Optogenetic Tools in the Microscopy of Cardiac Excitation-Contraction Coupling

  • Lars Kaestner
  • André Zeug
  • Qinghai Tian


Microscopy became a scientific investigation method in the seventeenth century with the application of the first build microscopes on biological samples [1, 2]. Soon it became a popular method to stain samples in order to visualise particular (cellular and subcellular) structures [3]. These stains, either based on absorption or fluorescence, have limitations in respect to their specificity and are often toxic to cells, which limits investigations to short intervals or even dead samples. In 1987 the idea came up to use a fluorescent protein that was discovered 25 years before [4], in particular a green fluorescent protein (GFP) form the medusa Aequorea victoria to label cells and cellular structures [5]. With the sequencing and cloning of GFP, a so-called ‘green revolution’ started, which led to regular usage of fluorescent proteins as markers or sensors (for details see below) in the majority of cellular research in physiology, microbiology, pharmacology, molecular biology, anatomy, cell biology, biophysics and many other biomedical fields. Although the expression of the fluorescent proteins and their optical investigation can already be regarded as optogenetic tools, this term was only applied when the optical properties of proteins were used to manipulate cells. The best-known example of such a protein is the channelrhodopsin, a light-gated ion channel [6, 7]. When this ion channel is expressed in a membrane and illuminated with light of the appropriate wavelength, the channel will be activated and opened, which results in passive transportation of ions across the membrane and a change of the membrane potential. However, within this chapter we consider both aspects, the observation and the manipulation as optogenetic tools. To use the optogenetic tool, the genes of these proteins need to be transferred into the cells to allow the expression of the protein. For an overview of gene delivery into target cells, see [8].


  1. 1.
    Swammerdam J. Bybel der natuur. London: C. G. Seyffert; 1737.Google Scholar
  2. 2.
    Scientists, A. C. O. D. The collected letters of Antoni Van Leeuwenhoek. Boca Raton, FL: CRC Press; 1996.Google Scholar
  3. 3.
    Milestones in light microscopy. Nat Cell Biol. 2009;11:1165.Google Scholar
  4. 4.
    Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan. Aequorea J Cell Compar Physiol. 1962;59:223–39.Google Scholar
  5. 5.
    Zimmer M. Glowing genes: a revolution in biotechnology. New York, NY: Prometheus Books; 2005.Google Scholar
  6. 6.
    Nagel G, et al. Channelrhodopsin-1: a light-gated proton channel in green algae. Science. 2002;296:2395–8.PubMedGoogle Scholar
  7. 7.
    Nagel G, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A. 2003;100:13940–5.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Kaestner L, Scholz A, Lipp P. Conceptual and technical aspects of transfection and gene delivery. Bioorg Med Chem Lett. 2015;25:1171–6.PubMedGoogle Scholar
  9. 9.
    Hu CD, Chinenov Y, Kerppola TK. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell. 2002;9:789–98.PubMedGoogle Scholar
  10. 10.
    Wang Q, Shui B, Kotlikoff MI, Sondermann H. Structural basis for calcium sensing by GCaMP2. Structure. 2008;16:1817–27.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–44.PubMedGoogle Scholar
  12. 12.
    Coutinho V, Mutoh H, Knöpfel T. Functional topology of the mossy fibre-granule cell--Purkinje cell system revealed by imaging of intrinsic fluorescence in mouse cerebellum. Eur J Neurosci. 2004;20:740–8.PubMedGoogle Scholar
  13. 13.
    Díez-García J, Akemann W, Knöpfel T. In vivo calcium imaging from genetically specified target cells in mouse cerebellum. Neuroimage. 2007;34:859–69.PubMedGoogle Scholar
  14. 14.
    Kaestner L, et al. Genetically encoded Ca2+ indicators in cardiac myocytes. Circ Res. 2014;114:1623–39.PubMedGoogle Scholar
  15. 15.
    Förster T. Intermolecular energy migration and fluorescence. Ann Phys. 1948;437:55–75.Google Scholar
  16. 16.
    Tsien RY, Bacskai BJ, Adams SR. FRET for studying intracellular signalling. Trends Cell Biol. 1993;3:242–5.PubMedGoogle Scholar
  17. 17.
    Heim R, Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996;6:178–82.Google Scholar
  18. 18.
    Youvan DC. Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads. Biotechnol Alia. 2006;3:1–18. Scholar
  19. 19.
    Berney C, Danuser G. FRET or no FRET: a quantitative comparison. Biophys J. 2003;84:3992–4010.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Gordon GW, Berry G, Liang XH, Levine B, Herman B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J. 1998;74:2702–13.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Xia Z, Liu Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J. 2001;81:2395–402.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Hoppe A, Christensen K, Swanson JA. Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J. 2002;83:3652–64.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Lakowicz JR. Principles of fluorescence spectroscopy. New York, NY: Springer Science & Business Media; 2013.Google Scholar
  24. 24.
    Wlodarczyk J, et al. Analysis of FRET signals in the presence of free donors and acceptors. Biophys J. 2008;94:986–1000.PubMedPubMedCentralGoogle Scholar
  25. 25.
    Thaler C, Koushik SV, Blank PS, Vogel SS. Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys J. 2005;89:2736–49.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:198–205.Google Scholar
  27. 27.
    Viero C, Kraushaar U, Ruppenthal S, Kaestner L, Lipp P. A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes. Cell Calcium. 2008;43:59–71.Google Scholar
  28. 28.
    Kaestner L. et al. Isolation and genetic manipulation of adult cardiac myocytes for confocal imaging. J Vis Exp. 2009; (31).Google Scholar
  29. 29.
    Tian Q, et al. Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement. J Mol Cell Cardiol. 2012;52:113–24.Google Scholar
  30. 30.
    Pahlavan S, et al. Gαq and Gα11 contribute to the maintenance of cellular electrophysiology and Ca2+ handling in ventricular cardiomyocytes. Cardiovasc Res. 2012;95:48–58.PubMedGoogle Scholar
  31. 31.
    Kang M, Walker J. Protein kinase C delta and epsilon mediate positive inotropy in adult ventricular myocytes. J Mol Cell Cardiol. 2005;38:753–64.PubMedGoogle Scholar
  32. 32.
    Chen T-W, et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013;499:295–300.PubMedPubMedCentralGoogle Scholar
  33. 33.
    Dana H, et al. Sensitive red protein calcium indicators for imaging neural activity. Elife. 2016;5:413.Google Scholar
  34. 34.
    Romoser VA, Hinkle PM, Persechini A. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. J Biol Chem. 1997;272:13270–4.PubMedGoogle Scholar
  35. 35.
    Miyawaki A, et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997;388:882–7.Google Scholar
  36. 36.
    Cox JA. Calcium-calmodulin interaction and cellular function. J Cardiovasc Pharmacol. 1986;8(Suppl 8):S48–51.PubMedGoogle Scholar
  37. 37.
    Garaschuk O, Griesbeck O, Konnerth A. Troponin C-based biosensors: a new family of genetically encoded indicators for in vivo calcium imaging in the nervous system. Cell Calcium. 2007;42:351–61.PubMedGoogle Scholar
  38. 38.
    Palmer AE, Jin C, Reed JC, Tsien RY. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A. 2004;101:17404–9.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Palmer AE, et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol. 2006;13:521–30.PubMedGoogle Scholar
  40. 40.
    Mank M, et al. A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change. Biophys J. 2006;90:1790–6.PubMedGoogle Scholar
  41. 41.
    Heim N, Griesbeck O. Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein. J Biol Chem. 2004;279:14280–6.PubMedGoogle Scholar
  42. 42.
    Kaestner L, et al. Genetically encoded voltage indicators in circulation research. Int J Mol Sci. 2015;16:21626–42.PubMedPubMedCentralGoogle Scholar
  43. 43.
    Siegel MS, Isacoff EY. A genetically encoded optical probe of membrane voltage. Neuron. 1997;19:735–41.PubMedGoogle Scholar
  44. 44.
    Sakai R, Repunte-Canonigo V, Raj CD, Knöpfel T. Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein. Eur J Neurosci. 2001;13:2314–8.PubMedGoogle Scholar
  45. 45.
    Knöpfel T, Tomita K, Shimazaki R, Sakai R. Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. Methods. 2003;30:42–8.PubMedGoogle Scholar
  46. 46.
    Ataka K, Pieribone VA. A genetically targetable fluorescent probe of channel gating with rapid kinetics. Biophys J. 2002;82:509–16.PubMedPubMedCentralGoogle Scholar
  47. 47.
    Baker BJ, et al. Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells. J Neurosci Methods. 2007;161:32–8.PubMedGoogle Scholar
  48. 48.
    Murata Y, Iwasaki H, Sasaki M, Inaba K, Okamura Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature. 2005;435:1239–43.PubMedGoogle Scholar
  49. 49.
    Ramsey IS, Moran MM, Chong JA, Clapham DE. A voltage-gated proton-selective channel lacking the pore domain. Nature. 2006;440:1213–6.PubMedPubMedCentralGoogle Scholar
  50. 50.
    Dimitrov D, et al. Engineering and characterization of an enhanced fluorescent protein voltage sensor. PLoS One. 2007;2:e440.PubMedPubMedCentralGoogle Scholar
  51. 51.
    Tsutsui H, Karasawa S, Okamura Y, Miyawaki A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat Methods. 2008;5:683–5.PubMedGoogle Scholar
  52. 52.
    Mutoh H, et al. Spectrally-resolved response properties of the three most advanced FRET based fluorescent protein voltage probes. PLoS One. 2009;4:e4555.PubMedPubMedCentralGoogle Scholar
  53. 53.
    Lam AJ, et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods. 2012;9:1005–12.PubMedPubMedCentralGoogle Scholar
  54. 54.
    Chang Liao M-L, et al. Sensing cardiac electrical activity with a cardiac myocyte--targeted optogenetic voltage indicator. Circ Res. 2015;117:401–12.PubMedGoogle Scholar
  55. 55.
    Tsutsui H, Higashijima S-I, Miyawaki A, Okamura Y. Visualizing voltage dynamics in zebrafish heart. J Physiol (Lond). 2010;588:2017–21.Google Scholar
  56. 56.
    Tallini YN, et al. Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci U S A. 2006;103:4753–8.PubMedPubMedCentralGoogle Scholar
  57. 57.
    Tian Q, et al. Optical action potential screening on adult ventricular myocytes as an alternative QT-screen. Cell Physiol Biochem. 2011;27:281–90.PubMedGoogle Scholar
  58. 58.
    Villalba-Galea CA, et al. Charge movement of a voltage-sensitive fluorescent protein. Biophys J. 2009;96:L19–21.PubMedPubMedCentralGoogle Scholar
  59. 59.
    Lundby A, Mutoh H, Dimitrov D, Akemann W, Knöpfel T. Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements. PLoS One. 2008;3:e2514.PubMedPubMedCentralGoogle Scholar
  60. 60.
    Baker BJ, et al. Genetically encoded fluorescent voltage sensors using the voltage-sensing domain of Nematostella and Danio phosphatases exhibit fast kinetics. J Neurosci Methods. 2012;208:190–6.PubMedPubMedCentralGoogle Scholar
  61. 61.
    Jin L, et al. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron. 2012;75:779–85.PubMedPubMedCentralGoogle Scholar
  62. 62.
    Miesenbock G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998;394:192–5.PubMedPubMedCentralGoogle Scholar
  63. 63.
    Hochbaum DR, et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat Methods. 2014;11:825–33.PubMedPubMedCentralGoogle Scholar
  64. 64.
    St-Pierre F, et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nat Neurosci. 2014;17:884–9.PubMedPubMedCentralGoogle Scholar
  65. 65.
    Zou P, et al. Bright and fast multicoloured voltage reporters via electrochromic FRET. Nat Commun. 2014;5:4625.PubMedPubMedCentralGoogle Scholar
  66. 66.
    Kralj JM, Hochbaum DR, Douglass AD, Cohen AE. Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science. 2011;333:345–8.PubMedPubMedCentralGoogle Scholar
  67. 67.
    Kralj JM, Douglass AD, Hochbaum DR, Maclaurin D, Cohen AE. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods. 2012;9:90–5.Google Scholar
  68. 68.
    Ernst OP, et al. Photoactivation of channelrhodopsin. J Biol Chem. 2008;283:1637–43.PubMedGoogle Scholar
  69. 69.
    Hou JH, Kralj JM, Douglass AD, Engert F, Cohen AE. Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents. Front Physiol. 2014;5:344.PubMedPubMedCentralGoogle Scholar
  70. 70.
    Gong Y, Wagner MJ, Zhong Li J, Schnitzer MJ. Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat Commun. 2014;5:3674.PubMedPubMedCentralGoogle Scholar
  71. 71.
    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. 2004;87:631–9.PubMedPubMedCentralGoogle Scholar
  72. 72.
    Bublitz G, King B, Boxer S. Electronic structure of the chromophore in green fluorescent protein. J Am Chem Soc. 1998;120:9370.Google Scholar
  73. 73.
    Rosell FI, Boxer SG. Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions. Biochemistry. 2003;42:177–83.PubMedGoogle Scholar
  74. 74.
    Khatchatouriants A, Lewis A, Rothman Z, Loew L, Treinin M. GFP is a selective non-linear optical sensor of electrophysiological processes in Caenorhabditis elegans. Biophys J. 2000;79:2345–52.PubMedPubMedCentralGoogle Scholar
  75. 75.
    Kaestner L, Tian Q, Lipp P. In: Jung G, editor. Action potentials in heart cells. New York, NY: Springer; 2012. p. 163–82.Google Scholar
  76. 76.
    Entcheva E. Cardiac optogenetics. AJP Heart Circ Physiol. 2013;304:H1179–91.Google Scholar
  77. 77.
    Bruegmann T, et al. Optogenetic control of heart muscle in vitro and in vivo. Nat Methods. 2010;7:897–900.PubMedGoogle Scholar
  78. 78.
    Vogt CC, et al. Systemic gene transfer enables optogenetic pacing of mouse hearts. Cardiovasc Res. 2015;106:338–43.PubMedGoogle Scholar
  79. 79.
    Ambrosi CM, Klimas A, Yu J, Entcheva E. Cardiac applications of optogenetics. Prog Biophys Mol Biol. 2014;115:294–304.PubMedPubMedCentralGoogle Scholar
  80. 80.
    Scheller A, Bai X, Kirchhoff F. The role of the oligodendrocyte lineage in acute brain trauma. Neurochem Res. 2017; Scholar
  81. 81.
    Wiesen K, et al. Cardiac remodeling in Gαq and Gα11 knock out mice. Int J Cardiol. 2016;202:836–45.PubMedGoogle Scholar
  82. 82.
    Prigge M, et al. Color-tuned channelrhodopsins for multiwavelength optogenetics. J Biol Chem. 2012;287:31804–12.PubMedPubMedCentralGoogle Scholar
  83. 83.
    Dempsey GT, et al. Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging. J Pharmacol Toxicol Methods. 2016;81:240. Scholar
  84. 84.
    Shang W, et al. Imaging Ca2+ nanosparks in heart with a new targeted biosensor. Circ Res. 2014;114(3):412. Scholar
  85. 85.
    Tian Q, et al. Optical measurement of action potential in adult ventricular myocytes. Biophys J. 2011;100:292a.Google Scholar
  86. 86.
    Haugland RP. Handbook of fluorescent probes and research products. Eugene, OR: Molecular Probes; 2002.Google Scholar
  87. 87.
    Kaestner L, Tian Q, Lipp P. Cardiac safety screens: molecular, cellular, and optical advancements. In: Lin CP, Ntziachistos V, editors. Biomedical optics III, vol. 8089. Munich: SPIE; 2011. p. 80890H-1–6.Google Scholar
  88. 88.
    Arrigoni C, Crivori P. Assessment of QT liabilities in drug development. Cell Biol Toxicol. 2007;23:1–13.PubMedGoogle Scholar
  89. 89.
    Sinnecker D, et al. Induced pluripotent stem cells in cardiovascular research. Rev Physiol Biochem Pharmacol. 2012;163:1. Scholar
  90. 90.
    Matsa E, Burridge PW, Wu JC. Human stem cells for modeling heart disease and for drug discovery. Sci Transl Med. 2014;6:239ps6.PubMedPubMedCentralGoogle Scholar
  91. 91.
    Dorn T, et al. Direct Nkx2-5 transcriptional repression of Isl1 controls cardiomyocyte subtype identity. Stem Cells. 2015;33:1113. Scholar
  92. 92.
    Leyton-Mange JS, et al. Rapid cellular phenotyping of human pluripotent stem cell-derived cardiomyocytes using a genetically encoded fluorescent voltage sensor. Stem Cell Rep. 2014;2:163–70.Google Scholar
  93. 93.
    Nagai T, Sawano A, Park ES, Miyawaki A. Circularly permuted green fluorescent proteins engineered to sense Ca2. Proc Natl Acad Sci U S A. 2001;98:3197–202.PubMedPubMedCentralGoogle Scholar
  94. 94.
    Chen Z, et al. Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes. Eur Heart J. 2017;38:292–301.PubMedGoogle Scholar
  95. 95.
    Tian Q, Kaestner L, Lipp P. Noise-free visualization of microscopic calcium signaling by pixel-wise fitting. Circ Res. 2012;111:17–27.Google Scholar
  96. 96.
    Tian Q, Kaestner L, Schröder L, Guo J, Lipp P. An adaptation of astronomical image processing enables characterization and functional 3D mapping of individual sites of excitation-contraction coupling in rat cardiac muscle. Elife. 2017;6:665.Google Scholar
  97. 97.
    Violin JD, Zhang JX, Tsien RY, Newton AC. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol. 2003;161:899–909.PubMedPubMedCentralGoogle Scholar
  98. 98.
    Schleifenbaum A, Stier G, Gasch A, Sattler M, Schultz C. Genetically encoded FRET probe for PKC activity based on pleckstrin. J Am Chem Soc. 2004;126:11786–7.PubMedGoogle Scholar
  99. 99.
    Knöpfel T, Gallero-Salas Y, Song C. Genetically encoded voltage indicators for large scale cortical imaging come of age. Curr Opin Chem Biol. 2015;27:75–83.PubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Lars Kaestner
    • 1
    • 2
  • André Zeug
    • 3
  • Qinghai Tian
    • 1
  1. 1.Theoretical Medicine and BiosciencesSaarland UniversityHomburg/SaarGermany
  2. 2.Experimental PhysicsSaarland UniversitySaarbrückenGermany
  3. 3.Institute for Neurophysiology, Hannover Medical SchoolHannoverGermany

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