Imaging in vivo: watching the brain in action

Key Points

  • In vivo imaging has been central to biology from its beginning, with optical imaging being the best compromise between damage and resolution.

  • Through the application of multi-photon excitation, high-resolution imaging has become possible in scattering tissue such as the brain.

  • This has enabled the imaging of cellular activity in the intact cerebral cortex both in single-cell compartments and populations of neurons.

  • Careful attention to experimental parameters is needed to reliably detect action potentials.

  • In addition to the detection of neural activation on the millisecond scale, multi-photon microscopy can be used to measure the morphological stability and plasticity of neuronal processes on the time scale of months right down to the single-spine and single-synaptic-bouton level.

  • In the future, multi-photon imaging should enable the detection of signalling in neuron populations distributed in all three spatial dimensions and in behaving animals.


The appeal of in vivo cellular imaging to any neuroscientist is not hard to understand: it is almost impossible to isolate individual neurons while keeping them and their complex interactions with surrounding tissue intact. These interactions lead to the complex network dynamics that underlie neural computation which, in turn, forms the basis of cognition, perception and consciousness. In vivo imaging allows the study of both form and function in reasonably intact preparations, often with subcellular spatial resolution, a time resolution of milliseconds and a purview of months. Recently, the limits of what can be achieved in vivo have been pushed into terrain that was previously only accessible in vitro, due to advances in both physical-imaging technology and the design of molecular contrast agents.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Measuring Ca2+ transients from dendrites and neuronal populations in vivo.
Figure 2: Spiking activity in populations of neurons can be inferred from Ca2+ transients, with single-action-potential resolution.
Figure 3: The persistence of dendritic spines in adult mice, revealed by in vivo imaging.


  1. 1

    Dobell, C. Antony van Leeuwenhoek and his “Little Animals” (John Bale, Sons and Danielsson, London, 1932).

    Google Scholar 

  2. 2

    Brown, R. A brief account of microscopical observations made in the months of June, July and August, 1827, on the particles contained in the pollen of plants; and on the general existence of active molecules in organic and inorganic bodies. Phil. Mag. 4, 161–173 (1828).

    Google Scholar 

  3. 3

    Callaghan, P. T. Principles of Nuclear Magnetic Resonance Microscopy (Clarendon, 1991).

    Google Scholar 

  4. 4

    Schmidt, K. C. & Smith, C. B. Resolution, sensitivity and precision with autoradiography and small animal positron emission tomography: implications for functional brain imaging in animal research. Nucl. Med. Biol. 32, 719–725 (2005).

    CAS  PubMed  Google Scholar 

  5. 5

    Romans, L. E. Introduction to Computed Tomography (Lippincott, Williams & Wilkins, London, 1995).

    Google Scholar 

  6. 6

    Fujimoto, J. G. Optical coherence tomography for ultrahigh resolution in vivo imaging. Nature Biotechnol. 21, 1361–1367 (2003).

    CAS  Google Scholar 

  7. 7

    Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990). This study provided the first demonstration that two-photon excitation can be used to image fluorescently stained living cells and cellular substructures.

    CAS  Google Scholar 

  8. 8

    Denk, W. et al. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy. J. Neurosci. Methods 54, 151–162 (1994).

    CAS  PubMed  Google Scholar 

  9. 9

    Denk, W. Two-photon excitation in functional biological imaging. J. Biomed. Opt. 1, 296–304 (1996).

    CAS  PubMed  Google Scholar 

  10. 10

    Denk, W. & Svoboda, K. Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357 (1997).

    CAS  PubMed  Google Scholar 

  11. 11

    Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nature Methods 2, 932–940 (2005). This reference and reference 10 are important technically orientated reviews of the application of two-photon excitation to neurobiology, and provide a good introduction to the technique for a general readership.

    CAS  Google Scholar 

  12. 12

    Williams, R. M., Piston, D. W. & Webb, W. W. 2-photon molecular-excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J. 8, 804–813 (1994).

    CAS  PubMed  Google Scholar 

  13. 13

    Denk, W., Piston, D. W. & Webb, W. W. in The Handbook of Confocal Microscopy (ed. Pawley, J.) 445–458 (Plenum, New York, 1995).

    Google Scholar 

  14. 14

    Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: multiphoton microscopy in the biosciences. Nature Biotechnol. 21, 1368–1376 (2003).

    Google Scholar 

  15. 15

    Diaspro, A., Chirico, G. & Collini, M. Two-photon fluorescence excitation and related techniques in biological microscopy. Q. Rev. Biophys. 38, 97–166 (2005).

    CAS  PubMed  Google Scholar 

  16. 16

    Oheim, M., Michael, D. J., Geisbauer, M., Madsen, D. & Chow, R. H. Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches. Adv. Drug Deliv. Rev. 58, 788–808 (2006).

    CAS  PubMed  Google Scholar 

  17. 17

    Svoboda, K. & Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).

    CAS  PubMed  Google Scholar 

  18. 18

    Helmchen, F., Svoboda, K., Denk, W. & Tank, D. W. In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nature Neurosci. 2, 989–996 (1999).

    CAS  PubMed  Google Scholar 

  19. 19

    Svoboda, K., Helmchen, F., Denk, W. & Tank, D. W. Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo. Nature Neurosci. 2, 65–73 (1999).

    CAS  PubMed  Google Scholar 

  20. 20

    Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).

    CAS  Google Scholar 

  21. 21

    Grutzendler, J., Kasthuri, N. & Gan, W. B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    CAS  Google Scholar 

  22. 22

    Trachtenberg, J. T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002). This reference and reference 21 used MPM in combination with genetically labelled neurons to follow changes in dendritic spine morphology over many months. This enabled the quantification of spine turnover rates (see also references 23–26 and 135).

    CAS  Google Scholar 

  23. 23

    Xu, H. T., Pan, F., Yang, G. & Gan, W. B. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nature Neurosci. 10, 549–551 (2007).

    CAS  PubMed  Google Scholar 

  24. 24

    Knott, G. W., Holtmaat, A., Wilbrecht, L., Welker, E. & Svoboda, K. Spine growth precedes synapse formation in the adult neocortex in vivo. Nature Neurosci. 9, 1117–1124 (2006).

    CAS  PubMed  Google Scholar 

  25. 25

    Grutzendler, J. & Gan, W. B. Two-photon imaging of synaptic plasticity and pathology in the living mouse brain. NeuroRx 3, 489–496 (2006).

    PubMed  PubMed Central  Google Scholar 

  26. 26

    Zuo, Y., Yang, G., Kwon, E. & Gan, W. B. Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex. Nature 436, 261–265 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Portera-Cailliau, C., Weimer, R. M., De Paola, V., Caroni, P. & Svoboda, K. Diverse modes of axon elaboration in the developing neocortex. PloS Biol. 3, 1473–1487 (2005).

    CAS  Google Scholar 

  28. 28

    Holtmaat, A. J. et al. Transient and persistent dendritic spines in the neocortex in vivo. Neuron 45, 279–291 (2005).

    CAS  Google Scholar 

  29. 29

    Stettler, D. D., Yamahachi, H., Li, W., Denk, W. & Gilbert, C. D. Axons and synaptic boutons are highly dynamic in adult visual cortex. Neuron 49, 877–887 (2006).

    CAS  PubMed  Google Scholar 

  30. 30

    Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl Acad. Sci. USA 100, 7319–7324 (2003). This was the first study to image Ca2+ transients in vivo from neuronal populations bulk-loaded with membrane-permeable Ca2+-indicator dyes. Most commercially available Ca2+-indicator dyes were tried.

    CAS  Google Scholar 

  31. 31

    Kerr, J. N., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl Acad. Sci. USA 102, 14063–14068 (2005). Using simultaneous imaging and targeted electrical recordings, this study showed that it was possible to infer electrical activity with single-cell and single-AP accuracy from Ca2+ transients measured in populations of bulk-loaded neurons (see also references 35, 103 and 155).

    CAS  Google Scholar 

  32. 32

    Ohki, K., Chung, S., Ch'ng, Y. H., Kara, P. & Reid, R. C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005). Using MPM and Ca2+ imaging, this study showed that neurons in the cat visual cortex are precisely arranged according to their preferred orientation and direction, but that neurons in the rat visual cortex are not (see also reference 33).

    CAS  PubMed  Google Scholar 

  33. 33

    Ohki, K. et al. Highly ordered arrangement of single neurons in orientation pinwheels. Nature 442, 925–928 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Mrsic-Flogel, T. D. et al. Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity. Neuron 54, 961–972 (2007).

    CAS  PubMed  Google Scholar 

  35. 35

    Kerr, J. N. et al. Spatial organization of neuronal population responses in layer 2/3 of rat barrel cortex. J. Neurosci. 27, 13316–13328 (2007).

    CAS  PubMed  Google Scholar 

  36. 36

    Gabriel, M. A system for multiple unit recording during avoidance behavior of the rabbit. Physiol. Behav. 12, 145–148 (1974).

    CAS  PubMed  Google Scholar 

  37. 37

    Kruger, J. & Bach, M. Simultaneous recording with 30 microelectrodes in monkey visual cortex. Exp. Brain Res. 41, 191–194 (1981).

    CAS  PubMed  Google Scholar 

  38. 38

    Shoham, S., O'Connor D, H. & Segev, R. How silent is the brain: is there a “dark matter” problem in neuroscience? J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192, 777–784 (2006).

    PubMed  Google Scholar 

  39. 39

    Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    CAS  Google Scholar 

  40. 40

    Frostig, R. D., Lieke, E. E., Ts'o, D. Y. & Grinvald, A. Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals. Proc. Natl Acad. Sci. USA 87, 6082–6086 (1990).

    CAS  Google Scholar 

  41. 41

    Grinvald, A., Frostig, R. D., Siegel, R. M. & Bartfeld, E. High-resolution optical imaging of functional brain architecture in the awake monkey. Proc. Natl Acad. Sci. USA 88, 11559–11563 (1991).

    CAS  PubMed  Google Scholar 

  42. 42

    Grinvald, A., Lieke, E., Frostig, R. D., Gilbert, C. D. & Wiesel, T. N. Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature 324, 361–364 (1986).

    CAS  Google Scholar 

  43. 43

    Huang, D. et al. Optical coherence tomography. Science 254, 1178–1181 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Mertz, J. Nonlinear microscopy: new techniques and applications. Curr. Opin. Neurobiol. 14, 610–616 (2004).

    CAS  PubMed  Google Scholar 

  45. 45

    Franken, P. A. & Ward, J. F. Optical harmonics and nonlinear phenomena. Rev. Mod. Phys. 35, 23–39 (1963).

    CAS  Google Scholar 

  46. 46

    Minsky, M. Microscopy apparatus. US patent 3013467 (1961).

  47. 47

    Centonze, V. E. & White, J. G. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys. J. 75, 2015–2024 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Squirrell, J. M., Wokosin, D. L., White, J. G. & Bavister, B. D. Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nature Biotechnol. 17, 763–767 (1999).

    CAS  Google Scholar 

  49. 49

    Voie, A. H., Burns, D. H. & Spelman, F. A. Orthogonal-plane fluorescence optical sectioning — 3-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

    CAS  PubMed  Google Scholar 

  50. 50

    Voie, A. H. Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy. Hear. Res. 171, 119–128 (2002).

    PubMed  Google Scholar 

  51. 51

    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    CAS  Google Scholar 

  52. 52

    Creutzfeldt, O. D., Watanabe, S. & Lux, H. D. Relations between EEG phenomena and potentials of single cortical cells. I. Evoked responses after thalamic and erpicortical stimulation. Electroencephalogr. Clin. Neurophysiol. 20, 1–18 (1966).

    CAS  PubMed  Google Scholar 

  53. 53

    Watanabe, S., Konishi, M. & Creutzfeldt, O. D. Postsynaptic potentials in the cat's visual cortex following electrical stimulation of afferent pathways. Exp. Brain Res. 1, 272–283 (1966).

    CAS  PubMed  Google Scholar 

  54. 54

    Eccles, J. C., Llinas, R. & Sasaki, K. The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum. J. Physiol. 182, 268–296 (1966).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Jagadeesh, B., Gray, C. M. & Ferster, D. Visually evoked oscillations of membrane potential in cells of cat visual cortex. Science 257, 552–554 (1992).

    CAS  PubMed  Google Scholar 

  56. 56

    Brecht, M. & Sakmann, B. Whisker maps of neuronal subclasses of the rat ventral posterior medial thalamus, identified by whole-cell voltage recording and morphological reconstruction. J. Physiol. 538, 495–515 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Margrie, T. W., Brecht, M. & Sakmann, B. In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Arch. 444, 491–498 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Fee, M. S. Active stabilization of electrodes for intracellular recording in awake behaving animals. Neuron 27, 461–468 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Crochet, S. & Petersen, C. C. Correlating whisker behavior with membrane potential in barrel cortex of awake mice. Nature Neurosci. 9, 608–610 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Lee, A. K., Manns, I. D., Sakmann, B. & Brecht, M. Whole-cell recordings in freely moving rats. Neuron 51, 399–407 (2006).

    CAS  PubMed  Google Scholar 

  61. 61

    Wilson, C. J. & Groves, P. M. Spontaneous firing patterns of identified spiny neurons in the rat neostriatum. Brain Res. 220, 67–80 (1981).

    CAS  PubMed  Google Scholar 

  62. 62

    Wilson, C. J. & Kawaguchi, Y. The origins of two-state spontaneous membrane potential fluctuations of neostriatal spiny neurons. J. Neurosci. 16, 2397–2410 (1996).

    CAS  Google Scholar 

  63. 63

    Markram, H., Lubke, J., Frotscher, M., Roth, A. & Sakmann, B. Physiology and anatomy of synaptic connections between thick tufted pyramidal neurones in the developing rat neocortex. J. Physiol. 500, 409–440 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Magee, J. C. & Johnston, D. A synaptically controlled, associative signal for Hebbian plasticity in hippocampal neurons. Science 275, 209–213 (1997).

    CAS  Google Scholar 

  65. 65

    Larkum, M. E., Zhu, J. J. & Sakmann, B. A new cellular mechanism for coupling inputs arriving at different cortical layers. Nature 398, 338–341 (1999).

    CAS  Google Scholar 

  66. 66

    Cossart, R., Lkegaya, Y. & Yuste, R. Calcium imaging of cortical networks dynamics. Cell Calcium 37, 451–457 (2005).

    CAS  PubMed  Google Scholar 

  67. 67

    Grinvald, A., Anglister, L., Freeman, J. A., Hildesheim, R. & Manker, A. Real-time optical imaging of naturally evoked electrical activity in intact frog brain. Nature 308, 848–850 (1984).

    CAS  PubMed  Google Scholar 

  68. 68

    Shoham, D. et al. Imaging cortical dynamics at high spatial and temporal resolution with novel blue voltage-sensitive dyes. Neuron 24, 791–802 (1999). This paper, together with reference 67, described the use of voltage-sensitive dyes for imaging activity from large areas of the cortex in both awake and anaesthetized animals. The approach was then complemented with simultaneous electrical recordings (see reference 69).

    CAS  PubMed  Google Scholar 

  69. 69

    Arieli, A., Sterkin, A., Grinvald, A. & Aertsen, A. Dynamics of ongoing activity: explanation of the large variability in evoked cortical responses. Science 273, 1868–1871 (1996).

    CAS  Google Scholar 

  70. 70

    Kenet, T., Bibitchkov, D., Tsodyks, M., Grinvald, A. & Arieli, A. Spontaneously emerging cortical representations of visual attributes. Nature 425, 954–956 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71

    Xu, W., Huang, X., Takagaki, K. & Wu, J. Y. Compression and reflection of visually evoked cortical waves. Neuron 55, 119–129 (2007).

    PubMed  PubMed Central  Google Scholar 

  72. 72

    Petersen, C. C., Grinvald, A. & Sakmann, B. 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, 1298–1309 (2003).

    CAS  PubMed  Google Scholar 

  73. 73

    Grinvald, A. & Hildesheim, R. VSDI: a new era in functional imaging of cortical dynamics. Nature Rev. Neurosci. 5, 874–885 (2004).

    CAS  Google Scholar 

  74. 74

    Arieli, A. & Grinvald, A. Optical imaging combined with targeted electrical recordings, microstimulation, or tracer injections. J. Neurosci. Methods 116, 15–28 (2002).

    PubMed  Google Scholar 

  75. 75

    Civillico, E. F. & Contreras, D. Comparison of responses to electrical stimulation and whisker deflection using two different voltage-sensitive dyes in mouse barrel cortex in vivo. J. Membr. Biol. 208, 171–182 (2005).

    CAS  PubMed  Google Scholar 

  76. 76

    Ogawa, S. et al. intrinsic signal changes accompanying sensory stimulation — functional brain mapping with magnetic-resonance-imaging. Proc. Natl Acad. Sci. USA 89, 5951–5955 (1992).

    CAS  PubMed  Google Scholar 

  77. 77

    Hubener, G., Lambacher, A. & Fromherz, P. Anellated hemicyanine dyes with large symmetrical solvatochromism of absorption and fluorescence. J. Phys. Chem. B 107, 7896–7902 (2003).

    Google Scholar 

  78. 78

    Kuhn, B., Fromherz, P. & Denk, W. High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge. Biophys. J. 87, 631–639 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79

    Waters, J. & Helmchen, F. Boosting of action potential backpropagation by neocortical network activity in vivo. J. Neurosci. 24, 11127–11136 (2004).

    CAS  PubMed  Google Scholar 

  80. 80

    Waters, J., Larkum, M., Sakmann, B. & Helmchen, F. Supralinear Ca2+ influx into dendritic tufts of layer 2/3 neocortical pyramidal neurons in vitro and in vivo. J. Neurosci. 23, 8558–8567 (2003).

    CAS  Google Scholar 

  81. 81

    Charpak, S., Mertz, J., Beaurepaire, E., Moreaux, L. & Delaney, K. Odor-evoked calcium signals in dendrites of rat mitral cells. Proc. Natl Acad. Sci. USA 98, 1230–1234 (2001).

    CAS  PubMed  Google Scholar 

  82. 82

    Stuart, G., Spruston, N., Sakmann, B. & Hausser, M. Action potential initiation and backpropagation in neurons of the mammalian CNS. Trends Neurosci. 20, 125–131 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. 83

    Hausselt, S. E., Euler, T., Detwiler, P. B. & Denk, W. A dendrite-autonomous mechanism for direction selectivity in retinal starburst amacrine cells. PLoS Biol. 5, e185 (2007).

    PubMed  PubMed Central  Google Scholar 

  84. 84

    Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L. & Tank, D. W. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 43–57 (2007). This was the first study to image activity in populations of neurons stained with Ca2+-sensitive dye in the awake, head-fixed mouse. The mice were trained to walk on a rotating sphere while neuronal populations were simultaneously imaged.

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85

    Helmchen, F., Fee, M. S., Tank, D. W. & Denk, W. A miniature head-mounted two-photon microscope. High-resolution brain imaging in freely moving animals. Neuron 31, 903–912 (2001). This paper introduced a new method for imaging that provided cellular resolution in the awake, behaving rodent.

    CAS  PubMed  Google Scholar 

  86. 86

    Flusberg, B. A., Jung, J. C., Cocker, E. D., Anderson, E. P. & Schnitzer, M. J. In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope. Opt. Lett. 30, 2272–2274 (2005).

    PubMed  Google Scholar 

  87. 87

    Piyawattanametha, W. et al. Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror. Opt. Lett. 31, 2018–2020 (2006).

    PubMed  PubMed Central  Google Scholar 

  88. 88

    Sawinski, J. & Denk, W. Miniature random-access fiber scanner for in vivo multiphoton imaging. J. Appl. Phys. 102, 034701 (2007).

    Google Scholar 

  89. 89

    Adelsberger, H., Garaschuk, O. & Konnerth, A. Cortical calcium waves in resting newborn mice. Nature Neurosci. 8, 988–990 (2005).

    CAS  PubMed  Google Scholar 

  90. 90

    Murayama, M., Perez-Garci, E., Luscher, H. R. & Larkum, M. E. Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats. J. Neurophysiol. 98, 1791–1805 (2007).

    PubMed  PubMed Central  Google Scholar 

  91. 91

    Sobel, E. C. & Tank, D. W. In vivo Ca2+ dynamics in a cricket auditory neuron: an example of chemical computation. Science 263, 823–826 (1994).

    CAS  PubMed  Google Scholar 

  92. 92

    Single, S. & Borst, A. Dendritic integration and its role in computing image velocity. Science 281, 1848–1850 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. 93

    Haag, J., Denk, W. & Borst, A. Fly motion vision is based on Reichardt detectors regardless of the signal-to-noise ratio. Proc. Natl Acad. Sci. USA 101, 16333–16338 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    Kerr, R. et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).

    CAS  PubMed  Google Scholar 

  95. 95

    Ng, M. et al. Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36, 463–474 (2002).

    CAS  PubMed  Google Scholar 

  96. 96

    Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B. & Axel, R. Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112, 271–282 (2003).

    CAS  PubMed  Google Scholar 

  97. 97

    Higashijima, S., Masino, M. A., Mandel, G. & Fetcho, J. R. Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator. J. Neurophysiol. 90, 3986–3997 (2003).

    PubMed  Google Scholar 

  98. 98

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997). This was the first study to demonstrate that genetically encoded fluorescent proteins targeted to specific intracellular locations can be used to report intracellular changes in [Ca2+].

    CAS  PubMed  PubMed Central  Google Scholar 

  99. 99

    Tsien, R. Y. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290, 527–528 (1981).

    CAS  PubMed  Google Scholar 

  100. 100

    Smetters, D., Majewska, A. & Yuste, R. Detecting action potentials in neuronal populations with calcium imaging. Methods 18, 215–221 (1999).

    CAS  PubMed  Google Scholar 

  101. 101

    Friedrich, R. W. & Korsching, S. I. Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging. Neuron 18, 737–752 (1997).

    CAS  PubMed  Google Scholar 

  102. 102

    Friedrich, R. W. & Korsching, S. I. Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage-sensitive axon tracer. J. Neurosci. 18, 9977–9988 (1998).

    CAS  PubMed  Google Scholar 

  103. 103

    Sato, T. R., Gray, N. W., Mainen, Z. F. & Svoboda, K. The functional microarchitecture of the mouse barrel cortex. PLoS Biol. 5, e189 (2007).

    PubMed  PubMed Central  Google Scholar 

  104. 104

    Sullivan, M. R., Nimmerjahn, A., Sarkisov, D. V., Helmchen, F. & Wang, S. S. In vivo calcium imaging of circuit activity in cerebellar cortex. J. Neurophysiol. 94, 1636–1644 (2005).

    CAS  PubMed  Google Scholar 

  105. 105

    Niell, C. M. & Smith, S. J. Functional imaging reveals rapid development of visual response properties in the zebrafish tectum. Neuron 45, 941–951 (2005).

    CAS  PubMed  Google Scholar 

  106. 106

    Wachowiak, M., Denk, W. & Friedrich, R. W. Functional organization of sensory input to the olfactory bulb glomerulus analyzed by two-photon calcium imaging. Proc. Natl Acad. Sci. USA 101, 9097–9102 (2004).

    CAS  PubMed  Google Scholar 

  107. 107

    Li, J. et al. Early development of functional spatial maps in the zebrafish olfactory bulb. J. Neurosci. 25, 5784–5795 (2005).

    CAS  PubMed  Google Scholar 

  108. 108

    Yaksi, E., Judkewitz, B. & Friedrich, R. W. Topological reorganization of odor representations in the olfactory bulb. PLoS Biol. 5, e178 (2007).

    PubMed  PubMed Central  Google Scholar 

  109. 109

    Bonhoeffer, T. & Grinvald, A. Iso-orientation domains in cat visual-cortex are arranged in pinwheel-like patterns. Nature 353, 429–431 (1991). Using intrinsic optical imaging of the cat visual cortex, this study was the first to show the existence of orientation pinwheels.

    CAS  Google Scholar 

  110. 110

    Brecht, M., Roth, A. & Sakmann, B. Dynamic receptive fields of reconstructed pyramidal cells in layers 3 and 2 of rat somatosensory barrel cortex. J. Physiol. 553, 243–265 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. 111

    de Kock, C. P., Bruno, R. M., Spors, H. & Sakmann, B. Layer- and cell-type-specific suprathreshold stimulus representation in rat primary somatosensory cortex. J. Physiol. 581, 139–154 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. 112

    Nimmerjahn, A., Kirchhoff, F., Kerr, J. N. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nature Methods 1, 31–37 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113

    Delaney, K., Davison, I. & Denk, W. Odour-evoked [Ca2+] transients in mitral cell dendrites of frog olfactory glomeruli. Eur. J. Neurosci. 13, 1658–1672 (2001).

    CAS  PubMed  Google Scholar 

  114. 114

    Yuste, R. & Denk, W. Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684 (1995).

    CAS  Google Scholar 

  115. 115

    Denk, W., Yuste, R., Svoboda, K. & Tank, D. W. Imaging calcium dynamics in dendritic spines. Curr. Opin. Neurobiol. 6, 372–378 (1996).

    CAS  PubMed  Google Scholar 

  116. 116

    Hasan, M. T. et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, e163 (2004).

    PubMed  PubMed Central  Google Scholar 

  117. 117

    Diez-Garcia, J. et al. Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein. Eur. J. Neurosci. 22, 627–635 (2005).

    PubMed  Google Scholar 

  118. 118

    Heim, N. et al. Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor. Nature Methods 4, 127–129 (2007).

    CAS  PubMed  Google Scholar 

  119. 119

    Diez-Garcia, J., Akemann, W. & Knopfel, T. In vivo calcium imaging from genetically specified target cells in mouse cerebellum. Neuroimage 34, 859–869 (2007).

    PubMed  Google Scholar 

  120. 120

    Osten, P., Grinevich, V. & Cetin, A. in Conditional Mutagenesis: An Approach to Disease Models (Handbook of Experimental Pharmacology) (eds Feil, R. & Metzger, D.) 177–202 (Springer, Berlin, 2007).

    Google Scholar 

  121. 121

    Margrie, T. W. et al. Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39, 911–918 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. 122

    Euler, T., Detwiler, P. B. & Denk, W. Directionally selective calcium signals in dendrites of starburst amacrine cells. Nature 418, 845–852 (2002).

    CAS  PubMed  Google Scholar 

  123. 123

    Stuart, G. J., Dodt, H. U. & Sakmann, B. Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflugers Arch. 423, 511–518 (1993).

    CAS  PubMed  Google Scholar 

  124. 124

    Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl Acad. Sci. USA 101, 18206–18211 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. 125

    Komai, S. et al. Postsynaptic excitability is necessary for strengthening of cortical sensory responses during experience-dependent development. Nature Neurosci. 9, 1125–1133 (2006).

    CAS  PubMed  Google Scholar 

  126. 126

    Niell, C. M., Meyer, M. P. & Smith, S. J. In vivo imaging of synapse formation on a growing dendritic arbor. Nature Neurosci. 7, 254–260 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. 127

    Calverley, R. K. & Jones, D. G. Contributions of dendritic spines and perforated synapses to synaptic plasticity. Brain Res. Brain Res. Rev. 15, 215–249 (1990).

    CAS  PubMed  Google Scholar 

  128. 128

    Balicegordon, R. J. & Lichtman, J. W. In vivo visualization of the growth of presynaptic and postsynaptic elements of neuromuscular-junctions in the mouse. J. Neurosci. 10, 894–908 (1990).

    CAS  Google Scholar 

  129. 129

    Sanes, J. R. & Lichtman, J. W. Development of the vertebrate neuromuscular junction. Annu. Rev. Neurosci. 22, 389–442 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. 130

    Javaherian, A. & Cline, H. T. Coordinated motor neuron axon growth and neuromuscular synaptogenesis are promoted by CPG15 in vivo. Neuron 45, 505–512 (2005).

    CAS  PubMed  Google Scholar 

  131. 131

    Hua, J. Y., Smear, M. C., Baier, H. & Smith, S. J. Regulation of axon growth in vivo by activity-based competition. Nature 434, 1022–1026 (2005).

    CAS  PubMed  Google Scholar 

  132. 132

    Wu, G. Y. & Cline, H. T. Time-lapse in vivo imaging of the morphological development of Xenopus optic tectal interneurons. J. Comp. Neurol. 459, 392–406 (2003).

    PubMed  Google Scholar 

  133. 133

    Kulesa, P. M. & Fraser, S. E. Cell dynamics during somite boundary formation revealed by time-lapse analysis. Science 298, 991–995 (2002).

    CAS  PubMed  Google Scholar 

  134. 134

    Feng, G. et al. Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers. Neuron 25, 295–306 (2000).

    CAS  Google Scholar 

  135. 135

    Zuo, Y., Lin, A., Chang, P. & Gan, W. B. Development of long-term dendritic spine stability in diverse regions of cerebral cortex. Neuron 46, 181–189 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. 136

    Holtmaat, A., Wilbrecht, L., Knott, G. W., Welker, E. & Svoboda, K. Experience-dependent and cell-type-specific spine growth in the neocortex. Nature 441, 979–983 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. 137

    Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).

    CAS  Google Scholar 

  138. 138

    De Paola, V. et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006).

    CAS  PubMed  Google Scholar 

  139. 139

    Mizrahi, A. Dendritic development and plasticity of adult-born neurons in the mouse olfactory bulb. Nature Neurosci. 10, 444–452 (2007).

    CAS  PubMed  Google Scholar 

  140. 140

    Theer, P., Hasan, M. T. & Denk, W. Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    CAS  PubMed  Google Scholar 

  141. 141

    Theer, P. & Denk, W. On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139–3149 (2006). This paper provides a thorough description, both theoretical and experimental, of the physical properties of two-photon imaging in light scattering media, with a focus on the imaging-depth limit.

    Google Scholar 

  142. 142

    Feierabend, M., Ruckel, M. & Denk, W. Coherence-gated wave-front sensing in strongly scattering samples. Opt. Lett. 29, 2255–2257 (2004).

    PubMed  Google Scholar 

  143. 143

    Rueckel, M., Mack-Bucher, J. A. & Denk, W. Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing. Proc. Natl Acad. Sci. USA 103, 17137–17142 (2006). This study showed how both imaging resolution and signal size were improved by measuring beam wavefront distortions and compensating for these distortions using adaptive optics. This technology will be of great benefit to imaging in preparations in which both intact dura and skull are required.

    CAS  PubMed  Google Scholar 

  144. 144

    Kralik, J. D. et al. Techniques for long-term multisite neuronal ensemble recordings in behaving animals. Methods 25, 121–150 (2001).

    CAS  PubMed  Google Scholar 

  145. 145

    Mehta, A. D., Jung, J. C., Flusberg, B. A. & Schnitzer, M. J. Fiber optic in vivo imaging in the mammalian nervous system. Curr. Opin. Neurobiol. 14, 617–628 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. 146

    Jung, J. C., Mehta, A. D., Aksay, E., Stepnoski, R. & Schnitzer, M. J. In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J. Neurophysiol. 92, 3121–3133 (2004).

    PubMed  PubMed Central  Google Scholar 

  147. 147

    Zucker, R. S. & Regehr, W. G. Short-term synaptic plasticity. Annu. Rev. Physiol. 64, 355–405 (2002).

    CAS  Google Scholar 

  148. 148

    Fan, G. Y. et al. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys. J. 76, 2412–2420 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. 149

    Lechleiter, J. D., Lin, D. T. & Sieneart, I. Multi-photon laser scanning microscopy using an acoustic optical deflector. Biophys. J. 83, 2292–2299 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. 150

    Vucinic, D. & Sejnowski, T. A compact multiphoton 3D imaging system for recording fast neuronal activity. PLoS ONE 2, e699 (2007).

    PubMed  PubMed Central  Google Scholar 

  151. 151

    Gobel, W., Kampa, B. M. & Helmchen, F. Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nature Methods 4, 73–79 (2007).

    PubMed  PubMed Central  Google Scholar 

  152. 152

    Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    CAS  Google Scholar 

  153. 153

    Levene, M. J., Dombeck, D. A., Kasischke, K. A., Molloy, R. P. & Webb, W. W. In vivo multiphoton microscopy of deep brain tissue. J. Neurophysiol. 91, 1908–1912 (2004).

    PubMed  Google Scholar 

  154. 154

    Gobel, W., Kerr, J. N., Nimmerjahn, A. & Helmchen, F. Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective. Opt. Lett. 29, 2521–2523 (2004).

    PubMed  Google Scholar 

  155. 155

    Yaksi, E. & Friedrich, R. W. Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging. Nature Methods 3, 377–383 (2006). This study describes a deconvolution-based method for estimating spike firing rates in neuronal populations bulk-loaded with Ca2+-indicator in the zebrafish tectum.

    CAS  PubMed  Google Scholar 

  156. 156

    Takano, T. et al. Astrocyte-mediated control of cerebral blood flow. Nature Neurosci. 9, 260–267 (2006).

    CAS  PubMed  Google Scholar 

  157. 157

    Winship, I. R., Plaa, N. & Murphy, T. H. Rapid astrocyte calcium signals correlate with neuronal activity and onset of the hemodynamic response in vivo. J. Neurosci. 27, 6268–6272 (2007).

    CAS  PubMed  Google Scholar 

  158. 158

    Takano, T., Han, X., Deane, R., Zlokovic, B. & Nedergaard, M. Two-photon imaging of astrocytic Ca2+ signaling and the microvasculature in experimental mice models of Alzheimer's disease. Ann. NY Acad. Sci. 1097, 40–50 (2007).

    CAS  Google Scholar 

  159. 159

    Zipfel, W. R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl Acad. Sci. USA 100, 7075–7080 (2003).

    CAS  PubMed  Google Scholar 

  160. 160

    Misgeld, T. & Kerschensteiner, M. In vivo imaging of the diseased nervous system. Nature Rev. Neurosci. 7, 449–463 (2006).

    CAS  Google Scholar 

  161. 161

    Kovacevic, N. et al. A three-dimensional MRI atlas of the mouse brain with estimates of the average and variability. Cereb. Cortex 15, 639–645 (2005).

    CAS  PubMed  Google Scholar 

  162. 162

    Gobel, W. & Helmchen, F. New angles on neuronal dendrites in vivo. J. Neurophysiol. 98, 3770–3779 (2007).

    PubMed  Google Scholar 

Download references

Author information



Ethics declarations

Competing interests

W.D. holds a patent on two-photon microscopy and receives royalties.

Related links

Related links


Winfried Denk's homepage

Jason Kerr's homepage



The deflection of light by particles with a deviating refractive index.

Refractive index

A property of materials that governs the speed of light as it travels through the material and the deflection of light as it crosses boundaries between materials.

Molecular absorption

A process by which the energy of a photon is used to elevate a molecule to a higher internal energy level. The photon itself is eliminated.


The emission of a photon by a molecule while the molecule undergoes a transition from an elevated energy state to a lower energy state.


The alteration or destruction of biological molecules as a result of photo-oxidative side effects of chromophore excitation.

Signal-to-noise ratio

The ratio between signal size and measurement noise.

High-resistance (sharp) micro electrode

A glass pipette with a tip diameter of less than 100 nm that is filled with saline and used to penetrate cells to gain electrical access to the cell interior.

Tight-seal electrodes

Patch pipettes that are sealed to the plasma membrane and used to carry out electrical recording of the intra-cellular voltage.

Functional MRI

(fMRI). The detection of changes in regional brain activity through their effects on blood flow and blood oxygenation which, in turn, affect the brightness of magnetic-resonance images.

Cell-attached recording

Extracellular electrical recording of a neuron's spiking using a glass patch pipette sealed to the outside of the plasma membrane, without gaining access to the cell interior.

Patch recordings

Electrical recordings made using a glass pipette with a tip diameter of approximately 1 micrometre that is filled with saline and used to form gigaohm seals on the cell membrane. By removing the membrane patch inside the pipette, electrical access to the cell interior can be gained.

Numerical aperture

A measure of the angular spread of the light rays that emerge from or are able to enter an objective lens.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kerr, J., Denk, W. Imaging in vivo: watching the brain in action. Nat Rev Neurosci 9, 195–205 (2008).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing