17(7), 5391–5401 (2009).
[CrossRef] [PubMed]

2008

P. J. Keller and E. H. 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]

P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008).
[CrossRef] [PubMed]

T. F. Holekamp, D. Turaga, and T. E. Holy, “Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy,” Neuron 57(5), 661–672 (2008).
[CrossRef] [PubMed]

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008).
[CrossRef]

2007

M. Takenaka, T. Horiuchi, and R. Yanagimachi, “Effects of light on development of mammalian zygotes,” Proc. Natl. Acad. Sci. U.S.A. 104(36), 14289–14293 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

2006

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing for axial scanning,” Opt. Express 14(25), 12243–12254 (2006).
[CrossRef] [PubMed]

2005

2004

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2003

2002

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
[CrossRef] [PubMed]

2001

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001).
[CrossRef] [PubMed]

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[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,” Neuron 26(3), 583–594 (2000).
[CrossRef] [PubMed]

A. Egner and S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000).
[CrossRef]

1999

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77(4), 2226–2236 (1999).
[CrossRef] [PubMed]

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999).
[CrossRef] [PubMed]

K. König, T. W. Becker, P. Fischer, I. Riemann, and K. J. Halbhuber, “Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,” Opt. Lett. 24(2), 113–115 (1999).
[CrossRef]

K. H. Kim, C. Buehler, and P. T. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38(28), 6004–6009 (1999).
[CrossRef]

1998

1996

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

1995

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
[CrossRef] [PubMed]

1994

E. H. K. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111(5-6), 536–547 (1994).
[CrossRef]

Antolini, R.

Araya, R.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008).
[CrossRef]

Athey, B.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

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,” Neuron 26(3), 583–594 (2000).
[CrossRef] [PubMed]

Baur, D.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77(4), 2226–2236 (1999).
[CrossRef] [PubMed]

Bavister, B. D.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999).
[CrossRef] [PubMed]

Becker, T. W.

Berns, M. W.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
[CrossRef] [PubMed]

Bliton, A. C.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Brakenhoff, G. J.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Buehler, C.

Cahalan, M. D.

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
[CrossRef] [PubMed]

Callamaras, N.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001).
[CrossRef] [PubMed]

Chapman, C. F.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
[CrossRef] [PubMed]

Cheng, D. K.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
[CrossRef] [PubMed]

Choudhury, A.

de Sars, V.

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Durst, M. E.

Egner, A.

Emiliani, V.

Fischer, P.

Froner, E.

Greger, K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Halbhuber, K. J.

Hell, S. W.

Holekamp, T. F.

T. F. Holekamp, D. Turaga, and T. E. Holy, “Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy,” Neuron 57(5), 661–672 (2008).
[CrossRef] [PubMed]

Holy, T. E.

T. F. Holekamp, D. Turaga, and T. E. Holy, “Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy,” Neuron 57(5), 661–672 (2008).
[CrossRef] [PubMed]

Hopt, A.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

Horiuchi, T.

M. Takenaka, T. Horiuchi, and R. Yanagimachi, “Effects of light on development of mammalian zygotes,” Proc. Natl. Acad. Sci. U.S.A. 104(36), 14289–14293 (2007).
[CrossRef] [PubMed]

Hsieh, C.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001).
[CrossRef] [PubMed]

Huisken, J.

P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Keller, P. J.

P. J. Keller and E. H. 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]

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,” Neuron 26(3), 583–594 (2000).
[CrossRef] [PubMed]

Kim, K. H.

Koester, H. J.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77(4), 2226–2236 (1999).
[CrossRef] [PubMed]

König, K.

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,” Neuron 26(3), 583–594 (2000).
[CrossRef] [PubMed]

Lindek, S.

E. H. K. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111(5-6), 536–547 (1994).
[CrossRef]

Liu, Y.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
[CrossRef] [PubMed]

Liu, Z.

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Miller, M. J.

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
[CrossRef] [PubMed]

Neher, E.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

Nguyen, Q. T.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001).
[CrossRef] [PubMed]

Nikolenko, V.

B. O. Watson, V. Nikolenko, and R. Yuste, “Two-photon imaging with diffractive optical elements,” Front Neural Circuits 3, 6 (2009).
[PubMed]

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008).
[CrossRef]

Norris, T.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Oron, D.

Pampaloni, F.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Papagiakoumou, E.

Parker, I.

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
[CrossRef] [PubMed]

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001).
[CrossRef] [PubMed]

Pavone, F. S.

Peterka, D. S.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008).
[CrossRef]

Riemann, I.

Sacconi, L.

Sahai-Hernandez, P.

P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008).
[CrossRef] [PubMed]

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,” Neuron 26(3), 583–594 (2000).
[CrossRef] [PubMed]

Scherz, P. J.

P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008).
[CrossRef] [PubMed]

Schönle, A.

Shi, K.

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006).
[CrossRef] [PubMed]

Silberberg, Y.

So, P. T.

Sonek, G. J.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995).
[CrossRef] [PubMed]

Squier, J.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996).
[CrossRef] [PubMed]

Squirrell, J. M.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999).
[CrossRef] [PubMed]

Stainier, D. Y.

P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008).
[CrossRef] [PubMed]

Stelzer, E. H.

P. J. Keller and E. H. 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]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Stelzer, E. H. K.

E. H. K. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111(5-6), 536–547 (1994).
[CrossRef]

Swoger, J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Taghizadeh, M. R.

Takenaka, M.

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J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999).
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Appl. Opt.

Biophys. J.

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[CrossRef] [PubMed]

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

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J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999).
[CrossRef] [PubMed]

Nat. Methods

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

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[CrossRef] [PubMed]

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Science

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002).
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Supplementary Material (1)

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Figures (5)

Fig. 1
Fig. 1

Schematic diagram of the 2p-SPIM experimental setup.

Fig. 2
Fig. 2

Calculated FWHM of TPEF axial and transversal PSF as a function of the illumination numerical aperture of cylindrical lens 2 (see Fig. 1) for two-photon excitation wavelength of 860 nm.

Fig. 3
Fig. 3

A: Pseudocolor 2p-SPIM fluorescence image of Coumarin 540 dye in methanol. Depicted also are the horizontal (B) and vertical (C) PSF obtained from cross-sectional intensity profiles (green lines). We measured the two-photon illumination length (δx) and height (δy) as 268 µm and 21.9 µm, respectively. D: Transversal PSF of the two-photon plane illumination obtained from z-section intensity profile of a typical XZ pseudocolor image (D, inset) of a 0.028 µm fluorescent bead excited. An average thickness (δz) of 3.4 ± 0.2 µm (n = 5) were measured. Image dimensions: (A) 590 µm × 150 µm (X × Y), (D, inset) 4 µm × 16 µm (X × Z).

Fig. 4
Fig. 4

Upper: 2p-SPIM fluorescence image section of cameleon-expressing in C. elegans pharyngeal muscles. Lower: Overlay of the 2p-SPIM fluorescence (cyan) and brightfield image of anterior of C. elegans depicting colocalization between the fluorescence signal and the pharynx. Also indicated are the distinct structures in the fluorescence image associated with the known anatomy of C. elegans. BC: bucal cavity, PC: procorpus, MC: metacarpus, IS: isthmus, TB: terminal bulb. Scale bars: 50 µm.

Fig. 5
Fig. 5

A to J: 2p-SPIM fluorescence optical sections of cameleon in C. elegans pharyngeal muscle at 4 µm depth intervals clearly depicting the optical sectioning capability of 2p-SPIM. K: Movie showing the reconstructed three-dimensional structure of the C. elegans pharynx (Media 1). Scale bars: 50 µm.

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