Intravital Two-Photon Excitation Microscopy in Neuroscience: General Concepts and Applications



Multiphoton excitation microscopy has revolutionized biomedical research during the last two decades by enabling high resolution fluorescent microscopy in intact tissues. This feature makes two-photon excitation (2PE) microscopy ideal for intravital imaging of neural tissue, permitting the observation of structural and functional neuronal dynamics in undisrupted neural circuits. Here we review the fundamental concepts of intravital 2PE microscopy, describe methods and techniques associated with it, and highlight the most significant findings reported on neuron and glia structure dynamics as well as on neuronal activity using this in vivo imaging technique.


Chronic in vivo imaging Calcium imaging Dendritic spine Axonal bouton Fluorophore GECI Cranial window Thinned-skull Pyramidal neuron Glia Deep tissue imaging Optogenetics 


  1. Akemann W, Sasaki M, Mutoh H, Imamura T, Honkura N, Knopfel T (2013) Two-photon voltage imaging using a genetically encoded voltage indicator. Sci Rep 3:2231. doi: 10.1038/srep02231 PubMedCentralPubMedGoogle Scholar
  2. Akerboom J, Carreras Calderón N, Tian L, Wabnig S, Prigge M, Tolö J, Gordus A, Orger M, Severi K, Macklin J, Patel R, Pulver S, Wardill T, Fischer E, Schüler C, Chen T-W, Sarkisyan K, Marvin J, Bargmann C, Kim D, Kügler S, Lagnado L, Hegemann P, Gottschalk A, Schreiter E, Looger L (2013) Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci 6:2. doi: 10.3389/fnmol.2013.00002 PubMedCentralPubMedGoogle Scholar
  3. Bardehle S, Krüger M, Buggenthin F, Schwausch J, Ninkovic J, Clevers H, Snippert H, Theis F, Meyer-Luehmann M, Bechmann I, Dimou L, Götz M (2013) Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation. Nat Neurosci 16(5):580–586. doi: 10.1038/nn.3371 PubMedGoogle Scholar
  4. Blinder P, Shih A, Rafie C, Kleinfeld D (2010) Topological basis for the robust distribution of blood to rodent neocortex. Proc Natl Acad Sci U S A 107(28):12670–12675. doi: 10.1073/pnas.1007239107 PubMedCentralPubMedGoogle Scholar
  5. Blinder P, Tsai P, Kaufhold J, Knutsen P, Suhl H, Kleinfeld D (2013) The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow. Nat Neurosci 16(7):889–897. doi: 10.1038/nn.3426 PubMedCentralPubMedGoogle Scholar
  6. Boyden E, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263–1268. doi: 10.1038/nn1525 PubMedGoogle Scholar
  7. Brown C, Li P, Boyd J, Delaney K, Murphy T (2007) Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke. J Neurosci 27(15):4101–4110. doi: 10.1523/jneurosci.4295-06.2007 PubMedGoogle Scholar
  8. Cai D, Cohen K, Luo T, Lichtman J, Sanes J (2013) Improved tools for the Brainbow toolbox. Nat Methods 10(6):540–547. doi: 10.1038/nmeth.2450 PubMedCentralGoogle Scholar
  9. Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D (1994) Green fluorescent protein as a marker for gene expression. Science (New York, NY) 263(5148):802–805. doi: 10.1126/science.8303295 Google Scholar
  10. Chen J, Nedivi E (2013) Highly specific structural plasticity of inhibitory circuits in the adult neocortex. Neuroscientist 19(4):384–393. doi: 10.1177/1073858413479824 PubMedGoogle Scholar
  11. Chen J, Flanders G, Lee W-CA, Lin W, Nedivi E (2011) Inhibitory dendrite dynamics as a general feature of the adult cortical microcircuit. J Neurosci 31(35):12437–12443. doi: 10.1523/JNEUROSCI.0420-11.2011 PubMedCentralPubMedGoogle Scholar
  12. Chen Q, Cichon J, Wang W, Qiu L, Lee S-JR, Campbell N, Destefino N, Goard M, Fu Z, Yasuda R, Looger L, Arenkiel B, Gan W-B, Feng G (2012) Imaging neural activity using Thy1-GCaMP transgenic mice. Neuron 76(2):297–308. doi: 10.1016/j.neuron.2012.07.011 PubMedCentralPubMedGoogle Scholar
  13. Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499(7458):295–300. doi: 10.1038/nature12354 PubMedCentralPubMedGoogle Scholar
  14. Cheng A, Gonçalves JT, Golshani P, Arisaka K, Portera-Cailliau C (2011) Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat Methods 8(2):139–142. doi: 10.1038/nmeth.1552 PubMedCentralPubMedGoogle Scholar
  15. Chow D, Groszer M, Pribadi M, Machniki M, Carmichael S, Liu X, Trachtenberg J (2009) Laminar and compartmental regulation of dendritic growth in mature cortex. Nat Neurosci 12(2):116–124. doi: 10.1038/nn.2255 PubMedCentralPubMedGoogle Scholar
  16. Cohen LB, Salzberg BM (1978) Optical measurement of membrane potential. Rev Physiol Biochem Pharmacol 83:35–88PubMedGoogle Scholar
  17. Cruz-Martin A, Crespo M, Portera-Cailliau C (2010) Delayed stabilization of dendritic spines in fragile X mice. J Neurosci 30(23):7793–8596. doi: 10.1523/jneurosci.0577-10.2010 PubMedCentralPubMedGoogle Scholar
  18. De Roo M, Klauser P, Muller D (2008) LTP promotes a selective long-term stabilization and clustering of dendritic spines. PLoS Biol 6(9):e219. doi: 10.1371/journal.pbio.0060219 PubMedCentralPubMedGoogle Scholar
  19. Deisseroth K (2011) Optogenetics. Nat Methods 8(1):26–29. doi: 10.1038/nmeth.f.324 PubMedGoogle Scholar
  20. Denk W (1994) Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions. Proc Natl Acad Sci U S A 91(14):6629–6633. doi: 10.1073/pnas.91.14.6629 PubMedCentralPubMedGoogle Scholar
  21. Denk W, Strickler J, Webb W (1990) Two-photon laser scanning fluorescence microscopy. Science (New York, NY) 248(4951):73–76. doi: 10.1126/science.2321027 Google Scholar
  22. Desai M, Kahn I, Knoblich U, Bernstein J, Atallah H, Yang A, Kopell N, Buckner R, Graybiel A, Moore C, Boyden E (2011) Mapping brain networks in awake mice using combined optical neural control and fMRI. J Neurophysiol 105(3):1393–1405. doi: 10.1152/jn.00828.2010 PubMedCentralPubMedGoogle Scholar
  23. Dittgen T, Nimmerjahn A, Komai S, Licznerski P, Waters J, Margrie T, Helmchen F, Denk W, Brecht M, Osten P (2004) Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci U S A 101(52):18206–18211. doi: 10.1073/pnas.0407976101 PubMedCentralPubMedGoogle Scholar
  24. Dixit R, Lu F, Cantrup R, Gruenig N, Langevin L, Kurrasch D, Schuurmans C (2011) Efficient gene delivery into multiple CNS territories using in utero electroporation. J Vis Exp (52). doi: 10.3791/2957
  25. Dong J, Revilla-Sanchez R, Moss S, Haydon P (2010) Multiphoton in vivo imaging of amyloid in animal models of Alzheimer’s disease. Neuropharmacology 59(4–5):268–275. doi: 10.1016/j.neuropharm.2010.04.007 PubMedCentralPubMedGoogle Scholar
  26. Drew P, Shih A, Driscoll J, Knutsen P, Blinder P, Davalos D, Akassoglou K, Tsai P, Kleinfeld D (2010) Chronic optical access through a polished and reinforced thinned skull. Nat Methods 7(12):981–984. doi: 10.1038/nmeth.1530 PubMedCentralPubMedGoogle Scholar
  27. Drew P, Shih A, Kleinfeld D (2011) Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity. Proc Natl Acad Sci U S A 108(20):8473–8478. doi: 10.1073/pnas.1100428108 PubMedCentralPubMedGoogle Scholar
  28. Farrar M, Bernstein I, Schlafer D, Cleland T, Fetcho J, Schaffer C (2012) Chronic in vivo imaging in the mouse spinal cord using an implanted chamber. Nat Methods 9(3):297–302. doi: 10.1038/nmeth.1856 PubMedCentralPubMedGoogle Scholar
  29. Feng G, Mellor R, Bernstein M, Keller-Peck C, Nguyen Q, Wallace M, Nerbonne J, Lichtman J, Sanes J (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28(1):41–92PubMedGoogle Scholar
  30. Fenno L, Yizhar O, Deisseroth K (2011) The development and application of optogenetics. Annu Rev Neurosci 34:389–412. doi: 10.1146/annurev-neuro-061010-113817 PubMedGoogle Scholar
  31. Fenrich K, Weber P, Hocine M, Zalc M, Rougon G, Debarbieux F (2012) Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows. J Physiol 590(Pt 16):3665–3675. doi: 10.1113/jphysiol.2012.230532 PubMedCentralPubMedGoogle Scholar
  32. Fisher JA, Salzberg BM, Yodh AG (2005) Near infrared two-photon excitation cross-sections of voltage-sensitive dyes. J Neurosci Methods 148(1):94–102. doi: 10.1016/j.jneumeth.2005.06.027 PubMedGoogle Scholar
  33. Flusberg B, Nimmerjahn A, Cocker E, Mukamel E, Barretto R, Ko T, Burns L, Jung J, Schnitzer M (2008) High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat Methods 5(11):935–943. doi: 10.1038/nmeth.1256 PubMedCentralPubMedGoogle Scholar
  34. Fu M, Yu X, Lu J, Zuo Y (2012) Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo. Nature 483(7387):92–97. doi: 10.1038/nature10844 PubMedCentralPubMedGoogle Scholar
  35. Ghiran I (2011) Introduction to fluorescence microscopy. Methods Mol Biol (Clifton, NJ) 689:93–136. doi: 10.1007/978-1-60761-950-5_7 Google Scholar
  36. Gobel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4(1):73–79. doi: 10.1038/nmeth989 PubMedGoogle Scholar
  37. Golshani P, Portera-Cailliau C (2008) In vivo 2-photon calcium imaging in layer 2/3 of mice. J Vis Exp (13). doi: 10.3791/681
  38. Golshani P, Gonçalves J, Khoshkhoo S, Mostany R, Smirnakis S, Portera-Cailliau C (2009) Internally mediated developmental desynchronization of neocortical network activity. J Neurosci 29(35):10890–10899. doi: 10.1523/jneurosci.2012-09.2009 PubMedCentralPubMedGoogle Scholar
  39. Gonçalves J, Anstey J, Golshani P, Portera-Cailliau C (2013) Circuit level defects in the developing neocortex of Fragile X mice. Nat Neurosci 16(7):903–909. doi: 10.1038/nn.3415 PubMedGoogle Scholar
  40. Grewe BF, Helmchen F (2009) Optical probing of neuronal ensemble activity. Curr Opin Neurobiol 19(5):520–529. doi: 10.1016/j.conb.2009.09.003 PubMedGoogle Scholar
  41. 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(5):399–405PubMedGoogle Scholar
  42. Grewe BF, Voigt FF, Van’t Hoff M, Helmchen F (2011) Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens. Biomed Opt Express 2(7):2035–2046PubMedCentralPubMedGoogle Scholar
  43. Grienberger C, Konnerth A (2012) Imaging calcium in neurons. Neuron 73(5):862–885. doi: 10.1016/j.neuron.2012.02.011 PubMedGoogle Scholar
  44. Grutzendler J, Kasthuri N, Gan W-B (2002) Long-term dendritic spine stability in the adult cortex. Nature 420(6917):812–818. doi: 10.1038/nature01276 PubMedGoogle Scholar
  45. Guo D, Arnspiger S, Rensing N, Wong M (2012) Brief seizures cause dendritic injury. Neurobiol Dis 45(1):348–355. doi: 10.1016/j.nbd.2011.08.020 PubMedCentralPubMedGoogle Scholar
  46. Harrison T, Ayling O, Murphy T (2012) Distinct cortical circuit mechanisms for complex forelimb movement and motor map topography. Neuron 74(2):397–409. doi: 10.1016/j.neuron.2012.02.028 PubMedGoogle Scholar
  47. Harvey C, Svoboda K (2007) Locally dynamic synaptic learning rules in pyramidal neuron dendrites. Nature 450(7173):1195–1200. doi: 10.1038/nature06416 PubMedCentralPubMedGoogle Scholar
  48. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–972. doi: 10.1038/nmeth818 PubMedGoogle Scholar
  49. Holtmaat A, Svoboda K (2009) Experience-dependent structural synaptic plasticity in the mammalian brain. Nat Rev Neurosci 10(9):647–705. doi: 10.1038/nrn2699 PubMedGoogle Scholar
  50. Holtmaat A, Trachtenberg J, Wilbrecht L, Shepherd G, Zhang X, Knott G, Svoboda K (2005) Transient and persistent dendritic spines in the neocortex in vivo. Neuron 45(2):279–370. doi: 10.1016/j.neuron.2005.01.003 PubMedGoogle Scholar
  51. Holtmaat A, Wilbrecht L, Knott G, Welker E, Svoboda K (2006) Experience-dependent and cell-type-specific spine growth in the neocortex. Nature 441(7096):979–1062. doi: 10.1038/nature04783 PubMedGoogle Scholar
  52. Holtmaat A, Bonhoeffer T, Chow D, Chuckowree J, De Paola V, Hofer S, Hübener M, Keck T, Knott G, Lee W-CA, Mostany R, Mrsic-Flogel T, Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg J, Wilbrecht L (2009) Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 4(8):1128–1172. doi: 10.1038/nprot.2009.89 PubMedCentralPubMedGoogle Scholar
  53. Horton NG, Wang K, Kobat D, Clark CG, Wise FW, Schaffer CB, Xu C (2013) In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat Photonics 7(3):205–209. doi: 10.1038/Nphoton.2012.336 Google Scholar
  54. Huber D, Petreanu L, Ghitani N, Ranade S, Hromádka T, Mainen Z, Svoboda K (2008) Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451(7174):61–64. doi: 10.1038/nature06445 PubMedCentralPubMedGoogle Scholar
  55. Hughes E, Kang S, Fukaya M, Bergles D (2013) Oligodendrocyte progenitors balance growth with self-repulsion to achieve homeostasis in the adult brain. Nat Neurosci 16(6):668–676. doi: 10.1038/nn.3390 PubMedGoogle Scholar
  56. Ji N, Sato TR, Betzig E (2012) Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex. Proc Natl Acad Sci U S A 109(1):22–27. doi: 10.1073/pnas.1109202108 PubMedCentralPubMedGoogle Scholar
  57. Jia H, Rochefort NL, Chen X, Konnerth A (2010) Dendritic organization of sensory input to cortical neurons in vivo. Nature 464(7293):1307–1312. doi: 10.1038/nature08947 PubMedGoogle Scholar
  58. Judkewitz B, Rizzi M, Kitamura K, Häusser M (2009) Targeted single-cell electroporation of mammalian neurons in vivo. Nat Protoc 4(6):862–869. doi: 10.1038/nprot.2009.56 PubMedGoogle Scholar
  59. Jung JC, Schnitzer MJ (2003) Multiphoton endoscopy. Opt Lett 28(11):902–904PubMedGoogle Scholar
  60. Kawakami R, Sawada K, Sato A, Hibi T, Kozawa Y, Sato S, Yokoyama H, Nemoto T (2013) Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser. Sci Rep 3:1014. doi: 10.1038/Srep01014 PubMedCentralPubMedGoogle Scholar
  61. Keck T, Mrsic-Flogel T, Vaz Afonso M, Eysel U, Bonhoeffer T, Hübener M (2008) Massive restructuring of neuronal circuits during functional reorganization of adult visual cortex. Nat Neurosci 11(10):1162–1169. doi: 10.1038/nn.2181 PubMedGoogle Scholar
  62. Kelly E, Majewska A (2010) Chronic imaging of mouse visual cortex using a thinned-skull preparation. Journal of visualized experiments. J Vis Exp (44). doi: 10.3791/2060
  63. Kerr JN, Greenberg D, Helmchen F (2005) Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci U S A 102(39):14063–14068. doi: 10.1073/pnas.0506029102 PubMedCentralPubMedGoogle Scholar
  64. Kerschensteiner M, Schwab M, Lichtman J, Misgeld T (2005) In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med 11(5):572–577. doi: 10.1038/nm1229 PubMedGoogle Scholar
  65. Kim J, Jiang N, Tadokoro C, Liu L, Ransohoff R, Lafaille J, Dustin M (2010) Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites. J Immunol Methods 352(1–2):89–100. doi: 10.1016/j.jim.2009.09.007 PubMedCentralPubMedGoogle Scholar
  66. Kleinfeld D, Delaney KR (1996) Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes. J Comp Neurol 375(1):89–108. doi: 10.1002/(SICI)1096-9861(19961104)375:1<89::AID-CNE6>3.0.CO;2-K PubMedGoogle Scholar
  67. Kleinfeld D, Mitra P, Helmchen F, Denk W (1998) 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):15741–15747PubMedCentralPubMedGoogle Scholar
  68. Kobat D, Horton N, Xu C (2011) In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J Biomed Opt 16(10):106014PubMedGoogle Scholar
  69. Kovalchuk Y, Garaschuk O (2012) Two-photon chloride imaging using MQAE in vitro and in vivo. Cold Spring Harb Protoc 2012(7):778–785. doi: 10.1101/pdb.prot070037 PubMedGoogle Scholar
  70. Kuhn B, Denk W, Bruno RM (2008) In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness. Proc Natl Acad Sci U S A 105(21):7588–7593. doi: 10.1073/pnas.0802462105 PubMedCentralPubMedGoogle Scholar
  71. Lai C, Franke T, Gan W-B (2012) Opposite effects of fear conditioning and extinction on dendritic spine remodelling. Nature 483(7387):87–91. doi: 10.1038/nature10792 PubMedGoogle Scholar
  72. Lakowicz JR (2010) Principles of fluorescence spectroscopy, 3rd edn. Springer, New YorkGoogle Scholar
  73. Lattarulo C, Thyssen D, Kuchibholta K, Hyman B, Bacskaiq B (2011) Microscopic imaging of intracellular calcium in live cells using lifetime-based ratiometric measurements of Oregon Green BAPTA-1. Methods Mol Biol (Clifton, NJ) 793:377–389. doi: 10.1007/978-1-61779-328-8_25 Google Scholar
  74. Lendvai B, Stern E, Chen B, Svoboda K (2000) Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature 404(6780):876–881. doi: 10.1038/35009107 PubMedGoogle Scholar
  75. Livet J, Weissman T, Kang H, Draft R, Lu J, Bennis R, Sanes J, Lichtman J (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450(7166):56–62. doi: 10.1038/nature06293 PubMedGoogle Scholar
  76. Majewska A, Yiu G, Yuste R (2000) A custom-made two-photon microscope and deconvolution system. Pflugers Arch 441(2–3):398–408. doi: 10.1007/s004240000435 PubMedGoogle Scholar
  77. Majewska A, Newton J, Sur M (2006) Remodeling of synaptic structure in sensory cortical areas in vivo. J Neurosci 26(11):3021–3029. doi: 10.1523/JNEUROSCI.4454-05.2006 PubMedGoogle Scholar
  78. Marshel JH, Kaye AP, Nauhaus I, Callaway EM (2012) Anterior-posterior direction opponency in the superficial mouse lateral geniculate nucleus. Neuron 76(4):713–720. doi: 10.1016/J.Neuron.2012.09.021 PubMedCentralPubMedGoogle Scholar
  79. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887. doi: 10.1038/42264 PubMedGoogle Scholar
  80. Mizrahi A, Katz L (2003) Dendritic stability in the adult olfactory bulb. Nat Neurosci 6(11):1201–1207. doi: 10.1038/nn1133 PubMedGoogle Scholar
  81. Mizrahi A, Crowley JC, Shtoyerman E, Katz LC (2004) High-resolution in vivo imaging of hippocampal dendrites and spines. J Neurosci 24(13):3147–3151. doi: 10.1523/Jneurosci.5218-03.2004 PubMedGoogle Scholar
  82. Mostany R, Portera-Cailliau C (2008a) A craniotomy surgery procedure for chronic brain imaging. Journal of visualized experiments. J Vis Exp (12). doi: 10.3791/680
  83. Mostany R, Portera-Cailliau C (2008b) A method for 2-photon imaging of blood flow in the neocortex through a cranial window. Journal of visualized experiments. J Vis Exp (12). doi: 10.3791/678
  84. Mostany R, Portera-Cailliau C (2011) Absence of large-scale dendritic plasticity of layer 5 pyramidal neurons in peri-infarct cortex. J Neurosci 31(5):1734–1742. doi: 10.1523/jneurosci.4386-10.2011 PubMedGoogle Scholar
  85. Mostany R, Chowdhury T, Johnston D, Portonovo S, Carmichael S, Portera-Cailliau C (2010) Local hemodynamics dictate long-term dendritic plasticity in peri-infarct cortex. J Neurosci 30(42):14116–14142. doi: 10.1523/jneurosci.3908-10.2010 PubMedGoogle Scholar
  86. Mostany R, Anstey J, Crump K, Maco B, Knott G, Portera-Cailliau C (2013) Altered synaptic dynamics during normal brain aging. J Neurosci 33(9):4094–4104. doi: 10.1523/JNEUROSCI.4825-12.2013 PubMedGoogle Scholar
  87. Nikolenko V, Nemet B, Yuste R (2003) A two-photon and second-harmonic microscope. Methods (San Diego, Calif) 30(1):3–18Google Scholar
  88. Nimchinsky E, Sabatini B, Svoboda K (2002) Structure and function of dendritic spines. Annu Rev Physiol 64:313–353. doi: 10.1146/annurev.physiol.64.081501.160008 PubMedGoogle Scholar
  89. Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science (New York, NY) 308(5726):1314–1318. doi: 10.1126/science.1110647 Google Scholar
  90. Nishimura N, Schaffer C, Friedman B, Tsai P, Lyden P, Kleinfeld D (2006) Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke. Nat Methods 3(2):99–207. doi: 10.1038/nmeth844 PubMedGoogle Scholar
  91. Oheim M, Beaurepaire E, Chaigneau E, Mertz J, Charpak S (2001) Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J Neurosci Methods 111(1):29–37PubMedGoogle Scholar
  92. Orbach HS, Cohen LB (1983) Optical monitoring of activity from many areas of the in vitro and in vivo salamander olfactory bulb: a new method for studying functional organization in the vertebrate central nervous system. J Neurosci 3(11):2251–2262PubMedGoogle Scholar
  93. Peterka DS, Takahashi H, Yuste R (2011) Imaging voltage in neurons. Neuron 69(1):9–21. doi: 10.1016/j.neuron.2010.12.010 PubMedCentralPubMedGoogle Scholar
  94. Petersen C, Grinvald A, Sakmann B (2003) Spatiotemporal dynamics of sensory responses in layer 2/3 of rat barrel cortex measured in vivo by voltage-sensitive dye imaging combined with whole-cell voltage recordings and neuron reconstructions. J Neurosci 23(4):1298–1607PubMedGoogle Scholar
  95. Petreanu L, Huber D, Sobczyk A, Svoboda K (2007) Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat Neurosci 10(5):663–668. doi: 10.1038/nn1891 PubMedGoogle Scholar
  96. Potter SM (2000) Two-photon microscopy for 4D imaging of living neurons. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons: a laboratory manual, 1st edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 20.21–20.16Google Scholar
  97. Poulet J, Fernandez L, Crochet S, Petersen C (2012) Thalamic control of cortical states. Nat Neurosci 15(3):370–372. doi: 10.1038/nn.3035 PubMedGoogle Scholar
  98. Rensing N, Ouyang Y, Yang X-F, Yamada K, Rothman S, Wong M (2005) In vivo imaging of dendritic spines during electrographic seizures. Ann Neurol 58(6):888–898. doi: 10.1002/ana.20658 PubMedGoogle Scholar
  99. Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240(1):237–246. doi: 10.1006/dbio.2001.0439 PubMedGoogle Scholar
  100. Sato T, Muroyama Y, Saito T (2013) Inducible gene expression in postmitotic neurons by an in vivo electroporation-based tetracycline system. J Neurosci 214(2):170–176. doi: 10.1016/j.jneumeth.2013.01.014 Google Scholar
  101. Schaffer C, Friedman B, Nishimura N, Schroeder L, Tsai P, Ebner F, Lyden P, Kleinfeld D (2006) Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. PLoS Biol 4(2):e22. doi: 10.1371/journal.pbio.0040022 PubMedCentralPubMedGoogle Scholar
  102. Shcherbakova DM, Subach OM, Verkhusha VV (2012) Red fluorescent proteins: advanced imaging applications and future design. Angew Chem Int Ed Engl 51(43):10724–10738. doi: 10.1002/anie.201200408 PubMedGoogle Scholar
  103. Shih A, Friedman B, Drew P, Tsai P, Lyden P, Kleinfeld D (2009) Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke. J Cereb Blood Flow Metab 29(4):738–789. doi: 10.1038/jcbfm.2008.166 PubMedCentralPubMedGoogle Scholar
  104. Shih A, Driscoll J, Drew P, Nishimura N, Schaffer C, Kleinfeld D (2012a) Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J Cereb Blood Flow Metab 32(7):1277–1309. doi: 10.1038/jcbfm.2011.196 PubMedCentralPubMedGoogle Scholar
  105. Shih A, Mateo C, Drew P, Tsai P, Kleinfeld D (2012b) A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J Vis Exp (61). doi: 10.3791/3742
  106. Shimomura O, Johnson F, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239. doi: 10.1002/jcp.1030590302 PubMedGoogle Scholar
  107. Silasi G, Boyd J, Ledue J, Murphy T (2013) Improved methods for chronic light-based motor mapping in mice: automated movement tracking with accelerometers, and chronic EEG recording in a bilateral thin-skull preparation. Front Neural Circuits 7:123. doi: 10.3389/fncir.2013.00123 PubMedCentralPubMedGoogle Scholar
  108. Spence MTZ, Johnson ID (2010) The molecular probes handbook: a guide to fluorescent probes and labeling technologies, 11th edn. Live Technologies Corporation, CarlsbadGoogle Scholar
  109. Spires-Jones T, Meyer-Luehmann M, Osetek J, Jones P, Stern E, Bacskai B, Hyman B (2007) Impaired spine stability underlies plaque-related spine loss in an Alzheimer’s disease mouse model. Am J Pathol 171(4):1304–1315. doi: 10.2353/ajpath.2007.070055 PubMedCentralPubMedGoogle Scholar
  110. Stettler D, Yamahachi H, Li W, Denk W, Gilbert C (2006) Axons and synaptic boutons are highly dynamic in adult visual cortex. Neuron 49(6):877–887. doi: 10.1016/j.neuron.2006.02.018 PubMedGoogle Scholar
  111. Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100(12):7319–7343. doi: 10.1073/pnas.1232232100 PubMedCentralPubMedGoogle Scholar
  112. Svoboda K, Denk W, Kleinfeld D, Tank D (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385(6612):161–165. doi: 10.1038/385161a0 PubMedGoogle Scholar
  113. Sword J, Masuda T, Croom D, Kirov S (2013) Evolution of neuronal and astroglial disruption in the peri-contusional cortex of mice revealed by in vivo two-photon imaging. Brain 136(Pt 5):1446–1461. doi: 10.1093/brain/awt026 PubMedCentralPubMedGoogle Scholar
  114. Tian L, Hires SA, Looger LL (2012) Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harb Protoc 2012(6):647–656. doi: 10.1101/pdb.top069609 PubMedGoogle Scholar
  115. Trachtenberg J, Chen B, Knott G, Feng G, Sanes J, Welker E, Svoboda K (2002) Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420(6917):788–882. doi: 10.1038/nature01273 PubMedGoogle Scholar
  116. Tsai PS, Kleinfeld D (2009) In vivo two-photon laser scanning microscopy with concurrent plasma-mediated ablation: principles and hardware realization. In: Frostig RD (ed) In vivo optical imaging of brain function, 2nd edn. CRC Press, Boca Raton, pp 59–115Google Scholar
  117. Tsien RY (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19(11):2396–2404PubMedGoogle Scholar
  118. Tsien R (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. doi: 10.1146/annurev.biochem.67.1.509 PubMedGoogle Scholar
  119. Weller T, Coons A (1954) Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc Soc Exp Biol Med 86(4):789–794. doi: 10.3181/00379727-86-21235 PubMedGoogle Scholar
  120. Xu T, Yu X, Perlik A, Tobin W, Zweig J, Tennant K, Jones T, Zuo Y (2009) Rapid formation and selective stabilization of synapses for enduring motor memories. Nature 462(7275):915–924. doi: 10.1038/nature08389 PubMedCentralPubMedGoogle Scholar
  121. Yang G, Pan F, Parkhurst C, Grutzendler J, Gan W-B (2010) Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5(2):201–208. doi: 10.1038/nprot.2009.222 PubMedGoogle Scholar
  122. Yizhar O, Fenno L, Davidson T, Mogri M, Deisseroth K (2011) Optogenetics in neural systems. Neuron 71(1):9–34. doi: 10.1016/j.neuron.2011.06.004 PubMedGoogle Scholar
  123. Zariwala H, Borghuis B, Hoogland T, Madisen L, Tian L, De Zeeuw C, Zeng H, Looger L, Svoboda K, Chen T-W (2012) A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo. J Neurosci 32(9):3131–3141. doi: 10.1523/JNEUROSCI.4469-11.2012 PubMedCentralPubMedGoogle Scholar
  124. Zhang F, Wang L-P, Brauner M, Liewald J, Kay K, Watzke N, Wood P, Bamberg E, Nagel G, Gottschalk A, Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature 446(7136):633–639. doi: 10.1038/nature05744 PubMedGoogle Scholar
  125. Zuo Y, Yang G, Kwon E, Gan W-B (2005) Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex. Nature 436(7048):261–266. doi: 10.1038/nature03715 PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Laboratory of GeneticsSalk Institute for Biological StudiesLa JollaUSA
  2. 2.Department of PharmacologyTulane University School of MedicineNew OrleansUSA

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