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Multicolor Superresolution Microscopy: Revealing the Nano World of Astrocytes In Situ

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Part of the book series: Neuromethods ((NM,volume 155))

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Abstract

Astroglia are essential to the development, homeostasis, and metabolic support of the brain but also to the formation and regulation of synaptic circuits. Experimental evidence has been emerging that astrocytes undergo substantial structural plasticity associated with age- and use-dependent changes in neural circuitries. The underlying cellular mechanisms are poorly understood, mainly due to the extraordinary complex, essentially nanoscopic morphology of astroglia. It appears that key morphological changes occur in fine astrocytic processes that are in the vicinity of synapses. However, the characteristic size of these compartment falls below the diffraction limit of conventional optical microscopy, making the deciphering of their molecular nanostructure a challenge.

Here we detail a superresolution microscopy approach that relies on direct stochastic optical reconstruction microscopy (dSTORM) to visualize astroglial organization on the nanoscale (in fixed brain tissue). We also provide a protocol for viral infection of astroglia in vivo (aimed at monitoring the cell activity with the genetically encoded calcium indicator GCaMP), followed by tissue sectioning, immunolabeling, and the subsequent dSTORM analysis. The presented workflow can be extended to a correlational-study protocol to reconstruct the nanoscopic morphology of the imaged cells.

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References

  1. Agulhon C, Petravicz J, McMullen AB, Sweger EJ, Minton SK, Taves SR, Casper KB, Fiacco TA, McCarthy KD (2008) What is the role of astrocyte calcium in neurophysiology? Neuron 59(6):932–946. https://doi.org/10.1016/j.neuron.2008.09.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dityatev A, Rusakov DA (2011) Molecular signals of plasticity at the tetrapartite synapse. Curr Opin Neurobiol 21(2):353–359. https://doi.org/10.1016/j.conb.2010.12.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Halassa MM, Haydon PG (2010) Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. Annu Rev Physiol 72:335–355. https://doi.org/10.1146/annurev-physiol-021909-135843

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Haydon PG (2001) GLIA: listening and talking to the synapse. Nat Rev Neurosci 2(3):185–193. https://doi.org/10.1038/35058528

    Article  CAS  PubMed  Google Scholar 

  5. Matyash V, Kettenmann H (2010) Heterogeneity in astrocyte morphology and physiology. Brain Res Rev 63(1–2):2–10. https://doi.org/10.1016/j.brainresrev.2009.12.001

    Article  CAS  PubMed  Google Scholar 

  6. Porter JT, McCarthy KD (1997) Astrocytic neurotransmitter receptors in situ and in vivo. Prog Neurobiol 51(4):439–455

    Article  CAS  Google Scholar 

  7. Seifert G, Carmignoto G, Steinhauser C (2010) Astrocyte dysfunction in epilepsy. Brain Res Rev 63(1–2):212–221. https://doi.org/10.1016/j.brainresrev.2009.10.004

    Article  CAS  PubMed  Google Scholar 

  8. Verkhratsky A, Sofroniew MV, Messing A, deLanerolle NC, Rempe D, Rodriguez JJ, Nedergaard M (2012) Neurological diseases as primary gliopathies: a reassessment of neurocentrism. ASN Neuro 4(3). https://doi.org/10.1042/AN20120010

  9. Volterra A, Meldolesi J (2005) Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neurosci 6(8):626–640. https://doi.org/10.1038/nrn1722

    Article  CAS  PubMed  Google Scholar 

  10. Zheng K, Bard L, Reynolds JP, King C, Jensen TP, Gourine AV, Rusakov DA (2015) Time-resolved imaging reveals heterogeneous landscapes of nanomolar Ca(2+) in neurons and astroglia. Neuron 88(2):277–288. https://doi.org/10.1016/j.neuron.2015.09.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Araque A, Carmignoto G, Haydon PG, Oliet SH, Robitaille R, Volterra A (2014) Gliotransmitters travel in time and space. Neuron 81(4):728–739. https://doi.org/10.1016/j.neuron.2014.02.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bazargani N, Attwell D (2016) Astrocyte calcium signaling: the third wave. Nat Neurosci 19(2):182–189. https://doi.org/10.1038/nn.4201

    Article  CAS  PubMed  Google Scholar 

  13. Khakh BS, Sofroniew MV (2015) Diversity of astrocyte functions and phenotypes in neural circuits. Nat Neurosci 18(7):942–952. https://doi.org/10.1038/nn.4043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rusakov DA (2015) Disentangling calcium-driven astrocyte physiology. Nat Rev Neurosci 16(4):226–233. https://doi.org/10.1038/nrn3878

    Article  CAS  PubMed  Google Scholar 

  15. Volterra A, Liaudet N, Savtchouk I (2014) Astrocyte Ca(2)(+) signalling: an unexpected complexity. Nat Rev Neurosci 15(5):327–335. https://doi.org/10.1038/nrn3725

    Article  CAS  PubMed  Google Scholar 

  16. Zorec R, Araque A, Carmignoto G, Haydon PG, Verkhratsky A, Parpura V (2012) Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route. ASN Neuro 4(2). https://doi.org/10.1042/AN20110061

  17. Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65(1):1–105

    Article  CAS  Google Scholar 

  18. Heller JP, Rusakov DA (2015) Morphological plasticity of astroglia: understanding synaptic microenvironment. Glia 63(12):2133–2151. https://doi.org/10.1002/glia.22821

    Article  PubMed  PubMed Central  Google Scholar 

  19. Rusakov DA, Bard L, Stewart MG, Henneberger C (2014) Diversity of astroglial functions alludes to subcellular specialisation. Trends Neurosci 37(4):228–242. https://doi.org/10.1016/j.tins.2014.02.008

    Article  CAS  PubMed  Google Scholar 

  20. Bernardinelli Y, Randall J, Janett E, Nikonenko I, Konig S, Jones EV, Flores CE, Murai KK, Bochet CG, Holtmaat A, Muller D (2014) Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability. Curr Biol 24(15):1679–1688. https://doi.org/10.1016/j.cub.2014.06.025

    Article  CAS  PubMed  Google Scholar 

  21. Haber M, Zhou L, Murai KK (2006) Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses. J Neurosci 26(35):8881–8891. https://doi.org/10.1523/JNEUROSCI.1302-06.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hirrlinger J, Hulsmann S, Kirchhoff F (2004) Astroglial processes show spontaneous motility at active synaptic terminals in situ. Eur J Neurosci 20(8):2235–2239. https://doi.org/10.1111/j.1460-9568.2004.03689.x

    Article  PubMed  Google Scholar 

  23. Perez-Alvarez A, Navarrete M, Covelo A, Martin ED, Araque A (2014) Structural and functional plasticity of astrocyte processes and dendritic spine interactions. J Neurosci 34(38):12738–12744. https://doi.org/10.1523/JNEUROSCI.2401-14.2014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bernardinelli Y, Muller D, Nikonenko I (2014) Astrocyte-synapse structural plasticity. Neural Plast 2014:232105. https://doi.org/10.1155/2014/232105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Theodosis DT, Poulain DA, Oliet SH (2008) Activity-dependent structural and functional plasticity of astrocyte-neuron interactions. Physiol Rev 88(3):983–1008. https://doi.org/10.1152/physrev.00036.2007

    Article  CAS  PubMed  Google Scholar 

  26. Medvedev N, Popov V, Henneberger C, Kraev I, Rusakov DA, Stewart MG (2014) Glia selectively approach synapses on thin dendritic spines. Philos Trans R Soc Lond B Biol Sci 369(1654):20140047. https://doi.org/10.1098/rstb.2014.0047

    Article  PubMed  PubMed Central  Google Scholar 

  27. Lushnikova I, Skibo G, Muller D, Nikonenko I (2009) Synaptic potentiation induces increased glial coverage of excitatory synapses in CA1 hippocampus. Hippocampus 19(8):753–762. https://doi.org/10.1002/hipo.20551

    Article  PubMed  Google Scholar 

  28. Patrushev I, Gavrilov N, Turlapov V, Semyanov A (2013) Subcellular location of astrocytic calcium stores favors extrasynaptic neuron-astrocyte communication. Cell Calcium 54(5):343–349. https://doi.org/10.1016/j.ceca.2013.08.003

    Article  CAS  PubMed  Google Scholar 

  29. Witcher MR, Kirov SA, Harris KM (2007) Plasticity of perisynaptic astroglia during synaptogenesis in the mature rat hippocampus. Glia 55(1):13–23. https://doi.org/10.1002/glia.20415

    Article  PubMed  Google Scholar 

  30. Medvedev NI, Popov VI, Rodriguez Arellano JJ, Dallerac G, Davies HA, Gabbott PL, Laroche S, Kraev IV, Doyere V, Stewart MG (2010) The N-methyl-D-aspartate receptor antagonist CPP alters synapse and spine structure and impairs long-term potentiation and long-term depression induced morphological plasticity in dentate gyrus of the awake rat. Neuroscience 165(4):1170–1181. https://doi.org/10.1016/j.neuroscience.2009.11.047

    Article  CAS  PubMed  Google Scholar 

  31. Popov VI, Davies HA, Rogachevsky VV, Patrushev IV, Errington ML, Gabbott PL, Bliss TV, Stewart MG (2004) Remodelling of synaptic morphology but unchanged synaptic density during late phase long-term potentiation (LTP): a serial section electron micrograph study in the dentate gyrus in the anaesthetised rat. Neuroscience 128(2):251–262. https://doi.org/10.1016/j.neuroscience.2004.06.029

    Article  CAS  PubMed  Google Scholar 

  32. Sherpa AD, Xiao F, Joseph N, Aoki C, Hrabetova S (2016) Activation of beta-adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume. Synapse 70(8):307–316. https://doi.org/10.1002/syn.21908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Witcher MR, Park YD, Lee MR, Sharma S, Harris KM, Kirov SA (2010) Three-dimensional relationships between perisynaptic astroglia and human hippocampal synapses. Glia 58(5):572–587. https://doi.org/10.1002/glia.20946

    Article  PubMed  PubMed Central  Google Scholar 

  34. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645. https://doi.org/10.1126/science.1127344

    Article  PubMed  Google Scholar 

  35. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813. https://doi.org/10.1126/science.1153529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97(15):8206–8210

    Article  CAS  Google Scholar 

  37. Patton BR, Burke D, Owald D, Gould TJ, Bewersdorf J, Booth MJ (2016) Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics. Opt Express 24(8):8862–8876. https://doi.org/10.1364/OE.24.008862

    Article  PubMed  Google Scholar 

  38. Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317(5845):1749–1753. https://doi.org/10.1126/science.1146598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Panatier A, Arizono M, Nagerl UV (2014) Dissecting tripartite synapses with STED microscopy. Philos Trans R Soc Lond B Biol Sci 369(1654):20130597. https://doi.org/10.1098/rstb.2013.0597

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rossi A, Moritz TJ, Ratelade J, Verkman AS (2012) Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes. J Cell Sci 125(Pt 18):4405–4412. https://doi.org/10.1242/jcs.109603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Smith AJ, Verkman AS (2015) Superresolution imaging of aquaporin-4 cluster size in antibody-stained paraffin brain sections. Biophys J 109(12):2511–2522. https://doi.org/10.1016/j.bpj.2015.10.047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gucek A, Jorgacevski J, Singh P, Geisler C, Lisjak M, Vardjan N, Kreft M, Egner A, Zorec R (2016) Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state. Cell Mol Life Sci 73(19):3719–3731. https://doi.org/10.1007/s00018-016-2213-2

    Article  CAS  PubMed  Google Scholar 

  43. Sakers K, Lake AM, Khazanchi R, Ouwenga R, Vasek MJ, Dani A, Dougherty JD (2017) Astrocytes locally translate transcripts in their peripheral processes. Proc Natl Acad Sci U S A. https://doi.org/10.1073/pnas.1617782114

  44. van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6(7):991–1009. https://doi.org/10.1038/nprot.2011.336

    Article  CAS  PubMed  Google Scholar 

  45. Endesfelder U, Heilemann M (2015) Direct stochastic optical reconstruction microscopy (dSTORM). Methods Mol Biol 1251:263–276. https://doi.org/10.1007/978-1-4939-2080-8_14

    Article  CAS  PubMed  Google Scholar 

  46. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47(33):6172–6176. https://doi.org/10.1002/anie.200802376

    Article  CAS  PubMed  Google Scholar 

  47. Oberheim NA, Goldman SA, Nedergaard M (2012) Heterogeneity of astrocytic form and function. Methods Mol Biol 814:23–45. https://doi.org/10.1007/978-1-61779-452-0_3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Grosche A, Grosche J, Tackenberg M, Scheller D, Gerstner G, Gumprecht A, Pannicke T, Hirrlinger PG, Wilhelmsson U, Huttmann K, Hartig W, Steinhauser C, Pekny M, Reichenbach A (2013) Versatile and simple approach to determine astrocyte territories in mouse neocortex and hippocampus. PLoS One 8(7):e69143. https://doi.org/10.1371/journal.pone.0069143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nishiyama H, Knopfel T, Endo S, Itohara S (2002) Glial protein S100B modulates long-term neuronal synaptic plasticity. Proc Natl Acad Sci U S A 99(6):4037–4042. https://doi.org/10.1073/pnas.052020999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Heller JP, Michaluk P, Sugao K, Rusakov DA (2017) Probing nano-organization of astroglia with multi-color super-resolution microscopy. J Neurosci Res. https://doi.org/10.1002/jnr.24026

  51. Jiang R, Haustein MD, Sofroniew MV, Khakh BS (2014) Imaging intracellular Ca(2)(+) signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators. J Vis Exp 93:e51972. https://doi.org/10.3791/51972

    Article  CAS  Google Scholar 

  52. Shigetomi E, Patel S, Khakh BS (2016) Probing the complexities of astrocyte calcium signaling. Trends Cell Biol 26(4):300–312. https://doi.org/10.1016/j.tcb.2016.01.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Srinivasan R, Lu TY, Chai H, Xu J, Huang BS, Golshani P, Coppola G, Khakh BS (2016) New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo. Neuron 92(6):1181–1195. https://doi.org/10.1016/j.neuron.2016.11.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Holtmaat A, de Paola V, Wilbrecht L, Trachtenberg JT, Svoboda K, Portera-Cailliau C (2012) Imaging neocortical neurons through a chronic cranial window. Cold Spring Harb Protoc 2012(6):694–701. https://doi.org/10.1101/pdb.prot069617

    Article  PubMed  Google Scholar 

  55. Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14(11):1481–1488. https://doi.org/10.1038/nn.2928

    Article  CAS  PubMed  Google Scholar 

  56. Juette MF, Gould TJ, Lessard MD, Mlodzianoski MJ, Nagpure BS, Bennett BT, Hess ST, Bewersdorf J (2008) Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5(6):527–529. https://doi.org/10.1038/nmeth.1211

    Article  CAS  PubMed  Google Scholar 

  57. Mlodzianoski MJ, Juette MF, Beane GL, Bewersdorf J (2009) Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy. Opt Express 17(10):8264–8277

    Article  CAS  Google Scholar 

  58. Metcalf DJ, Edwards R, Kumarswami N, Knight AE (2013) Test samples for optimizing STORM super-resolution microscopy. J Vis Exp 79. https://doi.org/10.3791/50579

  59. Packer AM, Russell LE, Dalgleish HW, Hausser M (2015) Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo. Nat Methods 12(2):140–146. https://doi.org/10.1038/nmeth.3217

    Article  CAS  PubMed  Google Scholar 

  60. Srinivasan R, Huang BS, Venugopal S, Johnston AD, Chai H, Zeng H, Golshani P, Khakh BS (2015) Ca(2+) signaling in astrocytes from Ip3r2(−/−) mice in brain slices and during startle responses in vivo. Nat Neurosci 18(5):708–717. https://doi.org/10.1038/nn.4001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Bishop D, Nikic I, Brinkoetter M, Knecht S, Potz S, Kerschensteiner M, Misgeld T (2011) Near-infrared branding efficiently correlates light and electron microscopy. Nat Methods 8(7):568–570. https://doi.org/10.1038/nmeth.1622

    Article  CAS  PubMed  Google Scholar 

  62. Hausser M, Margrie TW (2014) Two-photon targeted patching and electroporation in vivo. Cold Spring Harb Protoc 2014(1):78–85. https://doi.org/10.1101/pdb.prot080143

    Article  PubMed  Google Scholar 

  63. Blazquez-Llorca L, Hummel E, Zimmerman H, Zou C, Burgold S, Rietdorf J, Herms J (2015) Correlation of two-photon in vivo imaging and FIB/SEM microscopy. J Microsc 259(2):129–136. https://doi.org/10.1111/jmi.12231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Henriques R, Lelek M, Fornasiero EF, Valtorta F, Zimmer C, Mhlanga MM (2010) QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7(5):339–340. https://doi.org/10.1038/nmeth0510-339

    Article  CAS  PubMed  Google Scholar 

  65. Ovesny M, Krizek P, Borkovec J, Svindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30(16):2389–2390. https://doi.org/10.1093/bioinformatics/btu202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Herrmannsdorfer F, Flottmann B, Nanguneri S, Venkataramani V, Horstmann H, Kuner T, Heilemann M (2017) 3D d STORM imaging of fixed brain tissue. Methods Mol Biol 1538:169–184. https://doi.org/10.1007/978-1-4939-6688-2_13

    Article  CAS  PubMed  Google Scholar 

  67. Pleiner T, Bates M, Trakhanov S, Lee CT, Schliep JE, Chug H, Bohning M, Stark H, Urlaub H, Gorlich D (2015) Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. elife 4:e11349. https://doi.org/10.7554/eLife.11349

    Article  PubMed  PubMed Central  Google Scholar 

  68. de Castro MA, Rammner B, Opazo F (2016) Aptamer stainings for super-resolution microscopy. Methods Mol Biol 1380:197–210. https://doi.org/10.1007/978-1-4939-3197-2_17

    Article  CAS  PubMed  Google Scholar 

  69. Chamma I, Rossier O, Giannone G, Thoumine O, Sainlos M (2017) Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin. Nat Protoc 12(4):748–763. https://doi.org/10.1038/nprot.2017.010

    Article  CAS  PubMed  Google Scholar 

  70. Teng KW, Ishitsuka Y, Ren P, Youn Y, Deng X, Ge P, Belmont AS, Selvin PR (2016) Labeling proteins inside living cells using external fluorophores for microscopy. elife 5. https://doi.org/10.7554/eLife.20378

  71. Teng KW, Ishitsuka Y, Ren P, Youn Y, Deng X, Ge P, Lee SH, Belmont AS, Selvin PR (2017) Labeling proteins inside living cells using external fluorophores for fluorescence microscopy. elife 6. https://doi.org/10.7554/eLife.25460

  72. Wieneke R, Raulf A, Kollmannsperger A, Heilemann M, Tampe R (2015) SLAP: small labeling pair for single-molecule super-resolution imaging. Angew Chem Int Ed Engl 54(35):10216–10219. https://doi.org/10.1002/anie.201503215

    Article  CAS  PubMed  Google Scholar 

  73. Lotze J, Reinhardt U, Seitz O, Beck-Sickinger AG (2016) Peptide-tags for site-specific protein labelling in vitro and in vivo. Mol BioSyst 12(6):1731–1745. https://doi.org/10.1039/c6mb00023a

    Article  CAS  PubMed  Google Scholar 

  74. Raulf A, Spahn CK, Zessin PJ, Finan K, Bernhardt S, Heckel A, Heilemann M (2014) Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteinsdaggerElectronic supplementary information (ESI) available. RSC Adv 4(57):30462–30466. https://doi.org/10.1039/c4ra01027b

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Mateos-Gil P, Letschert S, Doose S, Sauer M (2016) Super-resolution imaging of plasma membrane proteins with click chemistry. Front Cell Dev Biol 4:98. https://doi.org/10.3389/fcell.2016.00098

    Article  PubMed  PubMed Central  Google Scholar 

  76. Lowery RL, Majewska AK (2010) Intracranial injection of adeno-associated viral vectors. J Vis Exp 45. https://doi.org/10.3791/2140

  77. Furstenberg A, Heilemann M (2013) Single-molecule localization microscopy-near-molecular spatial resolution in light microscopy with photoswitchable fluorophores. Phys Chem Chem Phys 15(36):14919–14930. https://doi.org/10.1039/c3cp52289j

    Article  CAS  PubMed  Google Scholar 

  78. Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8(12):1027–1036. https://doi.org/10.1038/nmeth.1768

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795. https://doi.org/10.1038/nmeth929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Chozinski TJ, Gagnon LA, Vaughan JC (2014) Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging. FEBS Lett 588(19):3603–3612. https://doi.org/10.1016/j.febslet.2014.06.043

    Article  CAS  PubMed  Google Scholar 

  81. Turkowyd B, Virant D, Endesfelder U (2016) From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem 408(25):6885–6911. https://doi.org/10.1007/s00216-016-9781-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Minoshima M, Kikuchi K (2017) Photostable and photoswitching fluorescent dyes for super-resolution imaging. J Biol Inorg Chem. https://doi.org/10.1007/s00775-016-1435-y

  83. Olivier N, Keller D, Gonczy P, Manley S (2013) Resolution doubling in 3D-STORM imaging through improved buffers. PLoS One 8(7):e69004. https://doi.org/10.1371/journal.pone.0069004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Nahidiazar L, Agronskaia AV, Broertjes J, van den Broek B, Jalink K (2016) Optimizing imaging conditions for demanding multi-color super resolution localization microscopy. PLoS One 11(7):e0158884. https://doi.org/10.1371/journal.pone.0158884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Kwakwa K, Savell A, Davies T, Munro I, Parrinello S, Purbhoo MA, Dunsby C, Neil MA, French PM (2016) easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy. J Biophotonics 9(9):948–957. https://doi.org/10.1002/jbio.201500324

    Article  PubMed  Google Scholar 

  86. Herbert S, Soares H, Zimmer C, Henriques R (2012) Single-molecule localization super-resolution microscopy: deeper and faster. Microsc Microanal 18(6):1419–1429. https://doi.org/10.1017/S1431927612013347

    Article  CAS  PubMed  Google Scholar 

  87. Kao HP, Verkman AS (1994) Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys J 67(3):1291–1300. https://doi.org/10.1016/S0006-3495(94)80601-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Pavani SR, Thompson MA, Biteen JS, Lord SJ, Liu N, Twieg RJ, Piestun R, Moerner WE (2009) Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci U S A 106(9):2995–2999. https://doi.org/10.1073/pnas.0900245106

    Article  PubMed  PubMed Central  Google Scholar 

  89. Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, Hess HF (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106(9):3125–3130. https://doi.org/10.1073/pnas.0813131106

    Article  PubMed  PubMed Central  Google Scholar 

  90. Lampe A, Haucke V, Sigrist SJ, Heilemann M, Schmoranzer J (2012) Multi-colour direct STORM with red emitting carbocyanines. Biol Cell 104(4):229–237. https://doi.org/10.1111/boc.201100011

    Article  CAS  PubMed  Google Scholar 

  91. Smith CS, Joseph N, Rieger B, Lidke KA (2010) Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7(5):373–375. https://doi.org/10.1038/nmeth.1449

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783. https://doi.org/10.1016/S0006-3495(02)75618-X

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11):4258–4272. https://doi.org/10.1529/biophysj.106.091116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This research was supported by European Union's Horizon 2020 research and innovation program (Marie Skłodowska-Curie grant agreement 798644-AstroMiRimage), Wellcome Trust Principal Fellowship (101896), European Research Council Advanced Grant (323113-NETSIGNAL), FP7 ITN (606950 EXTRABRAIN), and European Commission NEUROTWIN Grant (857562).

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Authors and Affiliations

  1. UCL Queen Square Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK

    Janosch P. Heller, James P. Reynolds & Dmitri A. Rusakov

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  1. Janosch P. Heller
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  2. James P. Reynolds
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  3. Dmitri A. Rusakov
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Correspondence to Janosch P. Heller or Dmitri A. Rusakov .

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Editors and Affiliations

  1. Cryo EM, Centre for Advanced Materials (CAM), Universität Heidelberg, Heidelberg, Germany

    Irene Wacker

  2. Sector Life Science, Carl Zeiss Microscopy GmbH, Oberkochen, Germany, Carl Zeiss, Oberkochen, Germany

    Eric Hummel

  3. BioQuant, Universität Heidelberg, Heidelberg, Germany, Cryo EM, BioQuant, Universitätsklinikum Heidelberg, Heidelberg, Germany

    Steffen Burgold

  4. Cryo EM, Centre for Advanced Materials (CAM), Universität Heidelberg, Heidelberg, Germany, Cryo EM, BioQuant, Universitätsklinikum Heidelberg, Heidelberg, Germany

    Rasmus Schröder

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Heller, J.P., Reynolds, J.P., Rusakov, D.A. (2020). Multicolor Superresolution Microscopy: Revealing the Nano World of Astrocytes In Situ. In: Wacker, I., Hummel, E., Burgold, S., Schröder, R. (eds) Volume Microscopy . Neuromethods, vol 155. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0691-9_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0691-9_2

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