Quantitative Super-Resolution Microscopy of Cardiomyocytes

  • Christian SoellerEmail author
  • Izzy D. Jayasinghe


Cardiomyocytes are among the largest of animal cell types. At ~20 μm in typical width and ~100 μm in length, these cells are intrinsically organised to contract rapidly and in synchrony in response to electrical activation. This excitation-contraction coupling (EC coupling) process is achieved through the synchronised opening of the primary calcium (Ca2+) release channels of the sarcoplasmic reticulum (SR)—the ryanodine receptors (RyRs) [1]. A series of tubular membrane invaginations of the surface sarcolemma, known as the t-tubules, are the primary sites of this Ca2+ release deeper within the cell where RyRs are organised into clusters in quasi-crystalline patterns [2, 3]. Flanked between the t-tubule membrane and the SR membrane at the dyadic cleft, the cytoplasmic portions of these giant (29 nm × 29 nm) Ca2+ channel [4] are opened by the Ca2+ that enters the cleft through the voltage-gated L-type Ca2+ channel (LCC). This process, known as Ca2+ induced Ca2+ release (CICR) crucially relies on the restricted diffusion and the consequently elevated concentration of the cleft Ca2+ [5]. Described in the theory of local control of EC coupling, the Ca2+ released via RyRs is likely a steep function of the dimensions of the dyadic cleft and the trigger Ca2+ concentration [6]. The synchronisation of the contraction also relies heavily on the effectiveness of this Ca2+ in reaching and activating the contractile machinery (which forms the myofibrils). Early light and electron micrographs have demonstrated that t-tubules and dyads are, to this end, organised all around the myofibrils to minimise the typical diffusional distance of the released calcium [7–10].


  1. 1.
    Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415(6868):198–205.Google Scholar
  2. 2.
    Asghari P, Scriven DR, Sanatani S, Gandhi SK, Campbell AI, Moore ED. Nonuniform and variable arrangements of ryanodine receptors within mammalian ventricular couplons. Circ Res. 2014;115(2):252–62.PubMedGoogle Scholar
  3. 3.
    Franzini-Armstrong C, Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev. 1997;77(3):699–729.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Yin CC, Lai FA. Intrinsic lattice formation by the ryanodine receptor calcium-release channel. Nat Cell Biol. 2000;2(9):669–71.PubMedGoogle Scholar
  5. 5.
    Soeller C, Cannell MB. Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad. Biophys J. 1997;73(1):97–111.PubMedPubMedCentralGoogle Scholar
  6. 6.
    Stern MD. Theory of excitation-contraction coupling in cardiac muscle. Biophys J. 1992;63(2):497–517.PubMedPubMedCentralGoogle Scholar
  7. 7.
    Fawcett DW, McNutt NS. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969;42(1):1–45.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Franzini-Armstrong C, Protasi F, Ramesh V. Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles. Biophys J. 1999;77(3):1528–39.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Soeller C, Cannell MB. Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques. Circ Res. 1999;84(3):266–75.PubMedGoogle Scholar
  10. 10.
    Soeller C, Crossman D, Gilbert R, Cannell MB. Analysis of ryanodine receptor clusters in rat and human cardiac myocytes. Proc Natl Acad Sci U S A. 2007;104(38):14958–63.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Hou Y, Jayasinghe I, Crossman DJ, Baddeley D, Soeller C. Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes. J Mol Cell Cardiol. 2015;80:45–55.PubMedGoogle Scholar
  12. 12.
    Song LS, Sobie EA, McCulle S, Lederer WJ, Balke CW, Cheng H. Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci U S A. 2006;103(11):4305–10.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Wei S, Guo A, Chen B, Kutschke W, Xie YP, Zimmerman K, Weiss RM, Anderson ME, Cheng H, Song LS. T-tubule remodeling during transition from hypertrophy to heart failure. Circ Res. 2010;107(4):520–31.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Crossman DJ, Ruygrok PN, Soeller C, Cannell MB. Changes in the organization of excitation-contraction coupling structures in failing human heart. PLoS One. 2011;6(3):e17901.PubMedPubMedCentralGoogle Scholar
  15. 15.
    Veratti E. Investigations on the fine structure of striated muscle fiber read before the Reale Istituto Lombardo, 13 March 1902. J Biophys Biochem Cytol. 1961;10(4, Suppl):1–59.PubMedPubMedCentralGoogle Scholar
  16. 16.
    Guo A, Zhang C, Wei S, Chen B, Song LS. Emerging mechanisms of T-tubule remodelling in heart failure. Cardiovasc Res. 2013;98(2):204–15.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Scriven DR, Dan P, Moore ED. Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. Biophys J. 2000;79(5):2682–91.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Jayasinghe ID, Cannell MB, Soeller C. Organization of ryanodine receptors, transverse tubules, and sodium-calcium exchanger in rat myocytes. Biophys J. 2009;97(10):2664–73.PubMedPubMedCentralGoogle Scholar
  19. 19.
    Scriven DR, Asghari P, Schulson MN, Moore ED. Analysis of Cav1.2 and ryanodine receptor clusters in rat ventricular myocytes. Biophys J. 2010;99(12):3923–9.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Soeller C, Jayasinghe ID, Li P, Holden AV, Cannell MB. Three-dimensional high-resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes. Exp Physiol. 2009;94(5):496–508.PubMedGoogle Scholar
  21. 21.
    Abbe E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch Mikrosk Anat. 1873;9(1):413–8.Google Scholar
  22. 22.
    Landstrom AP, Kellen CA, Dixit SS, van Oort RJ, Garbino A, Weisleder N, Ma J, Wehrens XH, Ackerman MJ. Junctophilin-2 expression silencing causes cardiocyte hypertrophy and abnormal intracellular calcium-handling. Circ Heart Fail. 2011;4(2):214–23.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Jost A, Heintzmann R. Superresolution multidimensional imaging with structured illumination microscopy. Annu Rev Mat Res. 2013;43(1):261–82.Google Scholar
  24. 24.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006;313(5793):1642–5.PubMedPubMedCentralGoogle Scholar
  25. 25.
    Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006;3(10):793–5.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Betzig E. Single molecules, cells, and super-resolution optics (Nobel Lecture). Angew Chem Int Ed. 2015;54(28):8034–53.Google Scholar
  27. 27.
    Klein T, Proppert S, Sauer M. Eight years of single-molecule localization microscopy. Histochem Cell Biol. 2014;141(6):561–75.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Toomre D, Bewersdorf J. A new wave of cellular imaging. Annu Rev Cell Dev Biol. 2010;26:285–314.PubMedGoogle Scholar
  29. 29.
    Baddeley D, Jayasinghe ID, Cremer C, Cannell MB, Soeller C. Light-induced dark states of organic fluochromes enable 30 nm resolution imaging in standard media. Biophys J. 2009a;96(2):L22–4.PubMedPubMedCentralGoogle Scholar
  30. 30.
    Baddeley D, Jayasinghe ID, Lam L, Rossberger S, Cannell MB, Soeller C. Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes. Proc Natl Acad Sci U S A. 2009b;106(52):22275–80.PubMedPubMedCentralGoogle Scholar
  31. 31.
    Wagner E, Lauterbach MA, Kohl T, Westphal V, Williams GS, Steinbrecher JH, Streich JH, Korff B, Tuan HT, Hagen B, Luther S, Hasenfuss G, Parlitz U, Jafri MS, Hell SW, Lederer WJ, Lehnart SE. Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction. Circ Res. 2012;111(4):402–14.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Jayasinghe ID, Clowsley AH, Munro M, Hou Y, Crossman DJ, Soeller C. Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques. Eur J Transl Myol. 2014a;25:4747.PubMedPubMedCentralGoogle Scholar
  33. 33.
    Jayasinghe ID, Munro M, Baddeley D, Launikonis BS, Soeller C. Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging. J R Soc Interface. 2014b;11(99)PubMedCentralGoogle Scholar
  34. 34.
    Chen-Izu Y, McCulle SL, Ward CW, Soeller C, Allen BM, Rabang C, Cannell MB, Balke CW, Izu LT. Three-dimensional distribution of ryanodine receptor clusters in cardiac myocytes. Biophys J. 2006;91(1):1–13.PubMedPubMedCentralGoogle Scholar
  35. 35.
    Crossman DJ, Hou Y, Jayasinghe I, Baddeley D, Soeller C. Combining confocal and single molecule localisation microscopy: a correlative approach to multi-scale tissue imaging. Methods. 2015;88:98–108.PubMedGoogle Scholar
  36. 36.
    Folling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods. 2008;5(11):943–5.PubMedGoogle Scholar
  37. 37.
    Sobie EA, Guatimosim S, Gomez-Viquez L, Song LS, Hartmann H, Saleet Jafri M, Lederer WJ. The Ca2+ leak paradox and rogue ryanodine receptors: SR Ca2+ efflux theory and practice. Prog Biophys Mol Biol. 2006;90(1-3):172–85.PubMedGoogle Scholar
  38. 38.
    Hayashi T, Martone ME, Yu Z, Thor A, Doi M, Holst MJ, Ellisman MH, Hoshijima M. Three-dimensional electron microscopy reveals new details of membrane systems for Ca2+ signaling in the heart. J Cell Sci. 2009;122(Pt 7):1005–13.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Bers DM, Stiffel VM. Ratio of ryanodine to dihydropyridine receptors in cardiac and skeletal muscle and implications for E-C coupling. Am J Physiol. 1993;264(6 Pt 1):C1587–93.PubMedGoogle Scholar
  40. 40.
    Baddeley D, Cannell M, Soeller C. Three-dimensional sub-100 nm super-resolution imaging of biological samples using a phase ramp in the objective pupil. Nano Res. 2011a;4(6):589–98.Google Scholar
  41. 41.
    Baddeley D, Crossman D, Rossberger S, Cheyne JE, Montgomery JM, Jayasinghe ID, Cremer C, Cannell MB, Soeller C. 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS One. 2011b;6(5):e20645.PubMedPubMedCentralGoogle Scholar
  42. 42.
    Murphy RM, Dutka TL, Horvath D, Bell JR, Delbridge LM, Lamb GD. Ca2+-dependent proteolysis of junctophilin-1 and junctophilin-2 in skeletal and cardiac muscle. J Physiol. 2013;591(Pt 3):719–29.PubMedGoogle Scholar
  43. 43.
    Wang W, Landstrom AP, Wang Q, Munro ML, Beavers D, Ackerman MJ, Soeller C, Wehrens XH. Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice. Am J Physiol Heart Circ Physiol. 2014;307(9):H1317–26.PubMedPubMedCentralGoogle Scholar
  44. 44.
    Guo A, Zhang X, Iyer VR, Chen B, Zhang C, Kutschke WJ, Weiss RM, Franzini-Armstrong C, Song L-S. Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress. Proc Natl Acad Sci. 2014;111(33):12240–5.PubMedGoogle Scholar
  45. 45.
    Franzini-Armstrong C, Peachey LD. A modified Golgi black reaction method for light and electron microscopy. J Histochem Cytochem. 1982;30(2):99–105.PubMedGoogle Scholar
  46. 46.
    Cannell MB, Crossman DJ, Soeller C. Effect of changes in action potential spike configuration, junctional sarcoplasmic reticulum micro-architecture and altered t-tubule structure in human heart failure. J Muscle Res Cell Motil. 2006;27(5-7):297–306.PubMedGoogle Scholar
  47. 47.
    Sachse FB, Torres NS, Savio-Galimberti E, Aiba T, Kass DA, Tomaselli GF, Bridge JH. Subcellular structures and function of myocytes impaired during heart failure are restored by cardiac resynchronization therapy. Circ Res. 2012;110(4):588–97.PubMedPubMedCentralGoogle Scholar
  48. 48.
    Shang W, Lu F, Sun T, Xu J, Li LL, Wang Y, Wang G, Chen L, Wang X, Cannell MB, Wang SQ, Cheng H. Imaging Ca2+ nanosparks in heart with a new targeted biosensor. Circ Res. 2014;114(3):412–20.PubMedGoogle Scholar
  49. 49.
    Guo A, Song LS. AutoTT: automated detection and analysis of T-tubule architecture in cardiomyocytes. Biophys J. 2014;106(12):2729–36.PubMedPubMedCentralGoogle Scholar
  50. 50.
    Jayasinghe I, Crossman D, Soeller C, Cannell M. Comparison of the organization of T-tubules, sarcoplasmic reticulum and ryanodine receptors in rat and human ventricular myocardium. Clin Exp Pharmacol Physiol. 2012a;39(5):469–76.PubMedGoogle Scholar
  51. 51.
    Jayasinghe ID, Baddeley D, Kong CH, Wehrens XH, Cannell MB, Soeller C. Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes. Biophys J. 2012b;102(5):L19–21.PubMedPubMedCentralGoogle Scholar
  52. 52.
    Wong J, Baddeley D, Bushong EA, Yu Z, Ellisman MH, Hoshijima M, Soeller C. Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions. Biophys J. 2013;104(11):L22–4.PubMedPubMedCentralGoogle Scholar
  53. 53.
    Pinali C, Kitmitto A. Serial block face scanning electron microscopy for the study of cardiac muscle ultrastructure at nanoscale resolutions. J Mol Cell Cardiol. 2014;76C:1–11.Google Scholar
  54. 54.
    Hess ST, Girirajan TP, Mason MD. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006;91(11):4258–72.PubMedPubMedCentralGoogle Scholar
  55. 55.
    Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed. 2008;47(33):6172–6.Google Scholar
  56. 56.
    van de Linde S, Sauer M. How to switch a fluorophore: from undesired blinking to controlled photoswitching. Chem Soc Rev. 2014;43(4):1076–87.PubMedGoogle Scholar
  57. 57.
    Vaughan JC, Dempsey GT, Sun E, Zhuang X. Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy. J Am Chem Soc. 2013;135(4):1197–200.PubMedPubMedCentralGoogle Scholar
  58. 58.
    Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods. 2011;8(12):1027–36.PubMedPubMedCentralGoogle Scholar
  59. 59.
    Tokunaga M, Imamoto N, Sakata-Sogawa K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods. 2008;5(2):159–61.PubMedGoogle Scholar
  60. 60.
    Huang F, Hartwich TMP, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, Uchil PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D, Bewersdorf J. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat Methods. 2013;10(7):653–8.PubMedPubMedCentralGoogle Scholar
  61. 61.
    Lin R, Clowsley AH, Jayasinghe I, Soeller C. Single-molecule localization microscopy with sCMOS cameras. Biophotonics. 2016;110:161a.Google Scholar
  62. 62.
    McGorty R, Kamiyama D, Huang B. Active microscope stabilization in three dimensions using image correlation. Optic Nanosc. 2013;2(1):3. Scholar
  63. 63.
    Cella Zanacchi F, Lavagnino Z, Faretta M, Furia L, Diaspro A. Light-sheet confined super-resolution using two-photon photoactivation. PLoS One. 2013;8(7):e67667.PubMedPubMedCentralGoogle Scholar
  64. 64.
    Huang B, Jones SA, Brandenburg B, Zhuang X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods. 2008;5(12):1047–52.PubMedPubMedCentralGoogle Scholar
  65. 65.
    Baddeley D. Efficient ROI selection for multi-emitter fitting approaches in single-molecule super-resolution microscopy. Biophys J. 2014;106(2):605a.Google Scholar
  66. 66.
    Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolkova K, Dlaskova A, Santorova J, Jezek P, Bewersdorf J. Sample drift correction in 3D fluorescence photoactivation localization microscopy. Opt Express. 2011;19(16):15009–19.PubMedGoogle Scholar
  67. 67.
    Baddeley D, Cannell MB, Soeller C. Visualization of localization microscopy data. Microsc Microanal. 2010;16(1):64–72.PubMedGoogle Scholar
  68. 68.
    Mortensen KI, Churchman LS, Spudich JA, Flyvbjerg H. Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods. 2010;7(5):377–81.PubMedPubMedCentralGoogle Scholar
  69. 69.
    Thompson RE, Larson DR, Webb WW. Precise nanometer localization analysis for individual fluorescent probes. Biophys J. 2002;82(5):2775–83.PubMedPubMedCentralGoogle Scholar
  70. 70.
    Jayasinghe ID, Launikonis BS. Three-dimensional reconstruction and analysis of the tubular system of vertebrate skeletal muscle. J Cell Sci. 2013;126:4048.PubMedGoogle Scholar
  71. 71.
    Hou Y, Crossman DJ, Rajagopal V, Baddeley D, Jayasinghe I, Soeller C. Super-resolution fluorescence imaging to study cardiac biophysics: alpha-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices. Prog Biophys Mol Biol. 2014;115:328.PubMedGoogle Scholar
  72. 72.
    Shim S-H, Xia C, Zhong G, Babcock HP, Vaughan JC, Huang B, Wang X, Xu C, Bi G-Q, Zhuang X. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc Natl Acad Sci. 2012;109(35):13978–83.PubMedGoogle Scholar
  73. 73.
    Banterle N, Bui KH, Lemke EA, Beck M. Fourier ring correlation as a resolution criterion for super-resolution microscopy. J Struct Biol. 2013;183(3):363–7.PubMedGoogle Scholar
  74. 74.
    Levet F, Hosy E, Kechkar A, Butler C, Beghin A, Choquet D, Sibarita JB. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nat Methods. 2015;12:1065.PubMedGoogle Scholar
  75. 75.
    Rossy J, Cohen E, Gaus K, Owen DM. Method for co-cluster analysis in multichannel single-molecule localisation data. Histochem Cell Biol. 2014;141(6):605–12.PubMedGoogle Scholar
  76. 76.
    Shivanandan A, Deschout H, Scarselli M, Radenovic A. Challenges in quantitative single molecule localization microscopy. FEBS Lett. 2014;588(19):3595–602.PubMedGoogle Scholar
  77. 77.
    Ulbrich MH, Isacoff EY. Subunit counting in membrane-bound proteins. Nat Methods. 2007;4(4):319–21.PubMedPubMedCentralGoogle Scholar
  78. 78.
    Jungmann R, Avendano MS, Woehrstein JB, Dai M, Shih WM, Yin P. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods. 2014;11(3):313–8.PubMedPubMedCentralGoogle Scholar
  79. 79.
    Jungmann R, Avendano MS, Dai M, Woehrstein JB, Agasti SS, Feiger Z, Rodal A, Yin P. Quantitative super-resolution imaging with qPAINT. Nat Methods. 2016;13(5):439–42.PubMedPubMedCentralGoogle Scholar
  80. 80.
    Loschberger A, van de Linde S, Dabauvalle MC, Rieger B, Heilemann M, Krohne G, Sauer M. Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J Cell Sci. 2012;125(Pt 3):570–5.PubMedGoogle Scholar
  81. 81.
    Müller CB, Enderlein J. Image scanning microscopy. Phys Rev Lett. 2010;104(19):198101.PubMedGoogle Scholar
  82. 82.
    Huff J. The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat Methods. 2015;12:1205.Google Scholar
  83. 83.
    Sengupta P, van Engelenburg SB, Lippincott-Schwartz J. Superresolution imaging of biological systems using photoactivated localization microscopy. Chem Rev. 2014;114(6):3189–202.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Living Systems InstituteUniversity of ExeterExeterUK
  2. 2.Department of PhysiologyUniversity of AucklandAucklandNew Zealand
  3. 3.Biomedical PhysicsUniversity of ExeterExeterUK
  4. 4.School of Biomedical SciencesUniversity of LeedsLeedsUK

Personalised recommendations