Abstract

Temporal focusing of ultrashort pulses has been shown to enable wide-field depth-resolved two-photon fluorescence microscopy. In this process, an entire plane in the sample is selectively excited by introduction of geometrical dispersion to an ultrashort pulse. Many applications, such as multiphoton lithography, uncaging or region-of-interest imaging, require, however, illumination patterns which significantly differ from homogeneous excitation of an entire plane in the sample. Here we consider the effects of such spatial modulation of a temporally focused excitation pattern on both the generated excitation pattern and on its axial confinement. The transition in the axial response between line illumination and wide-field illumination is characterized both theoretically and experimentally. For 2D patterning, we show that in the case of amplitude-only modulation the axial response is generally similar to that of wide-field illumination, while for phase-and-amplitude modulation the axial response slightly deteriorates when the phase variation is rapid, a regime which is shown to be relevant to excitation by beams shaped using spatial light modulators. Finally, general guidelines for the use of spatially modulated temporally focused excitation are presented.

© 2009 Optical Society of America

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References

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  1. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  2. R. R. Gattass and E. Mazur, "Femtosecond laser micromachining in transparent materials," Nature Photonics 2, 219-225 (2008).
    [CrossRef]
  3. X. Lv, C. Zhan, S. Zeng,W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Intr. 77, 046101 (2006) and refernces therein.
    [CrossRef]
  4. G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
    [CrossRef]
  5. B. Judkewitz, A. Roth, and M. Hausser, "Dendritic enlightenment: using patterned two-photon uncaging to reveal the secrets of the brain’s smallest dendrites," Neuron 50, 180-183 (2006)
    [CrossRef] [PubMed]
  6. A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
    [CrossRef]
  7. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998)
    [CrossRef]
  8. D. N. Fittinghoff, P. W. Wiseman, and J. A. Squier, "Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy," Opt. Express 7, 273-279 (2000).
    [CrossRef] [PubMed]
  9. T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
    [CrossRef] [PubMed]
  10. V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
    [CrossRef] [PubMed]
  11. C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  14. G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, "Simultaneous spatial and temporal focusing of femtosecond pulses," Opt. Express 13, 2153-2159 (2005).
    [CrossRef] [PubMed]
  15. E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses," Opt. Express 16, 22039-22047 (2008).
    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
  19. C. Ventalon and J. Mertz, "Quasi-confocal fluorescence sectioning with dynamic speckle illumination," Opt. Lett. 30, 3350-3352 (2005).
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    [CrossRef] [PubMed]
  22. H. Suchowski, D. Oron, and Y. Silberberg, "Generation of a dark nonlinear focus by spatiotemporal coherent control," Opt. Commun. 264, 482-487 (2006).
    [CrossRef]

2008

R. R. Gattass and E. Mazur, "Femtosecond laser micromachining in transparent materials," Nature Photonics 2, 219-225 (2008).
[CrossRef]

G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
[CrossRef]

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses," Opt. Express 16, 22039-22047 (2008).
[CrossRef] [PubMed]

2007

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

2006

H. Suchowski, D. Oron, and Y. Silberberg, "Generation of a dark nonlinear focus by spatiotemporal coherent control," Opt. Commun. 264, 482-487 (2006).
[CrossRef]

B. Judkewitz, A. Roth, and M. Hausser, "Dendritic enlightenment: using patterned two-photon uncaging to reveal the secrets of the brain’s smallest dendrites," Neuron 50, 180-183 (2006)
[CrossRef] [PubMed]

E. Tal and Y. Silberberg, "Transformation from an ultrashort pulse to a spatiotemporal speckle by a thin scattering surface," Opt. Lett. 31, 3529-3531 (2006).
[CrossRef] [PubMed]

2005

2001

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

2000

1998

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998)
[CrossRef]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

1995

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, 253-259 (1995).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Andresen, P.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

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, 253-259 (1995).
[CrossRef]

Bewersdorf, J.

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, 253-259 (1995).
[CrossRef]

Brakenhoff, G. J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

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, 253-259 (1995).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Charpak, S.

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

de Sars, V.

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses," Opt. Express 16, 22039-22047 (2008).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

DiGregorio, D. A.

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

Durst, M.

Emiliani, V.

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, "Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses," Opt. Express 16, 22039-22047 (2008).
[CrossRef] [PubMed]

Fink, R.

G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
[CrossRef]

Fittinghoff, D. N.

Fricke, M.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

Gattass, R. R.

R. R. Gattass and E. Mazur, "Femtosecond laser micromachining in transparent materials," Nature Photonics 2, 219-225 (2008).
[CrossRef]

Hausser, M.

B. Judkewitz, A. Roth, and M. Hausser, "Dendritic enlightenment: using patterned two-photon uncaging to reveal the secrets of the brain’s smallest dendrites," Neuron 50, 180-183 (2006)
[CrossRef] [PubMed]

Hell, S. W.

Hellweg, D.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

Judkewitz, B.

B. Judkewitz, A. Roth, and M. Hausser, "Dendritic enlightenment: using patterned two-photon uncaging to reveal the secrets of the brain’s smallest dendrites," Neuron 50, 180-183 (2006)
[CrossRef] [PubMed]

Kelleher, K.

G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
[CrossRef]

Lutz, C.

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

Lv, X.

X. Lv, C. Zhan, S. Zeng,W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Intr. 77, 046101 (2006) and refernces therein.
[CrossRef]

Mazur, E.

R. R. Gattass and E. Mazur, "Femtosecond laser micromachining in transparent materials," Nature Photonics 2, 219-225 (2008).
[CrossRef]

Mertz, J.

Muller, M.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Nielsen, T.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

Nikolenko, V.

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

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, 253-259 (1995).
[CrossRef]

Oron, D.

Otis, T. S.

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

Papagiakoumou, E.

Pick, R.

Poskanzer, K. E.

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

Reddy, G. D.

G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
[CrossRef]

Roth, A.

B. Judkewitz, A. Roth, and M. Hausser, "Dendritic enlightenment: using patterned two-photon uncaging to reveal the secrets of the brain’s smallest dendrites," Neuron 50, 180-183 (2006)
[CrossRef] [PubMed]

Saggau, P.

G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
[CrossRef]

Silberberg, Y.

Squier, J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

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, 253-259 (1995).
[CrossRef]

Squier, J. A.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Suchowski, H.

H. Suchowski, D. Oron, and Y. Silberberg, "Generation of a dark nonlinear focus by spatiotemporal coherent control," Opt. Commun. 264, 482-487 (2006).
[CrossRef]

Tal, E.

van Howe, J.

Ventalon, C.

Wade, M. H.

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, 253-259 (1995).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Wiseman, P. W.

Xu, C.

Yuste, R.

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

Zeng, S.

X. Lv, C. Zhan, S. Zeng,W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Intr. 77, 046101 (2006) and refernces therein.
[CrossRef]

Zhan, C.

X. Lv, C. Zhan, S. Zeng,W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Intr. 77, 046101 (2006) and refernces therein.
[CrossRef]

Zhu, G.

Zipfel, W.

J. Microsc.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real-time two-photon absorption microscopy using multipoint excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

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, 253-259 (1995).
[CrossRef]

J. Opt. Soc. Am. B

Nat. Methods

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

C. Lutz, T. S. Otis, V. de Sars, S. Charpak, D. A. DiGregorio, and V. Emiliani, "Holographic photolysis of caged neurotransmitters," Nat. Methods 5, 821-827 (2008)
[CrossRef]

Nature Neuroscience

G. D. Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nature Neuroscience 11, 713-720 (2008).
[CrossRef]

Nature Photonics

R. R. Gattass and E. Mazur, "Femtosecond laser micromachining in transparent materials," Nature Photonics 2, 219-225 (2008).
[CrossRef]

Neuron

B. Judkewitz, A. Roth, and M. Hausser, "Dendritic enlightenment: using patterned two-photon uncaging to reveal the secrets of the brain’s smallest dendrites," Neuron 50, 180-183 (2006)
[CrossRef] [PubMed]

Opt. Commun.

H. Suchowski, D. Oron, and Y. Silberberg, "Generation of a dark nonlinear focus by spatiotemporal coherent control," Opt. Commun. 264, 482-487 (2006).
[CrossRef]

Opt. Express

Opt. Lett.

Science

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Other

X. Lv, C. Zhan, S. Zeng,W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Intr. 77, 046101 (2006) and refernces therein.
[CrossRef]

J.W. Goodman, Introduction to Fourier Optics, third edition (Roberts and Company, Greenwood Village, 2005).

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Figures (7)

Fig. 1.
Fig. 1.

Setup used for temporal focusing. The grating is aligned perpendicular to the optic axis of a high magnification telescope comprised of a lens and a microscope objective. The excitation beam impinges on the grating at an angle α such that the center wavelength is diffracted towards the optical axis. An arbitrary pattern is projected on the grating, either via amplitude or phase-and-amplitude shaping. For the latter, the excitation beam is shaped by a spatial light modulator followed by a Fourier lens.

Fig. 2.
Fig. 2.

Depth response for illumination gratings with varying ratios of ∆y/2Mw 0: 5 (blue); 7.5 (green); 12.5 (red); 20 (cyan). Also plotted is the calculated response for an area (dashed black line) and for a single line (solid black line).

Fig. 3.
Fig. 3.

The effect of phase of lines in an array on the depth response for the case of ∆y/2Mw 0 = 7.5. a) Depth response for constant phase (blue), a holographically generated array (green) and a random phase (red), along with the calculated response for an area (dashed black line) and for a single line (solid black line). b)-d) YZ intensity plots for constant phase (b), a holographically generated array (c) and random phases (d).

Fig. 4.
Fig. 4.

Experimental realization of illumination by equally spaced lines. The depth response is presented for illumination with a holographically generated array of seven lines with varying ratios of ∆y/2Mw 0: 5 (blue) ; 7.5 (green) ; 12.5 (red) ; 20 (cyan). Also plotted is the measured response for a single line (solid black line).

Fig. 5.
Fig. 5.

2PEF intensity patterns (a,c) and calculated local FWHM (b,d) for two cases having constant spatial phase: A circle with sharp edges (a,b) and a circle with random intensity fluctuations (c,d).

Fig. 6.
Fig. 6.

2PEF intensity pattern (a) and calculated depth response (b) for an illumination pattern with random phase. The observed FWHM depth response of 4.5μm (solid blue line) is broader than the 3.3μm FWHM response observed for the case of a constant phase (dashed red line).

Fig. 7.
Fig. 7.

The origins of broadening of the depth response in temporal focusing with random phase. a) Two-dimensional probability histogram of the depth response FWHM and the signal intensity. Strong intensity is correlated with a narrow response. b) Two-dimensional probability histogram of the centroid of the depth response and the signal intensity. The peak of the signal is not necessarily centered at z=0 even for high signal intensities.

Equations (1)

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I 2 PEF ( x , y , z ) = dt A image ( x , y , z , t ) 4

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