Zebrafish Brain Development Monitored by Long-Term In Vivo Microscopy: A Comparison Between Laser Scanning Confocal and 2-Photon Microscopy

  • Nicolas Dross
  • Carlo Antonio Beretta
  • Peter Bankhead
  • Matthias Carl
  • Ulrike Engel
Protocol
Part of the Neuromethods book series (NM, volume 87)

Abstract

Zebrafish is an attractive model organism to study vertebrate brain development. Its transparency makes it possible to follow development using live imaging. In a transgenic line where a subset of neurons is labeled by GFP expression, their migration, proliferation and the extension of axons can be observed by laser scanning confocal microscopy (LSCM) or 2-photon microscopy (2PM). However, when the whole brain is imaged, LSCM might result in phototoxicity. In contrast, 2PM allows for image acquisition over several days at intervals shorter than an hour. In this article, we describe a method to image a large region of the brain (500 × 500 μm) spanning 300 μm in depth by 2PM over 2 days or more. The results are compared with those obtained by the more widespread LSCM. Visualization and analysis of the resulting data is challenging, as they exceed the size that can be loaded into standard rendering software. We propose a routine to reduce the size by maximum projection while keeping and displaying three-dimensional information by a color code within ImageJ.

Key words

Zebrafish Axon extension Laser scanning confocal microscopy 2-Photon microscopy Phototoxicity Photobleaching In vivo Long-term imaging 

References

  1. 1.
    Gan WB, Grutzendler J, Wong WT et al (2000) Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27:219–225PubMedCrossRefGoogle Scholar
  2. 2.
    Young P, Feng G (2004) Labeling neurons in vivo for morphological and functional studies. Curr Opin Neurobiol 14:642–646. doi:10.1016/j.conb.2004.08.007 PubMedCrossRefGoogle Scholar
  3. 3.
    Köster RW, Fraser SE (2004) Time-lapse microscopy of brain development. Methods Cell Biol 76:207–235PubMedCrossRefGoogle Scholar
  4. 4.
    Jiang YJ, Brand M, Heisenberg CP et al (1996) Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development 123:205–216PubMedGoogle Scholar
  5. 5.
    Driever W, Solnica-Krezel L, Schier AF et al (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123:37–46PubMedGoogle Scholar
  6. 6.
    Köster RW, Fraser SE (2001) Tracing transgene expression in living zebrafish embryos. Dev Biol 233:329–346PubMedCrossRefGoogle Scholar
  7. 7.
    Pan YA, Livet J, Sanes JR et al (2011) Multicolor Brainbow imaging in zebrafish. Cold Spring Harb Protoc 2011:pdb.prot5546. doi:10.1101/pdb.prot5546 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Köster RW, Fraser SE (2001) Direct imaging of in vivo neuronal migration in the developing cerebellum. Curr Biol 11:1858–1863PubMedCrossRefGoogle Scholar
  9. 9.
    Lowery LA, Sive H (2005) Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products. Development 132:2057–2067PubMedCrossRefGoogle Scholar
  10. 10.
    Graeden E, Sive H (2009) Live imaging of the zebrafish embryonic brain by confocal microscopy. J Vis Exp (26):1217. doi: 10.3791/1217
  11. 11.
    Amos W (2003) How the confocal laser scanning microscope entered biological research. Biol Cell 95:335–342. doi:10.1016/S0248-4900(03)00078-9 PubMedCrossRefGoogle Scholar
  12. 12.
    Squirrell JM, Wokosin DL, White JG, Bavister BD (1999) Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat Biotechnol 17:763–767. doi:10.1038/11698 PubMedCrossRefGoogle Scholar
  13. 13.
    Huisken J, Swoger J, Del Bene F et al (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305:1007–1009. doi:10.1126/science. 1100035 Google Scholar
  14. 14.
    Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EHK (2008) Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322:1065–1069PubMedCrossRefGoogle Scholar
  15. 15.
    Ahrens MB, Orger MB, Robson DN et al (2013) Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods 10:413–420. doi:10.1038/nmeth.2434 PubMedCrossRefGoogle Scholar
  16. 16.
    Minsky M (1961) Microscopy Apparatus.Google Scholar
  17. 17.
    Pawley JB (1995) Handbook of biological confocal microscopy, 2nd edn. Plenum Press, New York, NYCrossRefGoogle Scholar
  18. 18.
    Wilson T, Sheppard C (1984) Theory and practice of scanning optical microscopy. Academic, London, pp 1–213Google Scholar
  19. 19.
    Centonze VE, White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75:2015–2024PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Denk W, Strickler J, Webb W (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76. doi:10.1126/science.2321027 PubMedCrossRefGoogle Scholar
  21. 21.
    Conchello J, Lichtman JW (2005) Optical sectioning microscopy. Nat Methods 2:920–931. doi:10.1038/nmeth815 PubMedCrossRefGoogle Scholar
  22. 22.
    Kobat D, Durst ME, Nishimura N et al (2009) Deep tissue multiphoton microscopy using longer wavelength excitation. Opt Express 17:13354–13364PubMedCrossRefGoogle Scholar
  23. 23.
    Kamei M, Weinstein BM (2005) Long-term time-lapse fluorescence imaging of developing zebrafish. Zebrafish 2:113–123. doi:10.1089/zeb.2005.2.113 PubMedCrossRefGoogle Scholar
  24. 24.
    Göppert-Mayer M (1931) Über Elementarakte mit zwei Quantensprüngen. Ann Phys 401:273–294. doi:10.1002/andp.19314010303 CrossRefGoogle Scholar
  25. 25.
    Kaiser W, Garrett C (1961) Two-photon excitation in CaF2: Eu2+. Phys Rev Lett 7:229–231. doi:10.1103/PhysRevLett.7.229 CrossRefGoogle Scholar
  26. 26.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940. doi:10.1038/nmeth818 PubMedCrossRefGoogle Scholar
  27. 27.
    Diaspro A, Chirico G, Collini M (2005) Two-photon fluorescence excitation and related techniques in biological microscopy. Q Rev Biophys 38:97–166. doi:10.1017/S0033583505004129 PubMedCrossRefGoogle Scholar
  28. 28.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377. doi:10.1038/nbt899 PubMedCrossRefGoogle Scholar
  29. 29.
    Svoboda K, Yasuda R (2006) Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50:823–839. doi:10.1016/j.neuron.2006.05.019 PubMedCrossRefGoogle Scholar
  30. 30.
    Oheim M, Beaurepaire E, Chaigneau E et al (2001) Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J Neurosci Methods 111:29–37PubMedCrossRefGoogle Scholar
  31. 31.
    Yaroslavsky AN, Schulze PC, Yaroslavsky IV et al (2002) Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range. Phys Med Biol 47:2059–2073PubMedCrossRefGoogle Scholar
  32. 32.
    Denk W, Svoboda K (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18:351–357PubMedCrossRefGoogle Scholar
  33. 33.
    Xu C, Webb WW (1996) Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. J Opt Soc Am B 13:481. doi:10.1364/JOSAB.13.000481 CrossRefGoogle Scholar
  34. 34.
    Ahmed F, Wyckoff J, Lin EY et al (2002) GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res 62:7166–7169PubMedGoogle Scholar
  35. 35.
    Lawson N (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248:307–318. doi:10.1006/dbio.2002.0711 PubMedCrossRefGoogle Scholar
  36. 36.
    König K, So PT, Mantulin WW, Gratton E (1997) Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes. Opt Lett 22:135–136. doi:10.1016/j.biortech.2010.01.053 PubMedCrossRefGoogle Scholar
  37. 37.
    Beretta CA, Dross N, Guiterrez-Triana JA et al (2012) Habenula circuit development: past, present, and future. Front Neurosci 6:51. doi:10.3389/fnins.2012.00051 PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Westerfield M (1995) The zebrafish book. The University of Oregon Press, Eugene, ORGoogle Scholar
  39. 39.
    Sheppard CJR (1986) The spatial frequency cut-off in three-dimensional imaging. Optik 72:131–133Google Scholar
  40. 40.
    Sheppard CJR (1986) The spatial frequency cut-off in three-dimensional imaging II. Optik 74:128–129Google Scholar
  41. 41.
    SVI Scientific Volume Imaging: Nyquist rate and PSF calculator.Google Scholar
  42. 42.
    Schneider C, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. doi:10.1038/nmeth.2089 PubMedCrossRefGoogle Scholar
  43. 43.
    Beretta CA, Dross N, Bankhead P, Carl M (2013) The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function. Neural Dev 8:19. doi:10.1186/1749-8104-8-19 PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Terasaki M, Dailey ME (1995) Confocal microscopy of living cells. In: Pawley JB (ed) Handbook of biology confocal microscopy, 2nd edn. Plenum Press, New York, NY, pp 327–346CrossRefGoogle Scholar
  45. 45.
    Mahou P, Zimmerley M, Loulier K et al (2012) Multicolor two-photon tissue imaging by wavelength mixing. Nat Methods 9:815–818. doi:10.1038/nmeth.2098 PubMedCrossRefGoogle Scholar
  46. 46.
    Thomas JL, Ochocinska MJ, Hitchcock PF, Thummel R (2012) Using the Tg(nrd:egfp)/albino zebrafish line to characterize in vivo expression of neurod. PLoS One 7:e29128. doi:10.1371/journal.pone.0029128 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Nicolas Dross
    • 1
    • 2
  • Carlo Antonio Beretta
    • 3
  • Peter Bankhead
    • 1
    • 2
  • Matthias Carl
    • 3
  • Ulrike Engel
    • 1
    • 2
  1. 1.Nikon Imaging CenterUniversity of HeidelbergHeidelbergGermany
  2. 2.Center for Organismal StudiesHeidelberg UniversityHeidelbergGermany
  3. 3.Department of Cell and Molecular Biology, Medical Faculty MannheimHeidelberg UniversityMannheimGermany

Personalised recommendations