Imaging of Brain Slices with a Genetically Encoded Voltage Indicator

  • Peter Quicke
  • Samuel J. Barnes
  • Thomas KnöpfelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1563)


Functional fluorescence microscopy of brain slices using voltage sensitive fluorescent proteins (VSFPs) allows large scale electrophysiological monitoring of neuronal excitation and inhibition. We describe the equipment and techniques needed to successfully record functional responses optical voltage signals from cells expressing a voltage indicator such as VSFP Butterfly 1.2. We also discuss the advantages of voltage imaging and the challenges it presents.

Key words

Voltage imaging Fluorescence imaging Brain slices Voltage indicators Fluorescence microscopy Voltage sensitive fluorescent proteins 



This work was supported by the Engineering and Physical Sciences Research Council [grant number EP/L016737/1]. We would like to thank Elisa Ciglieri, Amanda Foust, Taylor Lyons, and Chenchen Song for their very helpful comments and advice on the manuscript.


  1. 1.
    Sakmann B, Neher E (1984) Patch clamp techniques for studying ionic channels in excitable membranes. Annu Rev Physiol 46:455–472CrossRefPubMedGoogle Scholar
  2. 2.
    Obien MEJ, Deligkaris K, Bullmann T et al (2015) Revealing neuronal function through microelectrode array recordings. Front Neurosci 8:423CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Knöpfel T, Díez-García J, Akemann W (2006) Optical probing of neuronal circuit dynamics: genetically encoded versus classical fluorescent sensors. Trends Neurosci 29:160–166CrossRefPubMedGoogle Scholar
  4. 4.
    Knöpfel T (2012) Genetically encoded optical indicators for the analysis of neuronal circuits. Nat Rev Neurosci 13:687–700PubMedGoogle Scholar
  5. 5.
    Franconville R, Revet G, Astorga G et al (2011) Somatic calcium level reports integrated spiking activity of cerebellar interneurons in vitro and in vivo. J Neurophysiol 106:1793–1805CrossRefPubMedGoogle Scholar
  6. 6.
    Antic SD, Empson RM, Knöpfel T (2016) Voltage imaging to understand connections and functions of neuronal circuits. J Neurophysiol. doi: 10.1152/jn.00226.2016 PubMedPubMedCentralGoogle Scholar
  7. 7.
    Hoover E, Squier J (2013) Advances in multiphoton microscopy technology. Nat Photonics 7:93–101CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Flusberg BA, Cocker ED, Piyawattanametha W et al (2005) Fiber-optic fluorescence imaging. Nat Methods 2:941–950CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Murray TA, Levene MJ (2012) Singlet gradient index lens for deep in vivo multiphoton microscopy. J Biomed Opt 17:021106CrossRefPubMedGoogle Scholar
  10. 10.
    Murayama M, Pérez-Garci E, Lüscher HR et al (2007) Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats. J Neurophysiol 98:1791–1805CrossRefPubMedGoogle Scholar
  11. 11.
    Carandini M, Shimaoka D, Rossi LF et al (2015) Imaging the awake visual cortex with a genetically encoded voltage indicator. J Neurosci 35:53–63CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76CrossRefPubMedGoogle Scholar
  13. 13.
    Akemann W, Mutoh H, Perron A et al (2012) Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. J Neurophysiol 108:2323–2337CrossRefPubMedGoogle Scholar
  14. 14.
    Kinosita K, Itoh H, Ishiwata S et al (1991) Dual-view microscopy with a single camera: Real-time imaging of molecular orientations and calcium. J Cell Biol 115:67–73CrossRefPubMedGoogle Scholar
  15. 15.
    Haga T, Takahashi S, Sonehara T et al (2011) Dual-view imaging system using a wide-range dichroic mirror for simultaneous four-color single-molecule detection. Anal Chem 83:6948–6955CrossRefPubMedGoogle Scholar
  16. 16.
    Ting JT, Daigle TL, Chen Q et al (2014) Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol Biol 1183:221–242CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Holtmaat A, Bonhoeffer T, Chow DK et al (2009) Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 4:1128–1144CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Peter Quicke
    • 1
    • 2
    • 3
  • Samuel J. Barnes
    • 3
  • Thomas Knöpfel
    • 2
    • 3
    Email author
  1. 1.Department of BioengineeringImperial College LondonLondonUK
  2. 2.Centre for NeurotechnologyImperial College LondonLondonUK
  3. 3.Division of Brain SciencesImperial College LondonLondonUK

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