Abstract

We demonstrate a compact, pulsed diode laser source suitable for multiphoton microscopy of biological samples. The center wavelength is 976 nm, near the peak of the two-photon cross section of common fluorescent markers such as genetically encoded green and yellow fluorescent proteins. The laser repetition rate is electrically tunable between 66.67 kHz and 10 MHz, with 2.3 ps pulse duration and peak powers >1 kW. The laser components are fiber-coupled and scalable to a compact package. We demonstrate >600 μm depth penetration in brain tissue, limited by laser power.

© 2016 Optical Society of America

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2016 (3)

2015 (3)

2014 (2)

2013 (4)

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photon. 7, 205–209 (2013).
[Crossref]

T. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499, 295–300 (2013).
[Crossref] [PubMed]

M. J. R. Heck, J. F. Bauters, M. L. Davenport, J. K. Doylend, S. Jain, G. Kurczveil, S. Srinivasan, Y. Tang, and J. E. Bowers, “Hybrid silicon photonic integrated circuit technology,” IEEE J. Sel. Topics Quantum Electron. 19, 6100117 (2013).
[Crossref]

A. J. Metcalf, V. Torres-Company, D. E. Leaird, and A. M. Weiner, “High-power broadly tunable electrooptic frequency comb generator,” IEEE J. Sel. Top. Quantum Electron. 19, 231–236 (2013).
[Crossref]

2011 (4)

2010 (2)

K. Wang, C. W. Freudiger, J. H. Lee, B. G. Saar, X. S. Xie, and C. Xu, “Synchronized time-lens source for coherent Raman scattering microscopy,” Opt. Express 18, 24019–24024 (2010).
[Crossref]

M. E. Llewellyn, K. R. Thompson, K. Deisseroth, and S. L. Delp, “Orderly recruitment of motor units under optical control in vivo,” Nat. Med. 16, 1161–1165 (2010).
[Crossref] [PubMed]

2009 (3)

2008 (2)

F. R. Ahmad, Y. W. Tseng, M. A. Kats, and F. Rana, “Energy limits imposed by two-photon absorption for pulse amplification in high-power semiconductor optical amplifiers,” Opt. Lett. 33, 1041–1043 (2008).
[Crossref] [PubMed]

D. Träutlein, F. Adler, K. Moutzouris, A. Jeromin, A. Leitenstorfer, and E. Ferrando-May, “Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source,” J. Biophoton. 1, 53–61 (2008).
[Crossref]

2007 (5)

H. Yokoyama, H. Tsubokawa, H. Guo, J. Shikata, K. Sato, K. Takashima, K. Kashiwagi, N. Saito, H. Taniguchi, and H. Ito, “Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source,” J. Biomed. Opt. 12, 054019 (2007).
[Crossref] [PubMed]

J. T. Gopinath, B. Chann, R. K. Huang, C. Harris, J. J. Plant, L. Missaggia, J. P. Donnelly, P. W. Juodawlkis, and D. J. Ripin, “980-nm monolithic passively mode-locked diode lasers with 62 pJ of pulse energy,” IEEE Photon. Tech. Lett. 19, 937–939 (2007).
[Crossref]

K. Taira, T. Hashimoto, and H. Yokoyama, “Two-photon fluorescence imaging with a pulse source based on a 980-nm gain-switched laser diode,” Opt. Express 15, 2454–2458 (2007).
[Crossref] [PubMed]

J. van Howe, J. H. Lee, and C. Xu, “Generation of 3.5 nJ femtosecond pulses from a continuous-wave laser without mode locking,” Opt. Lett. 32, 1408–1410 (2007).
[Crossref]

M. Kuramoto, N. Kitajima, H. Guo, Y. Furushima, M. Ikeda, and H. Yokoyama, “Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source,” Opt. Lett. 32, 2726–2728 (2007).
[Crossref] [PubMed]

2006 (1)

2005 (2)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2932 (2005).
[Crossref] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102, 16807–16812 (2005).
[Crossref]

2003 (2)

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

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 m in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28, 1022–1024 (2003).
[Crossref] [PubMed]

1999 (1)

T. Khayim, M. Yamauchi, D.-S. Kim, and T. Kobayashi, “Femtosecond optical pulse generation from a CW laser using an electrooptic phase modulator featuring lens modulation,” IEEE J. Quantum Electron. 35, 1412–1418 (1999).
[Crossref]

1996 (1)

1989 (1)

Adler, F.

D. Träutlein, F. Adler, K. Moutzouris, A. Jeromin, A. Leitenstorfer, and E. Ferrando-May, “Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source,” J. Biophoton. 1, 53–61 (2008).
[Crossref]

Ahmad, F. R.

Appel, B.

E. G. Hughes and B. Appel, “The cell biology of CNS myelination,” Curr. Opin. Neurobiol. 39, 93–100 (2016).
[Crossref]

Artigas, D.

Aviles-Espinosa, R.

Baohan, A.

T. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499, 295–300 (2013).
[Crossref] [PubMed]

Barbarin, Y.

Bauters, J. F.

M. J. R. Heck, J. F. Bauters, M. L. Davenport, J. K. Doylend, S. Jain, G. Kurczveil, S. Srinivasan, Y. Tang, and J. E. Bowers, “Hybrid silicon photonic integrated circuit technology,” IEEE J. Sel. Topics Quantum Electron. 19, 6100117 (2013).
[Crossref]

Bente, E. A. J. M.

Bernstein, J. G.

C. T. Wentz, J. G. Bernstein, P. Monahan, A. Guerra, A. Rodriguez, and E. S. Boyden, “A wirelessly powered and controlled device for optical neural control of freely-behaving animals,” J. Neural Eng. 8, 046021 (2011).
[Crossref] [PubMed]

Bowers, J. E.

M. J. R. Heck, J. F. Bauters, M. L. Davenport, J. K. Doylend, S. Jain, G. Kurczveil, S. Srinivasan, Y. Tang, and J. E. Bowers, “Hybrid silicon photonic integrated circuit technology,” IEEE J. Sel. Topics Quantum Electron. 19, 6100117 (2013).
[Crossref]

Boyden, E. S.

C. T. Wentz, J. G. Bernstein, P. Monahan, A. Guerra, A. Rodriguez, and E. S. Boyden, “A wirelessly powered and controlled device for optical neural control of freely-behaving animals,” J. Neural Eng. 8, 046021 (2011).
[Crossref] [PubMed]

Bright, V. M.

Cardin, J. A.

M. R. Warden, J. A. Cardin, and K. Deisseroth, “Optical neural interfaces,” Annu. Rev. Biomed. Eng. 16, 103–129 (2014).
[Crossref] [PubMed]

Chann, B.

J. T. Gopinath, B. Chann, R. K. Huang, C. Harris, J. J. Plant, L. Missaggia, J. P. Donnelly, P. W. Juodawlkis, and D. J. Ripin, “980-nm monolithic passively mode-locked diode lasers with 62 pJ of pulse energy,” IEEE Photon. Tech. Lett. 19, 937–939 (2007).
[Crossref]

Chen, T.

T. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499, 295–300 (2013).
[Crossref] [PubMed]

Chen, Z.

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14, 030508 (2009).
[Crossref] [PubMed]

Clark, C. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photon. 7, 205–209 (2013).
[Crossref]

Cormack, R.

Côté, D.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102, 16807–16812 (2005).
[Crossref]

Dai, Y.

Davenport, M. L.

M. J. R. Heck, J. F. Bauters, M. L. Davenport, J. K. Doylend, S. Jain, G. Kurczveil, S. Srinivasan, Y. Tang, and J. E. Bowers, “Hybrid silicon photonic integrated circuit technology,” IEEE J. Sel. Topics Quantum Electron. 19, 6100117 (2013).
[Crossref]

Deisseroth, K.

L. Grosenick, J. H. Marshel, and K. Deisseroth, “Closed-loop and activity-guided optogenetic control,” Neuron 86, 106–139 (2015).
[Crossref] [PubMed]

M. R. Warden, J. A. Cardin, and K. Deisseroth, “Optical neural interfaces,” Annu. Rev. Biomed. Eng. 16, 103–129 (2014).
[Crossref] [PubMed]

M. E. Llewellyn, K. R. Thompson, K. Deisseroth, and S. L. Delp, “Orderly recruitment of motor units under optical control in vivo,” Nat. Med. 16, 1161–1165 (2010).
[Crossref] [PubMed]

Delp, S. L.

M. E. Llewellyn, K. R. Thompson, K. Deisseroth, and S. L. Delp, “Orderly recruitment of motor units under optical control in vivo,” Nat. Med. 16, 1161–1165 (2010).
[Crossref] [PubMed]

Denk, W.

Donnelly, J. P.

J. T. Gopinath, B. Chann, R. K. Huang, C. Harris, J. J. Plant, L. Missaggia, J. P. Donnelly, P. W. Juodawlkis, and D. J. Ripin, “980-nm monolithic passively mode-locked diode lasers with 62 pJ of pulse energy,” IEEE Photon. Tech. Lett. 19, 937–939 (2007).
[Crossref]

Doylend, J. K.

M. J. R. Heck, J. F. Bauters, M. L. Davenport, J. K. Doylend, S. Jain, G. Kurczveil, S. Srinivasan, Y. Tang, and J. E. Bowers, “Hybrid silicon photonic integrated circuit technology,” IEEE J. Sel. Topics Quantum Electron. 19, 6100117 (2013).
[Crossref]

Dunn, A. K.

Durst, M. E.

Evans, C. L.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102, 16807–16812 (2005).
[Crossref]

Fernée, D. C.

Ferrando-May, E.

D. Träutlein, F. Adler, K. Moutzouris, A. Jeromin, A. Leitenstorfer, and E. Ferrando-May, “Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source,” J. Biophoton. 1, 53–61 (2008).
[Crossref]

Filippidis, G.

Freudiger, C. W.

Furushima, Y.

Gibson, E. A.

Goldak, J. R.

Gopinath, J. T.

B. N. Ozbay, J. T. Losacco, R. Cormack, R. Weir, V. M. Bright, J. T. Gopinath, D. Restrepo, and E. A. Gibson, “Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts,” Opt. Lett. 40, 2553–2556 (2015).
[Crossref]

J. T. Gopinath, B. Chann, R. K. Huang, C. Harris, J. J. Plant, L. Missaggia, J. P. Donnelly, P. W. Juodawlkis, and D. J. Ripin, “980-nm monolithic passively mode-locked diode lasers with 62 pJ of pulse energy,” IEEE Photon. Tech. Lett. 19, 937–939 (2007).
[Crossref]

Grosenick, L.

L. Grosenick, J. H. Marshel, and K. Deisseroth, “Closed-loop and activity-guided optogenetic control,” Neuron 86, 106–139 (2015).
[Crossref] [PubMed]

Guerra, A.

C. T. Wentz, J. G. Bernstein, P. Monahan, A. Guerra, A. Rodriguez, and E. S. Boyden, “A wirelessly powered and controlled device for optical neural control of freely-behaving animals,” J. Neural Eng. 8, 046021 (2011).
[Crossref] [PubMed]

Guo, H.

Guo, X.

Hamilton, C.

Harris, C.

J. T. Gopinath, B. Chann, R. K. Huang, C. Harris, J. J. Plant, L. Missaggia, J. P. Donnelly, P. W. Juodawlkis, and D. J. Ripin, “980-nm monolithic passively mode-locked diode lasers with 62 pJ of pulse energy,” IEEE Photon. Tech. Lett. 19, 937–939 (2007).
[Crossref]

Hasan, M. T.

Hashimoto, T.

Heck, M. J. R.

M. J. R. Heck, J. F. Bauters, M. L. Davenport, J. K. Doylend, S. Jain, G. Kurczveil, S. Srinivasan, Y. Tang, and J. E. Bowers, “Hybrid silicon photonic integrated circuit technology,” IEEE J. Sel. Topics Quantum Electron. 19, 6100117 (2013).
[Crossref]

Heiss, D.

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2932 (2005).
[Crossref] [PubMed]

Horton, N. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photon. 7, 205–209 (2013).
[Crossref]

Huang, L.

Huang, R. K.

J. T. Gopinath, B. Chann, R. K. Huang, C. Harris, J. J. Plant, L. Missaggia, J. P. Donnelly, P. W. Juodawlkis, and D. J. Ripin, “980-nm monolithic passively mode-locked diode lasers with 62 pJ of pulse energy,” IEEE Photon. Tech. Lett. 19, 937–939 (2007).
[Crossref]

Hughes, E. G.

E. G. Hughes and B. Appel, “The cell biology of CNS myelination,” Curr. Opin. Neurobiol. 39, 93–100 (2016).
[Crossref]

Ikeda, M.

Ishizawa, A.

Ito, H.

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Annu. Rev. Biomed. Eng. (1)

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Biomed. Opt. Express (3)

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IEEE Photon. Tech. Lett. (1)

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J. Biophoton. (1)

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

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W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
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Nat. Med. (1)

M. E. Llewellyn, K. R. Thompson, K. Deisseroth, and S. L. Delp, “Orderly recruitment of motor units under optical control in vivo,” Nat. Med. 16, 1161–1165 (2010).
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Nat. Methods (1)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2932 (2005).
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Nat. Photon. (1)

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photon. 7, 205–209 (2013).
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Nature (1)

T. Chen, T. J. Wardill, Y. Sun, S. R. Pulver, S. L. Renninger, A. Baohan, E. R. Schreiter, R. A. Kerr, M. B. Orger, V. Jayaraman, L. L. Looger, K. Svoboda, and D. S. Kim, “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature 499, 295–300 (2013).
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Neuron (1)

L. Grosenick, J. H. Marshel, and K. Deisseroth, “Closed-loop and activity-guided optogenetic control,” Neuron 86, 106–139 (2015).
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Opt. Express (6)

Opt. Lett. (8)

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Photon. Res. (1)

Proc. Natl. Acad. Sci. U.S.A. (1)

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102, 16807–16812 (2005).
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Figures (4)

Fig. 1
Fig. 1 Laser schematic. A gain-switched diode laser is used as the seed source. Electro-optic intensity and phase modulators (IM and PM) are driven at 18 GHz, generating bursts of chirped pulses. The pulse bursts are amplified in Yb:fiber amplifiers (Pre-amp: pre-amplifier; Power amp: power amplifier) and compressed with a double-passed grating compressor. A spectral filter reduces the amplified spontaneous emission from the pre-amp. Solid lines indicate the optical path; dashed lines indicate electrical paths.
Fig. 2
Fig. 2 (a) Optical spectrum of the laser source. The spectral bandwidth is 0.95 nm (−3 dB). The resolution bandwidth of the optical spectrum analyzer was 0.08 nm. (b) Autocorrelation of the laser source fitted to a sech2 pulse shape (dashed line). The wings of the pulse indicate uncompensated higher order dispersion. The extended autocorrelation (inset) shows the burst duration of ∼100 ps. Pulse bursts include several pulses of 2.3 ps duration.
Fig. 3
Fig. 3 Two-photon laser scanning microscope schematic. The laser beam is scanned across the sample using galvonometric mirrors and the combination of scan and tube lenses. The microscope objective focuses the the excitation laser and collects the fluorescence light. Fluorescence is separated from the excitation laser using a dichroic mirror and color filter and detected using a photon-counting photo-multiplier tube (PMT).
Fig. 4
Fig. 4 Two-photon fluorescence images of GFP-labeled oligodendrocytes in ex vivo mouse brain slice. (a) Three-dimensional reconstruction. The z-axis corresponds to depth into the tissue. (b) Individual images at specified depths. The laser repetition rate was 10 MHz, and the power at the sample was 26 mW. The dwell time was increased to compensate for the attenuation of the tissue. The images were individually normalized and processed with a median filter of 0.5 pixel radius. The yz-projection was bandpass filtered to remove horizontal striping. The full scale is ∼320 μm in x and y dimensions and 700 μm in the z-dimension. The scale bar is 80 μm.

Equations (1)

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N exc P ave 2 f τ ,

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