Multiphoton Brain Imaging

  • Anna DunaevskyEmail author
Part of the Springer Protocols Handbooks book series (SPH)


Imaging approaches have revolutionized neuroscience research. The discovery and development of dyes, fluorescent proteins, light-activated proteins (optogenetics), as well as transgenic animals with subset of cells expressing fluorescent proteins allows scientists to probe the structure and function of neurons with ever-increasing specificity. The ability to perform such investigations in intact animals affords linkages between cellular form and function to the dynamics of neural networks, animal behavior, and disease. Two-photon laser scanning microscopy (TPLSM) is amongst the best approaches available for high-resolution imaging of neurons and other cells that provides spatiotemporal information in the intact brain not feasible by other methods.

The objective of this chapter is to address the following questions:
  1. 1.

    Why is there a need for TPLSM for in vivo imaging?

  2. 2.

    How does TPLSM work?

  3. 3.

    What are its common applications?

  4. 4.

    How to perform in vivo imaging of the brain using TPLSM?



Multiphoton Confocal GFP In vivo Cranial window 


  1. Brown CE, Li P, Boyd JD, Delaney KR, Murphy TH (2007) Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke. J Neurosci 27:4101–4109CrossRefPubMedGoogle Scholar
  2. Christensen DJ, Nedergaard M (2011) Two-photon in vivo imaging of cells. Pediatr Nephrol 26:1483–1489CrossRefPubMedGoogle Scholar
  3. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8:752–758CrossRefPubMedGoogle Scholar
  4. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76CrossRefPubMedGoogle Scholar
  5. Drobizhev M, Makarov NS, Tillo SE, Hughes TE, Rebane A (2011) Two-photon absorption properties of fluorescent proteins. Nat Methods 8:393–399CrossRefPubMedPubMedCentralGoogle Scholar
  6. Grewe BF, Langer D, Kasper H, Kampa BM, Helmchen F (2010) High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat Methods 7:399–405CrossRefPubMedGoogle Scholar
  7. Grutzendler J, Kasthuri N, Gan WB (2002) Long-term dendritic spine stability in the adult cortex. Nature 420:812–816CrossRefPubMedGoogle Scholar
  8. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940CrossRefPubMedGoogle Scholar
  9. Hirase H, Qian L, Bartho P, Buzsaki G (2004) Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol 2:E96CrossRefPubMedPubMedCentralGoogle Scholar
  10. Holtmaat AJ, Trachtenberg JT, Wilbrecht L, Shepherd GM, Zhang X, Knott GW, Svoboda K (2005) Transient and persistent dendritic spines in the neocortex in vivo. Neuron 45:279–291CrossRefPubMedGoogle Scholar
  11. Lee WC, Huang H, Feng G, Sanes JR, Brown EN, So PT, Nedivi E (2006) Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex. PLoS Biol 4:e29CrossRefPubMedGoogle Scholar
  12. Marker DF, Tremblay ME, Lu SM, Majewska AK, Gelbard HA (2010) A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation. J Vis Exp Jove Sep 19;(43).Google Scholar
  13. Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai H (2001) Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4:1086–1092CrossRefPubMedPubMedCentralGoogle Scholar
  14. McGavern DB, Kang SS (2011) Illuminating viral infections in the nervous system. Nat Rev Immunol 11:318–329CrossRefPubMedPubMedCentralGoogle Scholar
  15. Noguchi J, Nagaoka A, Watanabe S, Ellis-Davies GC, Kitamura K, Kano M, Matsuzaki M, Kasai H (2011) In vivo two-photon uncaging of glutamate revealing the structure-function relationships of dendritic spines in the neocortex of adult mice. J Physiol 589:2447–2457CrossRefPubMedPubMedCentralGoogle Scholar
  16. Pawley J (2005) Handbook of biological confocal microscopy, 3rd edn. Springer, BerlinGoogle Scholar
  17. Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E, Svoboda K (2002) Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420:788–794CrossRefPubMedGoogle Scholar
  18. Ustione A, Piston DW (2011) A simple introduction to multiphoton microscopy. J Microsc 243:221–226CrossRefPubMedGoogle Scholar
  19. Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X, Kang J, Takano T, Nedergaard M (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nat Neurosci 9:816–823CrossRefPubMedGoogle Scholar
  20. Williams RM, Piston DW, Webb WW (1994) Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J 8:804–813CrossRefPubMedGoogle Scholar
  21. Xu HT, Pan F, Yang G, Gan WB (2007) Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat Neurosci 10:549–551CrossRefPubMedGoogle Scholar
  22. Xu T, Yu X, Perlik AJ, Tobin WF, Zweig JA, Tennant K, Jones T, Zuo Y (2009) Rapid formation and selective stabilization of synapses for enduring motor memories. Nature 462:915–919CrossRefPubMedPubMedCentralGoogle Scholar
  23. Yang G, Pan F, Parkhurst CN, Grutzendler J, Gan WB (2011) Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5:201–208CrossRefGoogle Scholar
  24. Yuste R, Denk W (1995) Dendritic spines as basic functional units of neuronal integration. Nature 375:682–684CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Developmental Neuroscience, Munroe Meyer Institute for Genetics and RehabilitationUniversity of Nebraska Medical CenterOmahaUSA

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