High-speed, low-photodamage nonlinear imaging using passive pulse splitters

Nature Methods volume 5, pages197–202(2008)Cite this article

A Corrigendum to this article was published on 01 February 2009

This article has been updated

Abstract

Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.

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Figure 1: Construction of pulse splitters.
Figure 2: Pulse splitting increases TPE signal rates.
Figure 3: Effect of pulse splitting on photobleaching of fixed GFP-labeled brain slices.
Figure 4: Effect of 64 × pulse splitting on in vivo photobleaching in a GFP-labeled C.elegans larva.
Figure 5: Effect of 64 × pulse splitting on photodamage during Ca2+ imaging of CA1 pyramidal neurons injected with Oregon Green BAPTA-1.

Change history

  • 31 December 2008

    NOTE: In the version of this article initially published, the traces in Figure 2 were incorrectly labeled. The error has been corrected in the HTML and PDF versions of the article.

References

  1. 1

    Chen, T.S. et al. High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy. Biochem. Biophys. Res. Commun. 291, 1272–1275 (2002).

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Patterson, G.H. & Piston, D.W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3

    Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Sizer, T., II Increase in laser repetition rate by spectral selection. IEEE J. Quantum Electron. 25, 97–103 (1989).

    CAS  Article  Google Scholar 

  6. 6

    MacFarlane, D.L., Strozewski, K.J. & Tatum, J.A. Mode-locked laser pulse train repetition frequency multiplication: the optical rattler. Appl. Opt. 30, 1042–1047 (1991).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Narayan, V., Dowling, E.M. & MacFarlane, D.L. Design of multimirror structures for high-frequency bursts and codes of ultrashort pulses. IEEE J. Quantum Electron. 30, 1671–1679 (1994).

    CAS  Article  Google Scholar 

  8. 8

    Guild, J.B., Xu, C. & Webb, W.W. Measurement of group delay dispersion of high numerical aperture objective lenses using two-photon excited fluorescence. Appl. Opt. 36, 397–401 (1997).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Squier, J. & Muller, M. High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging. Rev. Sci. Instrum. 72, 2855–2867 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Volkmer, A. et al. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 78, 1589–1598 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Schonle, A. & Hell, S.W. Heating by absorption in the focus of an objective lens. Opt. Lett. 23, 325–327 (1998).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Koester, H.J. et al. Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Magee, J.C. Dendritic Ih normalizes temporal summation in hippocampal CA1 neurons. Nat. Neurosci. 2, 508–514 (1999).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Campagnola, P.J. et al. High-resolution nonlinear optical imaging of live cells by second harmonic generation. Biophys. J. 77, 3341–3349 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Florsheimer, M., Brillert, C. & Fuchs, H. Chemical imaging of interfaces by sum frequency microscopy. Langmuir 15, 5437–5439 (1999).

    Article  Google Scholar 

  17. 17

    Ji, N. et al. Three-dimensional chiral imaging by sum-frequency generation. J. Am. Chem. Soc. 128, 3482–3483 (2006).

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Zumbusch, A., Holtom, G.R. & Xie, X.S. Three-dimensional vibrational imaging by coherent anti-stokes raman scattering. Phys. Rev. Lett. 82, 4142 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Mertz, J., Xu, C. & Webb, W.W. Single-molecule detection by two-photon-excited fluorescence. Opt. Lett. 20, 2532–2534 (1995).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Chu, S.W., Liu, T.M. & Sun, C.K. Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser. Opt. Exp. 11, 933–938 (2003).

    Article  Google Scholar 

  21. 21

    Bewersdorf, J., Pick, R. & Hell, S.W. Multifocal multiphoton microscopy. Opt. Lett. 23, 655–657 (1998).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Buist, A.H. et al. Real time two-photon absorption microscopy using multi point excitation. J. Microsc. (Oxf.) 192, 217–226 (1998).

    CAS  Article  Google Scholar 

  23. 23

    Fittinghoff, D.N. & Squier, J.A. Time-decorrelated multifocal array for multiphoton microscopy and micromachining. Opt. Lett. 25, 1213–1215 (2000).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Fricke, M. & Nielsen, T. Two-dimensional imaging without scanning by multifocal multiphoton microscopy. Appl. Opt. 44, 2984–2988 (2005).

    Article  PubMed  Google Scholar 

  25. 25

    Sacconi, L. et al. Multiphoton multifocal microscopy exploiting a diffractive optical element. Opt. Lett. 28, 1918–1920 (2003).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Kim, K.H. et al. Multifocal multiphoton microscopy based on multianode photomultiplier tubes. Opt. Exp. 15, 11658–11678 (2007).

    Article  Google Scholar 

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Acknowledgements

We thank our colleagues at Janelia Farm Research Campus, Howard Hughes Medical Institute, T. Sato and T.-Y. Mao for providing the GFP-labeled brain slice samples, V. Jayaraman and J. Seelig for sharing their Ti:sapphire laser, R. Kerr and H.-C. Peng for guidance with the C. elegans samples, and K. Svoboda for helpful suggestions.

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  1. Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, 20147, Virginia, USA

    Na Ji, Jeffrey C Magee & Eric Betzig

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  1. Na Ji
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  2. Jeffrey C Magee
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  3. Eric Betzig
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Correspondence to Na Ji.

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Supplementary Figures 1–6, Supplementary Methods (PDF 1900 kb)

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Ji, N., Magee, J. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat Methods 5, 197–202 (2008). https://doi.org/10.1038/nmeth.1175

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