Abstract

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.

© 2017 Optical Society of America

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2016 (7)

M. A. Gaafar, A. Rahimi-Iman, K. A. Fedorova, W. Stolz, E. U. Rafailov, and M. Koch, “Mode-locked semiconductor disk lasers,” Adv. Opt. Photonics 8(3), 370–400 (2016).
[Crossref]

E. P. Perillo, J. E. McCracken, D. C. Fernée, J. R. Goldak, F. A. Medina, D. R. Miller, H.-C. Yeh, and A. K. Dunn, “Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser,” Biomed. Opt. Express 7(2), 324–334 (2016).
[Crossref] [PubMed]

D. Waldburger, S. M. Link, M. Mangold, C. G. E. Alfieri, E. Gini, M. Golling, B. W. Tilma, and U. Keller, “High-power 100 fs semiconductor disk lasers,” Optica 3(8), 844–852 (2016).
[Crossref]

K. Podgorski and G. N. Ranganathan, “Brain heating induced by near infrared lasers during multi-photon microscopy,” J. Neurophysiol 116(3) 1012-1023 (2016).

J. L. Chen, F. F. Voigt, M. Javadzadeh, R. Krueppel, and F. Helmchen, “Long-range population dynamics of anatomically defined neocortical networks,” eLife 5, 14679 (2016).
[Crossref] [PubMed]

S. Caviglia, M. Brankatschk, E. J. Fischer, S. Eaton, and S. Luschnig, “Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis,” Nat. Cell Biol. 18(7), 727–739 (2016).
[PubMed]

G.-A. Pilz, S. Carta, A. Stäuble, A. Ayaz, S. Jessberger, and F. Helmchen, “Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus,” J. Neurosci. 36(28), 7407–7414 (2016).
[Crossref] [PubMed]

2015 (5)

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE Multiphoton Microscopy in the Biomedical Sciences XV 93292, 93292 (2015).

M. Mangold, M. Golling, E. Gini, B. W. Tilma, and U. Keller, “Sub-300-femtosecond operation from a MIXSEL,” Opt. Express 23(17), 22043–22059 (2015).
[Crossref] [PubMed]

K. Gürel, V. J. Wittwer, M. Hoffmann, C. J. Saraceno, S. Hakobyan, B. Resan, A. Rohrbacher, K. Weingarten, S. Schilt, and T. Südmeyer, “Green-diode-pumped femtosecond Ti:Sapphire laser with up to 450 mW average power,” Opt. Express 23(23), 30043–30048 (2015).
[Crossref] [PubMed]

A. Miyawaki and Y. Niino, “Molecular Spies for Bioimaging--Fluorescent Protein-Based Probes,” Mol. Cell 58(4), 632–643 (2015).
[Crossref] [PubMed]

B. W. Tilma, M. Mangold, C. A. Zaugg, S. M. Link, D. Waldburger, A. Klenner, A. S. Mayer, E. Gini, M. Golling, and U. Keller, “Recent advances in ultrafast semiconductor disk lasers,” Light Sci. Appl. 4(7), e310 (2015).
[Crossref]

2014 (6)

H. Wang, L. Kong, A. Forrest, D. Bajek, S. E. Haggett, X. Wang, B. Cui, J. Pan, Y. Ding, and M. A. Cataluna, “Ultrashort pulse generation by semiconductor mode-locked lasers at 760 nm,” Opt. Express 22(21), 25940–25946 (2014).
[Crossref] [PubMed]

Y. Kusama, Y. Tanushi, M. Yokoyama, R. Kawakami, T. Hibi, Y. Kozawa, T. Nemoto, S. Sato, and H. Yokoyama, “7-ps optical pulse generation from a 1064-nm gain-switched laser diode and its application for two-photon microscopy,” Opt. Express 22(5), 5746–5753 (2014).
[Crossref] [PubMed]

M. Mangold, S. M. Link, A. Klenner, C. A. Zaugg, M. Golling, B. W. Tilma, and U. Keller, “Amplitude noise and timing jitter characterization of a high-power Mode-Locked Integrated External-Cavity Surface Emitting Laser,” IEEE Photonics J. 6(1), 1–9 (2014).
[Crossref]

B. Resan, R. Aviles-Espinosa, S. Kurmulis, J. Licea-Rodriguez, F. Brunner, A. Rohrbacher, D. Artigas, P. Loza-Alvarez, and K. J. Weingarten, “Two-photon fluorescence imaging with 30 fs laser system tunable around 1 micron,” Opt. Express 22(13), 16456–16461 (2014).
[Crossref] [PubMed]

C. Wang, R. Liu, D. E. Milkie, W. Sun, Z. Tan, A. Kerlin, T. W. Chen, D. S. Kim, and N. Ji, “Multiplexed aberration measurement for deep tissue imaging in vivo,” Nat. Methods 11(10), 1037–1040 (2014).
[Crossref] [PubMed]

C. A. Zaugg, A. Klenner, M. Mangold, A. S. Mayer, S. M. Link, F. Emaury, M. Golling, E. Gini, C. J. Saraceno, B. W. Tilma, and U. Keller, “Gigahertz self-referenceable frequency comb from a semiconductor disk laser,” Opt. Express 22(13), 16445–16455 (2014).
[Crossref] [PubMed]

2013 (2)

2012 (4)

P. G. Antal and R. Szipőcs, “Tunable, low-repetition-rate, cost-efficient femtosecond Ti:sapphire laser for nonlinear microscopy,” Appl. Phys. B 107(1), 17–22 (2012).
[Crossref]

C. G. Durfee, T. Storz, J. Garlick, S. Hill, J. A. Squier, M. Kirchner, G. Taft, K. Shea, H. Kapteyn, M. Murnane, and S. Backus, “Direct diode-pumped Kerr-lens mode-locked Ti:sapphire laser,” Opt. Express 20(13), 13677–13683 (2012).
[Crossref] [PubMed]

C. Grienberger and A. Konnerth, “Imaging Calcium in Neurons,” Neuron 73(5), 862–885 (2012).
[Crossref] [PubMed]

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

2011 (5)

H. Studier, H. G. Breunig, and K. König, “Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear femtosecond-laser scanning microscopy,” J. Biophotonics 4(1), 84–91 (2011).
[Crossref] [PubMed]

R. Aviles-Espinosa, G. Filippidis, C. Hamilton, G. Malcolm, K. J. Weingarten, T. Südmeyer, Y. Barbarin, U. Keller, S. I. C. O. Santos, D. Artigas, and P. Loza-Alvarez, “Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms,” Biomed. Opt. Express 2(4), 739–747 (2011).
[Crossref] [PubMed]

P. Klopp, U. Griebner, M. Zorn, and M. Weyers, “Pulse repetition rate up to 92 GHz or pulse duration shorter than 110 fs from a mode-locked semiconductor disk laser,” Appl. Phys. Lett. 98(7), 071103 (2011).
[Crossref]

X. Chen, U. Leischner, N. L. Rochefort, I. Nelken, and A. Konnerth, “Functional mapping of single spines in cortical neurons in vivo,” Nature 475(7357), 501–505 (2011).
[Crossref] [PubMed]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

2010 (1)

K. G. Wilcox, A. H. Quarterman, H. Beere, D. A. Ritchie, and A. C. Tropper, “High Peak Power Femtosecond Pulse Passively Mode-Locked Vertical-External-Cavity Surface-Emitting Laser,” IEEE Photonics Technol. Lett. 22(14), 1021–1023 (2010).
[Crossref]

2009 (3)

P. Golshani, J. T. Gonçalves, S. Khoshkhoo, R. Mostany, S. Smirnakis, and C. Portera-Cailliau, “Internally mediated developmental desynchronization of neocortical network activity,” J. Neurosci. 29(35), 10890–10899 (2009).
[Crossref] [PubMed]

B. A. Wilt, L. D. Burns, E. T. Wei Ho, K. K. Ghosh, E. A. Mukamel, and M. J. Schnitzer, “Advances in Light Microscopy for Neuroscience,” Annu. Rev. Neurosci. 32(1), 435–506 (2009).
[Crossref] [PubMed]

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).

2008 (2)

2007 (2)

A. Ehlers, I. Riemann, S. Martin, R. Le Harzic, A. Bartels, C. Janke, and K. König, “High (1 GHz) repetition rate compact femtosecond laser: A powerful multiphoton tool for nanomedicine and nanobiotechnology,” J. Appl. Phys. 102(1), 014701 (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(18), 2726–2728 (2007).
[Crossref] [PubMed]

2006 (3)

H. Yokoyama, H. Guo, T. Yoda, K. Takashima, K. Sato, H. Taniguchi, and H. Ito, “Two-photon bioimaging with picosecond optical pulses from a semiconductor laser,” Opt. Express 14(8), 3467–3471 (2006).
[Crossref] [PubMed]

U. Keller and A. C. Tropper, “Passively modelocked surface-emitting semiconductor lasers,” Phys. Rep. 429(2), 67–120 (2006).
[Crossref]

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, “Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy,” J. Biomed. Opt. 11, 020501 (2006).

2005 (2)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

S. Hoogland, A. Garnache, I. Sagnes, J. S. Roberts, and A. C. Tropper, “10-GHz Train of Sub-500-fs Optical Soliton-Like Pulses From a Surface-Emitting Semiconductor Laser,” IEEE Photonics Technol. Lett. 17(2), 267–269 (2005).
[Crossref]

2003 (3)

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

S.-W. Chu, T.-M. Liu, C.-K. Sun, C.-Y. Lin, and H.-J. Tsai, “Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser,” Opt. Express 11(8), 933–938 (2003).
[Crossref] [PubMed]

C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003).
[Crossref] [PubMed]

2002 (1)

A. Garnache, S. Hoogland, A. C. Tropper, I. Sagnes, G. Saint-Girons, and J. S. Roberts, “Sub-500-fs soliton pulse in a passively mode-locked broadband surface-emitting laser with 100-mW average power,” Appl. Phys. Lett. 80(21), 3892–3894 (2002).
[Crossref]

2001 (4)

R. Häring, R. Paschotta, E. Gini, F. Morier-Genoud, D. Martin, H. Melchior, and U. Keller, “Picosecond surface-emitting semiconductor laser with >200 mW average power,” Electron. Lett. 37(12), 766–767 (2001).
[Crossref]

S.-W. Chu, I. H. Chen, T.-M. Liu, P. C. Chen, C.-K. Sun, and B.-L. Lin, “Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser,” Opt. Lett. 26(23), 1909–1911 (2001).
[Crossref] [PubMed]

T.-M. Liu, S.-W. Chu, C.-K. Sun, B.-L. Lin, P.-C. Cheng, and I. Johnson, “Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser,” Scanning 23(4), 249–254 (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 (1)

S. Hoogland, S. Dhanjal, A. C. Tropper, J. S. Roberts, R. Haring, R. Paschotta, F. Morier-Genoud, and U. Keller, “Passively mode-locked diode-pumped surface-emitting semiconductor laser,” IEEE Photonics Technol. Lett. 12(9), 1135–1137 (2000).
[Crossref]

1998 (1)

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[Crossref] [PubMed]

1997 (1)

1996 (1)

1994 (1)

A. Giesen, H. Hügel, A. Voss, K. Wittig, U. Brauch, and H. Opower, “Scalable Concept for Diode-Pumped High-Power Solid-State Lasers,” Appl. Phys. B 58(5), 365–372 (1994).
[Crossref]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

1986 (1)

Alfieri, C. G. E.

Antal, P. G.

P. G. Antal and R. Szipőcs, “Tunable, low-repetition-rate, cost-efficient femtosecond Ti:sapphire laser for nonlinear microscopy,” Appl. Phys. B 107(1), 17–22 (2012).
[Crossref]

Arganda-Carreras, I.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Artigas, D.

Aviles-Espinosa, R.

Ayaz, A.

G.-A. Pilz, S. Carta, A. Stäuble, A. Ayaz, S. Jessberger, and F. Helmchen, “Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus,” J. Neurosci. 36(28), 7407–7414 (2016).
[Crossref] [PubMed]

Backus, S.

Bajek, D.

Barbarin, Y.

Bartels, A.

A. Ehlers, I. Riemann, S. Martin, R. Le Harzic, A. Bartels, C. Janke, and K. König, “High (1 GHz) repetition rate compact femtosecond laser: A powerful multiphoton tool for nanomedicine and nanobiotechnology,” J. Appl. Phys. 102(1), 014701 (2007).
[Crossref]

Beere, H.

K. G. Wilcox, A. H. Quarterman, H. Beere, D. A. Ritchie, and A. C. Tropper, “High Peak Power Femtosecond Pulse Passively Mode-Locked Vertical-External-Cavity Surface-Emitting Laser,” IEEE Photonics Technol. Lett. 22(14), 1021–1023 (2010).
[Crossref]

Beere, H. E.

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] [PubMed]

Brankatschk, M.

S. Caviglia, M. Brankatschk, E. J. Fischer, S. Eaton, and S. Luschnig, “Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis,” Nat. Cell Biol. 18(7), 727–739 (2016).
[PubMed]

Brauch, U.

A. Giesen, H. Hügel, A. Voss, K. Wittig, U. Brauch, and H. Opower, “Scalable Concept for Diode-Pumped High-Power Solid-State Lasers,” Appl. Phys. B 58(5), 365–372 (1994).
[Crossref]

Breunig, H. G.

H. Studier, H. G. Breunig, and K. König, “Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear femtosecond-laser scanning microscopy,” J. Biophotonics 4(1), 84–91 (2011).
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H. Studier, H. G. Breunig, and K. König, “Two-photon imaging with 80 MHz and 1-GHz repetition rate Ti:sapphire oscillators,” Proc. SPIE Multiphoton Microscopy in the Biomedical Sciences X7569 (2010).

Brunner, F.

Burns, L. D.

B. A. Wilt, L. D. Burns, E. T. Wei Ho, K. K. Ghosh, E. A. Mukamel, and M. J. Schnitzer, “Advances in Light Microscopy for Neuroscience,” Annu. Rev. Neurosci. 32(1), 435–506 (2009).
[Crossref] [PubMed]

Cardona, A.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Carriles, R.

Carta, S.

G.-A. Pilz, S. Carta, A. Stäuble, A. Ayaz, S. Jessberger, and F. Helmchen, “Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus,” J. Neurosci. 36(28), 7407–7414 (2016).
[Crossref] [PubMed]

Cataluna, M. A.

Caviglia, S.

S. Caviglia, M. Brankatschk, E. J. Fischer, S. Eaton, and S. Luschnig, “Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis,” Nat. Cell Biol. 18(7), 727–739 (2016).
[PubMed]

Chen, I. H.

Chen, J. L.

J. L. Chen, F. F. Voigt, M. Javadzadeh, R. Krueppel, and F. Helmchen, “Long-range population dynamics of anatomically defined neocortical networks,” eLife 5, 14679 (2016).
[Crossref] [PubMed]

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE Multiphoton Microscopy in the Biomedical Sciences XV 93292, 93292 (2015).

Chen, P. C.

Chen, T. W.

C. Wang, R. Liu, D. E. Milkie, W. Sun, Z. Tan, A. Kerlin, T. W. Chen, D. S. Kim, and N. Ji, “Multiplexed aberration measurement for deep tissue imaging in vivo,” Nat. Methods 11(10), 1037–1040 (2014).
[Crossref] [PubMed]

Chen, X.

X. Chen, U. Leischner, N. L. Rochefort, I. Nelken, and A. Konnerth, “Functional mapping of single spines in cortical neurons in vivo,” Nature 475(7357), 501–505 (2011).
[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).

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, “Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy,” J. Biomed. Opt. 11, 020501 (2006).

Cheng, P.-C.

T.-M. Liu, S.-W. Chu, C.-K. Sun, B.-L. Lin, P.-C. Cheng, and I. Johnson, “Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser,” Scanning 23(4), 249–254 (2001).
[Crossref] [PubMed]

Chu, S.-W.

Cui, B.

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[Crossref] [PubMed]

K. Svoboda, W. H. Knox, S. Tsuda, and W. Denk, “Two-photon-excitation scanning microscopy of living neurons with a saturable Bragg reflector mode-locked diode-pumped Cr:LiSrAlFl laser,” Opt. Lett. 21(17), 1411–1413 (1996).
[Crossref] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Deutsch, M.

Dhanjal, S.

S. Hoogland, S. Dhanjal, A. C. Tropper, J. S. Roberts, R. Haring, R. Paschotta, F. Morier-Genoud, and U. Keller, “Passively mode-locked diode-pumped surface-emitting semiconductor laser,” IEEE Photonics Technol. Lett. 12(9), 1135–1137 (2000).
[Crossref]

Ding, Y.

Drobizhev, M.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Dunn, A. K.

Durfee, C.

M. D. Young, S. Backus, C. Durfee, and J. Squier, “Multiphoton imaging with a direct-diode pumped femtosecond Ti:sapphire laser,” J. Microsc. 249(2), 83–86 (2013).
[Crossref] [PubMed]

Durfee, C. G.

Eaton, S.

S. Caviglia, M. Brankatschk, E. J. Fischer, S. Eaton, and S. Luschnig, “Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis,” Nat. Cell Biol. 18(7), 727–739 (2016).
[PubMed]

Ehlers, A.

A. Ehlers, I. Riemann, S. Martin, R. Le Harzic, A. Bartels, C. Janke, and K. König, “High (1 GHz) repetition rate compact femtosecond laser: A powerful multiphoton tool for nanomedicine and nanobiotechnology,” J. Appl. Phys. 102(1), 014701 (2007).
[Crossref]

Eliceiri, K.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Emaury, F.

Fedorova, K. A.

M. A. Gaafar, A. Rahimi-Iman, K. A. Fedorova, W. Stolz, E. U. Rafailov, and M. Koch, “Mode-locked semiconductor disk lasers,” Adv. Opt. Photonics 8(3), 370–400 (2016).
[Crossref]

Fernée, D. C.

Filippidis, G.

Fischer, E. J.

S. Caviglia, M. Brankatschk, E. J. Fischer, S. Eaton, and S. Luschnig, “Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis,” Nat. Cell Biol. 18(7), 727–739 (2016).
[PubMed]

Forrest, A.

Freund, I.

Frise, E.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Furushima, Y.

Gaafar, M. A.

M. A. Gaafar, A. Rahimi-Iman, K. A. Fedorova, W. Stolz, E. U. Rafailov, and M. Koch, “Mode-locked semiconductor disk lasers,” Adv. Opt. Photonics 8(3), 370–400 (2016).
[Crossref]

Garaschuk, O.

C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003).
[Crossref] [PubMed]

Garlick, J.

Garnache, A.

S. Hoogland, A. Garnache, I. Sagnes, J. S. Roberts, and A. C. Tropper, “10-GHz Train of Sub-500-fs Optical Soliton-Like Pulses From a Surface-Emitting Semiconductor Laser,” IEEE Photonics Technol. Lett. 17(2), 267–269 (2005).
[Crossref]

A. Garnache, S. Hoogland, A. C. Tropper, I. Sagnes, G. Saint-Girons, and J. S. Roberts, “Sub-500-fs soliton pulse in a passively mode-locked broadband surface-emitting laser with 100-mW average power,” Appl. Phys. Lett. 80(21), 3892–3894 (2002).
[Crossref]

Ghosh, K. K.

B. A. Wilt, L. D. Burns, E. T. Wei Ho, K. K. Ghosh, E. A. Mukamel, and M. J. Schnitzer, “Advances in Light Microscopy for Neuroscience,” Annu. Rev. Neurosci. 32(1), 435–506 (2009).
[Crossref] [PubMed]

Giesen, A.

A. Giesen, H. Hügel, A. Voss, K. Wittig, U. Brauch, and H. Opower, “Scalable Concept for Diode-Pumped High-Power Solid-State Lasers,” Appl. Phys. B 58(5), 365–372 (1994).
[Crossref]

Gini, E.

Girkin, J. M.

J. M. Girkin and D. L. Wokosin, “Novel compact sources for multiphoton microscopy,” Proc. SPIE Multiphoton Microscopy in the Biomedical Sciences4262, 186 (2001).

Goldak, J. R.

Golling, M.

D. Waldburger, S. M. Link, M. Mangold, C. G. E. Alfieri, E. Gini, M. Golling, B. W. Tilma, and U. Keller, “High-power 100 fs semiconductor disk lasers,” Optica 3(8), 844–852 (2016).
[Crossref]

M. Mangold, M. Golling, E. Gini, B. W. Tilma, and U. Keller, “Sub-300-femtosecond operation from a MIXSEL,” Opt. Express 23(17), 22043–22059 (2015).
[Crossref] [PubMed]

B. W. Tilma, M. Mangold, C. A. Zaugg, S. M. Link, D. Waldburger, A. Klenner, A. S. Mayer, E. Gini, M. Golling, and U. Keller, “Recent advances in ultrafast semiconductor disk lasers,” Light Sci. Appl. 4(7), e310 (2015).
[Crossref]

M. Mangold, S. M. Link, A. Klenner, C. A. Zaugg, M. Golling, B. W. Tilma, and U. Keller, “Amplitude noise and timing jitter characterization of a high-power Mode-Locked Integrated External-Cavity Surface Emitting Laser,” IEEE Photonics J. 6(1), 1–9 (2014).
[Crossref]

C. A. Zaugg, A. Klenner, M. Mangold, A. S. Mayer, S. M. Link, F. Emaury, M. Golling, E. Gini, C. J. Saraceno, B. W. Tilma, and U. Keller, “Gigahertz self-referenceable frequency comb from a semiconductor disk laser,” Opt. Express 22(13), 16445–16455 (2014).
[Crossref] [PubMed]

Golshani, P.

P. Golshani, J. T. Gonçalves, S. Khoshkhoo, R. Mostany, S. Smirnakis, and C. Portera-Cailliau, “Internally mediated developmental desynchronization of neocortical network activity,” J. Neurosci. 29(35), 10890–10899 (2009).
[Crossref] [PubMed]

Gonçalves, J. T.

P. Golshani, J. T. Gonçalves, S. Khoshkhoo, R. Mostany, S. Smirnakis, and C. Portera-Cailliau, “Internally mediated developmental desynchronization of neocortical network activity,” J. Neurosci. 29(35), 10890–10899 (2009).
[Crossref] [PubMed]

Griebner, U.

P. Klopp, U. Griebner, M. Zorn, and M. Weyers, “Pulse repetition rate up to 92 GHz or pulse duration shorter than 110 fs from a mode-locked semiconductor disk laser,” Appl. Phys. Lett. 98(7), 071103 (2011).
[Crossref]

Grienberger, C.

C. Grienberger and A. Konnerth, “Imaging Calcium in Neurons,” Neuron 73(5), 862–885 (2012).
[Crossref] [PubMed]

Guo, H.

Gürel, K.

Haggett, S. E.

Hakobyan, S.

Hamilton, C.

Haring, R.

S. Hoogland, S. Dhanjal, A. C. Tropper, J. S. Roberts, R. Haring, R. Paschotta, F. Morier-Genoud, and U. Keller, “Passively mode-locked diode-pumped surface-emitting semiconductor laser,” IEEE Photonics Technol. Lett. 12(9), 1135–1137 (2000).
[Crossref]

Häring, R.

R. Häring, R. Paschotta, E. Gini, F. Morier-Genoud, D. Martin, H. Melchior, and U. Keller, “Picosecond surface-emitting semiconductor laser with >200 mW average power,” Electron. Lett. 37(12), 766–767 (2001).
[Crossref]

Hartenstein, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Heinen, B.

Helmchen, F.

G.-A. Pilz, S. Carta, A. Stäuble, A. Ayaz, S. Jessberger, and F. Helmchen, “Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus,” J. Neurosci. 36(28), 7407–7414 (2016).
[Crossref] [PubMed]

J. L. Chen, F. F. Voigt, M. Javadzadeh, R. Krueppel, and F. Helmchen, “Long-range population dynamics of anatomically defined neocortical networks,” eLife 5, 14679 (2016).
[Crossref] [PubMed]

F. F. Voigt, J. L. Chen, R. Krueppel, and F. Helmchen, “A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo,” Proc. SPIE Multiphoton Microscopy in the Biomedical Sciences XV 93292, 93292 (2015).

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[Crossref] [PubMed]

Hibi, T.

Hill, S.

Hoffmann, M.

Holthoff, K.

C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, “In vivo two-photon calcium imaging of neuronal networks,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7319–7324 (2003).
[Crossref] [PubMed]

Hoogland, S.

S. Hoogland, A. Garnache, I. Sagnes, J. S. Roberts, and A. C. Tropper, “10-GHz Train of Sub-500-fs Optical Soliton-Like Pulses From a Surface-Emitting Semiconductor Laser,” IEEE Photonics Technol. Lett. 17(2), 267–269 (2005).
[Crossref]

A. Garnache, S. Hoogland, A. C. Tropper, I. Sagnes, G. Saint-Girons, and J. S. Roberts, “Sub-500-fs soliton pulse in a passively mode-locked broadband surface-emitting laser with 100-mW average power,” Appl. Phys. Lett. 80(21), 3892–3894 (2002).
[Crossref]

S. Hoogland, S. Dhanjal, A. C. Tropper, J. S. Roberts, R. Haring, R. Paschotta, F. Morier-Genoud, and U. Keller, “Passively mode-locked diode-pumped surface-emitting semiconductor laser,” IEEE Photonics Technol. Lett. 12(9), 1135–1137 (2000).
[Crossref]

Hoover, E. E.

Hopt, A.

A. Hopt and E. Neher, “Highly Nonlinear Photodamage in Two-Photon Fluorescence Microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[Crossref] [PubMed]

Hügel, H.

A. Giesen, H. Hügel, A. Voss, K. Wittig, U. Brauch, and H. Opower, “Scalable Concept for Diode-Pumped High-Power Solid-State Lasers,” Appl. Phys. B 58(5), 365–372 (1994).
[Crossref]

Hughes, T. E.

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M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
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D. Waldburger, S. M. Link, M. Mangold, C. G. E. Alfieri, E. Gini, M. Golling, B. W. Tilma, and U. Keller, “High-power 100 fs semiconductor disk lasers,” Optica 3(8), 844–852 (2016).
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M. Mangold, M. Golling, E. Gini, B. W. Tilma, and U. Keller, “Sub-300-femtosecond operation from a MIXSEL,” Opt. Express 23(17), 22043–22059 (2015).
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B. W. Tilma, M. Mangold, C. A. Zaugg, S. M. Link, D. Waldburger, A. Klenner, A. S. Mayer, E. Gini, M. Golling, and U. Keller, “Recent advances in ultrafast semiconductor disk lasers,” Light Sci. Appl. 4(7), e310 (2015).
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S. Hoogland, S. Dhanjal, A. C. Tropper, J. S. Roberts, R. Haring, R. Paschotta, F. Morier-Genoud, and U. Keller, “Passively mode-locked diode-pumped surface-emitting semiconductor laser,” IEEE Photonics Technol. Lett. 12(9), 1135–1137 (2000).
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Pietzsch, T.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
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G.-A. Pilz, S. Carta, A. Stäuble, A. Ayaz, S. Jessberger, and F. Helmchen, “Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus,” J. Neurosci. 36(28), 7407–7414 (2016).
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K. Podgorski and G. N. Ranganathan, “Brain heating induced by near infrared lasers during multi-photon microscopy,” J. Neurophysiol 116(3) 1012-1023 (2016).

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P. Golshani, J. T. Gonçalves, S. Khoshkhoo, R. Mostany, S. Smirnakis, and C. Portera-Cailliau, “Internally mediated developmental desynchronization of neocortical network activity,” J. Neurosci. 29(35), 10890–10899 (2009).
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J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
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Rafailov, E. U.

M. A. Gaafar, A. Rahimi-Iman, K. A. Fedorova, W. Stolz, E. U. Rafailov, and M. Koch, “Mode-locked semiconductor disk lasers,” Adv. Opt. Photonics 8(3), 370–400 (2016).
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M. A. Gaafar, A. Rahimi-Iman, K. A. Fedorova, W. Stolz, E. U. Rafailov, and M. Koch, “Mode-locked semiconductor disk lasers,” Adv. Opt. Photonics 8(3), 370–400 (2016).
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M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
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Riemann, I.

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S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, “Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy,” J. Biomed. Opt. 11, 020501 (2006).

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K. G. Wilcox, A. C. Tropper, H. E. Beere, D. A. Ritchie, B. Kunert, B. Heinen, and W. Stolz, “4.35 kW peak power femtosecond pulse mode-locked VECSEL for supercontinuum generation,” Opt. Express 21(2), 1599–1605 (2013).
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K. G. Wilcox, A. C. Tropper, H. E. Beere, D. A. Ritchie, B. Kunert, B. Heinen, and W. Stolz, “4.35 kW peak power femtosecond pulse mode-locked VECSEL for supercontinuum generation,” Opt. Express 21(2), 1599–1605 (2013).
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S. Mirkhanov, A. H. Quarterman, S. Swift, B. B. Praveen, C. J. C. Smyth, and K. G. Wilcox, “Multiphoton imaging with high peak power VECSELs,” Proc. SPIE Vertical External Cavity Surface Emitting Lasers9734, 973412 (2016)

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W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
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M. D. Young, S. Backus, C. Durfee, and J. Squier, “Multiphoton imaging with a direct-diode pumped femtosecond Ti:sapphire laser,” J. Microsc. 249(2), 83–86 (2013).
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M. Mangold, S. M. Link, A. Klenner, C. A. Zaugg, M. Golling, B. W. Tilma, and U. Keller, “Amplitude noise and timing jitter characterization of a high-power Mode-Locked Integrated External-Cavity Surface Emitting Laser,” IEEE Photonics J. 6(1), 1–9 (2014).
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W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
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P. Klopp, U. Griebner, M. Zorn, and M. Weyers, “Pulse repetition rate up to 92 GHz or pulse duration shorter than 110 fs from a mode-locked semiconductor disk laser,” Appl. Phys. Lett. 98(7), 071103 (2011).
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Adv. Opt. Photonics (1)

M. A. Gaafar, A. Rahimi-Iman, K. A. Fedorova, W. Stolz, E. U. Rafailov, and M. Koch, “Mode-locked semiconductor disk lasers,” Adv. Opt. Photonics 8(3), 370–400 (2016).
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P. G. Antal and R. Szipőcs, “Tunable, low-repetition-rate, cost-efficient femtosecond Ti:sapphire laser for nonlinear microscopy,” Appl. Phys. B 107(1), 17–22 (2012).
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A. Giesen, H. Hügel, A. Voss, K. Wittig, U. Brauch, and H. Opower, “Scalable Concept for Diode-Pumped High-Power Solid-State Lasers,” Appl. Phys. B 58(5), 365–372 (1994).
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A. Garnache, S. Hoogland, A. C. Tropper, I. Sagnes, G. Saint-Girons, and J. S. Roberts, “Sub-500-fs soliton pulse in a passively mode-locked broadband surface-emitting laser with 100-mW average power,” Appl. Phys. Lett. 80(21), 3892–3894 (2002).
[Crossref]

P. Klopp, U. Griebner, M. Zorn, and M. Weyers, “Pulse repetition rate up to 92 GHz or pulse duration shorter than 110 fs from a mode-locked semiconductor disk laser,” Appl. Phys. Lett. 98(7), 071103 (2011).
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IEEE Photonics J. (1)

M. Mangold, S. M. Link, A. Klenner, C. A. Zaugg, M. Golling, B. W. Tilma, and U. Keller, “Amplitude noise and timing jitter characterization of a high-power Mode-Locked Integrated External-Cavity Surface Emitting Laser,” IEEE Photonics J. 6(1), 1–9 (2014).
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IEEE Photonics Technol. Lett. (3)

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[Crossref]

K. G. Wilcox, A. H. Quarterman, H. Beere, D. A. Ritchie, and A. C. Tropper, “High Peak Power Femtosecond Pulse Passively Mode-Locked Vertical-External-Cavity Surface-Emitting Laser,” IEEE Photonics Technol. Lett. 22(14), 1021–1023 (2010).
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S. Hoogland, S. Dhanjal, A. C. Tropper, J. S. Roberts, R. Haring, R. Paschotta, F. Morier-Genoud, and U. Keller, “Passively mode-locked diode-pumped surface-emitting semiconductor laser,” IEEE Photonics Technol. Lett. 12(9), 1135–1137 (2000).
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Supplementary Material (5)

NameDescription
» Visualization 1: AVI (6323 KB)      Multiphoton imaging in a Drosophila larva using the ultrafast SDL.
» Visualization 2: AVI (10380 KB)      In vivo two-photon imaging of blood vessels filled with Texas Red Dextran using the ultrafast SDL.
» Visualization 3: AVI (4485 KB)      In vivo imaging of R-CaMP1.07-expressing neurons in mouse neocortex using the ultrafast SDL.
» Visualization 4: AVI (3029 KB)      In vivo imaging of R-CaMP1.07-expressing neurons in mouse neocortex using the ultrafast SDL.
» Visualization 5: AVI (11298 KB)      In vivo calcium imaging using OGB-1 in a young (P8) mouse with the ultrafast SDL.

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Figures (12)

Fig. 1
Fig. 1

Overview of the two-photon absorption cross-sections of various fluorescent proteins [8] used in two-photon microscopy combined with the spectral coverage of existing ultrafast lasers. Ti:Sapphire lasers have a large tuning range of >300 nm. In contrast, other commercial ultrafast lasers (blue lines) cannot be tuned to match the absorption peaks of dyes. Ultrafast semiconductor disk lasers (SDLs, orange lines) have the potential to become less expensive sources and can be designed with emission wavelengths between 650 and 2800 nm (typical lasers with a center wavelength at 920-nm, 970-nm and 1080-nm are shown here, but more options are possible) [9, 10]. The SDL laser presented in this paper operates at 1027 nm (indicated as “This work”).

Fig. 2
Fig. 2

A) Typical laser cavity of a pulsed Semiconductor Disk Laser (SDL) or Vertical External Cavity Surface Emitting Laser (VECSEL) consisting of an optically pumped gain chip, a SESAM to enable pulse formation with passive mode-locking and an output coupler. The cavity length is set to reach a pulse repetition rate of ≈1-2 GHz with an (unfolded) length of 10-15 cm. B) Overview of the evolution of pulse durations from SESAM-mode-locked SDLs around 1000 nm since the first demonstration in 2000 [34, 36–41], recently reaching sub-100 fs [42].

Fig. 3
Fig. 3

Multiphoton microscopy (MPM) imaging at high repetition rates: A) Overlay of the pulse trains from a Ti:Sapphire laser and a SDL set to generate the same two-photon excited fluorescence signal at comparable pulse duration, center wavelength, and optical resolution. We use the parameters of the two lasers compared in this study as examples. B) In order to achieve constant signal, the ratio of average powers has to be set to the square root of the ratio of repetition rates.

Fig. 4
Fig. 4

A) Overview of the microscope setup: Excitation light from either the SDL or a Ti:Sapphire laser (Spectra-Physics Mai Tai DeepSee) can be coupled into the microscope via a Pockels cell (PC) and a beam expander (BE). After the scan mirrors, the beam is directed into the objective by a scan and tube lens. The emission light is sent to a two-channel detection system via dichroic mirrors (DCs), emission filters (EF) and then collected by hybrid photo-detectors (HPDs). For small, transparent samples, a transmission detector with a single HPD can be added. B) Maximum intensity projections of images of the same 200 nm bead taken with the Ti:Sapphire and SDL. C) Gaussian fit of the data in B) with full width at half maximum (FWHM) values for both lasers.

Fig. 5
Fig. 5

A) Pulse width measurements of the ultrafast SDL in combination with the 20x microscope objective. B) The SDL has a spectrum with 9.5 nm full width at half maximum (FWHM) (i.e the spectrum is supporting 118 fs pulses). C-D) Fundamental mode-locking is demonstrated with the Radio Frequency (RF) traces taken at the main laser frequency of 1.63 GHz and on its harmonics. The drop of the power in the harmonics is due to the limited bandwidth of the RF amplifier used to measure it. The resolution bandwidth (RBW) is the smallest frequency increment that can be resolved.

Fig. 6
Fig. 6

A Ti:Sapphire laser (Mai-Tai HP from Spectra-Physics) was used for comparison with the SDL. A) Using the prechirping unit in the Ti:Sapphire laser, the pulses were adjusted to a length of 165 fs after the objective (20x). B) The spectrum was centered at 1026 nm to overlap with the spectrum of the SDL. Both spectra were measured after the microscope objective. C-D) Fundamental mode-locking was confirmed by the Radio Frequency (RF) traces taken at the main laser frequency of 80.65 MHz and on its harmonics. The resolution bandwidth (RBW) is the smallest frequency increment that can be resolved.

Fig. 7
Fig. 7

Imaging comparison between the SDL and the Ti:Sapphire laser: The power levels were set to a ratio of ≈4.5 to create approximately the same signal based on the ratio of repetition rates [48]. The images are sum projections over a stack of 10 slices (taken at 2048 x 2048 pixel with 10 µs pixel dwell time) using 1 µm z-spacing. A) Comparison images of fixed BPAE cells (similar histogram settings). Green channel: Microtubules, red channel: F-Actin. B) Comparison of the signal counts created by the SDL and the Ti:Sapphire laser along the line profiles highlighted in A.

Fig. 8
Fig. 8

Comparison of bleaching rates in a Drosophila larva labeled with mKate2: A) Raw bleaching time courses for the SDL (blue) and for the Ti:Sapphire laser (red) at a power ratio of 4.6 giving a similar generated signal as expected by theory. B) Tri-exponential function used to fit bleaching curves in A. As shown in C), this function was the most adequate to obtain an accurate and robust fit because a single or bi-exponential function cannot fully fit the data. D) Formula to calculate the photodamage coefficient β using the approach in [48]. E) Taking into account the three time constants of the bleaching curves (τ1, τ2 and τ3), β can be obtained in each case. The average β value across all three extracted time constants is centered at 2.10 with a standard deviation of 0.51.

Fig. 9
Fig. 9

A) Multiphoton imaging in a Drosophila larva using the ultrafast SDL: Maximum intensity projection of a stack covering a range of 160 µm (Average laser power: 17 mW, 1696 x 1142 pixel, 10 µs dwell time). Red channel: Fluorescence from mKate2, Green channel: SHG signal. The SHG signal is predominantly originating from sarcomeres in the muscles. B) Image of the sarcomeres at higher zoom showing the characteristic double-band structure (6 mW, 1024 x 1024 pixel, 10 µs dwell time, 4x averaging).

Fig. 10
Fig. 10

In vivo two-photon imaging of blood vessels filled with Texas Red Dextran using the ultrafast SDL. A) Selected frames from a z-stack at different depths as measured from the brain surface (512 x 512 pixel, 10 µs pixel dwell time). For each z-plane, the histogram was adjusted for better visibility. The top right insets show the same z-positions with the identical histogram settings throughout to compare signal strength. The SHG signal from the dura mater is visible at 20 µm. B) YZ-side-projection of the same data set (red channel only).

Fig. 11
Fig. 11

In vivo imaging of R-CaMP1.07-expressing neurons in mouse neocortex using the ultrafast SDL. A) Single z-planes from a stack starting at the dura down to a depth of 360 µm (72 mW laser power, 1024 x 1042 pixel, 10 µs dwelltime). For each z-plane, the histogram was adapted for better visibility. The insets show the same z-positions with the same histogram settings. Neurons are visible as hollow rings as the calcium indicator does not enter the cell nucleus. The yellow frame at a depth of 125 µm indicates the functional imaging region in B). B) Calcium imaging in a subset of neurons (acquired at 72 mW laser power, 100 x 100 pixel, 6 µs dwelltime). Average projection and example traces of three neurons and a neuropil (n.p.) region of interest are shown with a frame rate of 10.68 Hz. For this experiment, an HPD recorded all available signal without additional filters except an IR rejection filter.

Fig. 12
Fig. 12

In vivo calcium imaging in a young (P8) mouse with the ultrafast SDL: A) Overview image (924 μm x 465 μm) of tdTomato-expressing VIP-positive interneurons (red) and the surrounding cell population labeled with the calcium indicator OGB-1 (green) at a depth of 140 µm (51.4 mW average power, 3492 x 1598 pixel, 10 µs pixel dwelltime, 4x average). Shadows in the image originate from superficial blood vessels. B) Field-of-view selected for calcium imaging (164 μm x 89 μm, 155 µm depth, 55.8 mW laser power, 200 x 100 pixel, 5 µs pixel dwell time) and calcium transients recorded from several neurons (6.42 Hz frame rate). Cell 9 is a VIP-positive interneuron co-labeled with tdTomato. n.p.: Neuropil.

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