Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Advanced imaging and labelling methods to decipher brain cell organization and function

Abstract

The brain is arguably the most complex organ. The branched and extended morphology of nerve cells, their subcellular complexity, the multiplicity of brain cell types as well as their intricate connectivity and the scattering properties of brain tissue present formidable challenges to the understanding of brain function. Neuroscientists have often been at the forefront of technological and methodological developments to overcome these hurdles to visualize, quantify and modify cell and network properties. Over the last few decades, the development of advanced imaging methods has revolutionized our approach to explore the brain. Super-resolution microscopy and tissue imaging approaches have recently exploded. These instrumentation-based innovations have occurred in parallel with the development of new molecular approaches to label protein targets, to evolve new biosensors and to target them to appropriate cell types or subcellular compartments. We review the latest developments for labelling and functionalizing proteins with small localization and functionalized reporters. We present how these molecular tools are combined with the development of a wide variety of imaging methods that break either the diffraction barrier or the tissue penetration depth limits. We put these developments in perspective to emphasize how they will enable step changes in our understanding of the brain.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Multiscale imaging in neuroscience.
Fig. 2: Comparison of probes used for protein labelling.
Fig. 3: Comparison of common protein labelling strategies.
Fig. 4: Main strategies used to control the emitting state of fluorescent probes for SRI applications.
Fig. 5: High-resolution and super-resolution imaging principles.
Fig. 6: Single-molecule-based super-resolution and tracking examples.

References

  1. 1.

    Chen, H., Tang, A. H. & Blanpied, T. A. Subsynaptic spatial organization as a regulator of synaptic strength and plasticity. Curr. Opin. Neurobiol. 51, 147–153 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Bourne, J. N. & Harris, K. M. Balancing structure and function at hippocampal dendritic spines. Annu. Rev. Neurosci. 31, 47–67 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Choquet, D. Linking nanoscale dynamics of AMPA receptor organization to plasticity of excitatory synapses and learning. J. Neurosci. 38, 9318–9329 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Biederer, T., Kaeser, P. S. & Blanpied, T. A. Transcellular nanoalignment of synaptic function. Neuron 96, 680–696 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Tang, A. H. et al. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature https://doi.org/10.1038/nature19058 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Haas, K. T. et al. Pre-post synaptic alignment through neuroligin-1 tunes synaptic transmission efficiency. eLife https://doi.org/10.7554/eLife.31755 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Hruska, M., Henderson, N., Le Marchand, S. J., Jafri, H. & Dalva, M. B. Synaptic nanomodules underlie the organization and plasticity of spine synapses. Nat. Neurosci. 21, 671–682 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Leterrier, C. The axon initial segment: an updated viewpoint. J. Neurosci. 38, 2135–2145 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452–456 (2013). This article describes one of the key breakthrough discoveries in the neuroscience field achieved by super-resolution microscopy.

    CAS  PubMed  Google Scholar 

  10. 10.

    Leterrier, C., Dubey, P. & Roy, S. The nano-architecture of the axonal cytoskeleton. Nat. reviews. Neurosci. 18, 713–726 (2017).

    CAS  Google Scholar 

  11. 11.

    Zhou, R., Han, B., Xia, C. & Zhuang, X. Membrane-associated periodic skeleton is a signaling platform for RTK transactivation in neurons. Science 365, 929–934 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Vassilopoulos, S., Gibaud, S., Jimenez, A., Caillol, G. & Leterrier, C. Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings. Nat. Commun. 10, 5803 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Theodosis, D. T., Poulain, D. A. & Oliet, S. H. Activity-dependent structural and functional plasticity of astrocyte-neuron interactions. Physiol. Rev. 88, 983–1008 (2008).

    CAS  PubMed  Google Scholar 

  14. 14.

    Ramon y Cajal, S. Textura del Sistema Nervioso del Hombre y de los Vertebrados: Estudios Sobre el Plan Estructural y Composición Histológica de los Centros Nerviosos Adicionados de Consideraciones Fisiológicas Fundadas en los Nuevos Descubrimentos (Moya, 1899).

  15. 15.

    Betzig, E. Single molecules, cells, and super-resolution optics (Nobel Lecture). Angew. Chem. Int. Ed. 54, 8034–8053 (2015).

    CAS  Google Scholar 

  16. 16.

    Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 18, 685–701 (2017).

    CAS  PubMed  Google Scholar 

  17. 17.

    Saxton, M. J. & Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).

    CAS  PubMed  Google Scholar 

  18. 18.

    Bruchez, M. P. Quantum dots find their stride in single molecule tracking. Curr. Opin. Chem. Biol. 15, 775–780 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Balasubramanian, G., Lazariev, A., Arumugam, S. R. & Duan, D. W. Nitrogen-Vacancy color center in diamond-emerging nanoscale applications in bioimaging and biosensing. Curr. Opin. Chem. Biol. 20, 69–77 (2014).

    CAS  PubMed  Google Scholar 

  20. 20.

    Schlichthaerle, T., Strauss, M. T., Schueder, F., Woehrstein, J. B. & Jungmann, R. DNA nanotechnology and fluorescence applications. Curr. Opin. Biotechnol. 39, 41–47 (2016).

    CAS  PubMed  Google Scholar 

  21. 21.

    Lee, S. H. et al. Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes. eLife https://doi.org/10.7554/eLife.27744 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Godin, A. G. et al. Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain. Nat. Nanotechnol. 12, 238–243 (2017).

    CAS  PubMed  Google Scholar 

  23. 23.

    Godin, A. G. et al. Photoswitchable single-walled carbon nanotubes for super-resolution microscopy in the near-infrared. Sci. Adv. 5, eaax1166 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Wang, L., Frei, M. S., Salim, A. & Johnsson, K. Small-molecule fluorescent probes for live-cell super-resolution microscopy. J. Am. Chem. Soc. 141, 2770–2781 (2019).

    CAS  PubMed  Google Scholar 

  25. 25.

    Li, H. & Vaughan, J. C. Switchable fluorophores for single-molecule localization microscopy. Chem. Rev. 118, 9412–9454 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Koide, Y. et al. Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging. J. Am. Chem. Soc. 134, 5029–5031 (2012).

    CAS  PubMed  Google Scholar 

  28. 28.

    Lukinavicius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    CAS  PubMed  Google Scholar 

  29. 29.

    Uno, K., Bossi, M. L., Irie, M., Belov, V. N. & Hell, S. W. Reversibly photoswitchable fluorescent diarylethenes resistant against photobleaching in aqueous solutions. J. Am. Chem. Soc. 141, 16471–16478 (2019).

    CAS  PubMed  Google Scholar 

  30. 30.

    Podgorski, K., Terpetschnig, E., Klochko, O. P., Obukhova, O. M. & Haas, K. Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging. PLoS ONE 7, e51980 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Tsunoyama, T. A. et al. Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function. Nat. Chem. Biol. 14, 497–506 (2018).

    CAS  PubMed  Google Scholar 

  32. 32.

    Niekamp, S., Stuurman, N. & Vale, R. D. A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging. Nat. Methods https://doi.org/10.1038/s41592-020-0782-3 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Plamont, M. A. et al. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. Proc. Natl Acad. Sci. USA 113, 497–502 (2016).

    CAS  PubMed  Google Scholar 

  34. 34.

    Kozma, E. & Kele, P. Fluorogenic probes for super-resolution microscopy. Org. Biomol. Chem. 17, 215–233 (2019).

    CAS  PubMed  Google Scholar 

  35. 35.

    Zheng, Q. et al. Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging. ACS Cent. Sci. 5, 1602–1613 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Wang, L. et al. A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy. Nat. Chem. 12, 165–172 (2020).

    CAS  PubMed  Google Scholar 

  37. 37.

    Jradi, F. M. & Lavis, L. D. Chemistry of photosensitive fluorophores for single-molecule localization microscopy. ACS Chem. Biol. 14, 1077–1090 (2019).

    CAS  PubMed  Google Scholar 

  38. 38.

    Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47, 6172–6176 (2008).

    CAS  Google Scholar 

  39. 39.

    Grimm, J. B. et al. Bright photoactivatable fluorophores for single-molecule imaging. Nat. Methods 13, 985–988 (2016).

    CAS  PubMed  Google Scholar 

  40. 40.

    Frei, M. S. et al. Photoactivation of silicon rhodamines via a light-induced protonation. Nat. Commun. 10, 4580 (2019).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Uno, S. N. et al. A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging. Nat. Chem. 6, 681–689 (2014).

    CAS  PubMed  Google Scholar 

  42. 42.

    Uno, S. N., Kamiya, M., Morozumi, A. & Urano, Y. A green-light-emitting, spontaneously blinking fluorophore based on intramolecular spirocyclization for dual-colour super-resolution imaging. Chem. Commun. 54, 102–105 (2017).

    Google Scholar 

  43. 43.

    Arai, Y. et al. Spontaneously blinking fluorescent protein for simple single laser super-resolution live cell imaging. ACS Chem. Biol. 13, 1938–1943 (2018).

    CAS  PubMed  Google Scholar 

  44. 44.

    Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

    CAS  PubMed  Google Scholar 

  45. 45.

    Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Guo, S. M. et al. Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes. Nat. Commun. 10, 4377 (2019).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Kiuchi, T., Higuchi, M., Takamura, A., Maruoka, M. & Watanabe, N. Multitarget super-resolution microscopy with high-density labeling by exchangeable probes. Nat. Methods 12, 743–746 (2015).

    CAS  PubMed  Google Scholar 

  49. 49.

    Asanuma, D. et al. Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics. Angew. Chem. Int. Ed. 53, 6085–6089 (2014).

    CAS  Google Scholar 

  50. 50.

    Martineau, M. et al. Semisynthetic fluorescent pH sensors for imaging exocytosis and endocytosis. Nat. Commun. 8, 1412 (2017).

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Collot, M. et al. CaRuby-Nano: a novel high affinity calcium probe for dual color imaging. eLife https://doi.org/10.7554/eLife.05808 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Contractor, A. A. & Miller, E. W. Imaging Ca2+ with a fluorescent rhodol. Biochemistry 57, 237–240 (2018).

    CAS  PubMed  Google Scholar 

  53. 53.

    Boggess, S. C. et al. New molecular scaffolds for fluorescent voltage indicators. ACS Chem. Biol. 14, 390–396 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Deal, P. E. et al. Covalently tethered rhodamine voltage reporters for high speed functional imaging in brain tissue. J. Am. Chem. Soc. 142, 614–622 (2020).

    CAS  PubMed  Google Scholar 

  55. 55.

    Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).

    CAS  PubMed  Google Scholar 

  56. 56.

    Shaner, N. C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci. Rep. 6, 20889 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).

    CAS  PubMed  Google Scholar 

  59. 59.

    Oliinyk, O. S., Shemetov, A. A., Pletnev, S., Shcherbakova, D. M. & Verkhusha, V. V. Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing. Nat. Commun. 10, 279 (2019).

    PubMed  PubMed Central  Google Scholar 

  60. 60.

    Matlashov, M. E. et al. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nat. Commun. 11, 239 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Cranfill, P. J. et al. Quantitative assessment of fluorescent proteins. Nat. Methods 13, 557–562 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Balleza, E., Kim, J. M. & Cluzel, P. Systematic characterization of maturation time of fluorescent proteins in living cells. Nat. Methods 15, 47–51 (2018).

    CAS  PubMed  Google Scholar 

  63. 63.

    Lambert, T. J. FPbase: a community-editable fluorescent protein database. Nat. Methods 16, 277–278 (2019).

    CAS  PubMed  Google Scholar 

  64. 64.

    Shcherbakova, D. M. & Verkhusha, V. V. Chromophore chemistry of fluorescent proteins controlled by light. Curr. Opin. Chem. Biol. 20, 60–68 (2014).

    CAS  PubMed  Google Scholar 

  65. 65.

    Nienhaus, K. & Nienhaus, G. U. Fluorescent proteins for live-cell imaging with super-resolution. Chem. Soc. Rev. 43, 1088–1106 (2014).

    CAS  PubMed  Google Scholar 

  66. 66.

    Zhang, M. et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat. Methods 9, 727–729 (2012).

    CAS  PubMed  Google Scholar 

  67. 67.

    Wang, S., Moffitt, J. R., Dempsey, G. T., Xie, X. S. & Zhuang, X. Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proc. Natl Acad. Sci. USA 111, 8452–8457 (2014).

    CAS  PubMed  Google Scholar 

  68. 68.

    Zhang, M. et al. Fast super-resolution imaging technique and immediate early nanostructure capturing by a photoconvertible fluorescent protein. Nano Lett. https://doi.org/10.1021/acs.nanolett.9b02855 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Zhang, X. et al. Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy. Proc. Natl Acad. Sci. USA 113, 10364–10369 (2016).

    CAS  PubMed  Google Scholar 

  70. 70.

    Pennacchietti, F. et al. Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy. Nat. Methods 15, 601–604 (2018).

    CAS  PubMed  Google Scholar 

  71. 71.

    Bourgeois, D. Deciphering structural photophysics of fluorescent proteins by kinetic crystallography. Int. J. Mol. Sci. https://doi.org/10.3390/ijms18061187 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Duwe, S. & Dedecker, P. Optimizing the fluorescent protein toolbox and its use. Curr. Opin. Biotechnol. 58, 183–191 (2019).

    CAS  PubMed  Google Scholar 

  73. 73.

    Subach, F. V., Piatkevich, K. D. & Verkhusha, V. V. Directed molecular evolution to design advanced red fluorescent proteins. Nat. Methods 8, 1019–1026 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat. Methods 8, 1027–1036 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Hartwich, T. M. P. et al. A stable, high refractive index, switching buffer for super-resolution imaging. bioRxiv https://doi.org/10.1101/465492 (2018).

    Article  Google Scholar 

  76. 76.

    De Zitter, E. et al. Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM. Nat. Methods 16, 707–710 (2019).

    PubMed  Google Scholar 

  77. 77.

    Klevanski, M. et al. Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues. Nat. Commun. https://doi.org/10.1038/s41467-020-15362-1 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Chamma, I. et al. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin. Nat. Commun. 7, 10773 (2016). This article reports an engineered monomeric ligand to label specifically biotinylated proteins.

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Helma, J., Cardoso, M. C., Muyldermans, S. & Leonhardt, H. Nanobodies and recombinant binders in cell biology. J. Cell Biol. 209, 633–644 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Bedford, R. et al. Alternative reagents to antibodies in imaging applications. Biophys. Rev. 9, 299–308 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Fukata, Y. et al. Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J. Cell Biol. 202, 145–161 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82.

    Gross, G. G. et al. Recombinant probes for visualizing endogenous synaptic proteins in living neurons. Neuron 78, 971–985 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. 83.

    Dong, J. X. et al. A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons. eLife https://doi.org/10.7554/eLife.48750 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  84. 84.

    Gerdes, C. et al. A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol. bioRxiv https://doi.org/10.1101/846014 (2019).

    Article  Google Scholar 

  85. 85.

    Maidorn, M., Olichon, A., Rizzoli, S. O. & Opazo, F. Nanobodies reveal an extra-synaptic population of SNAP-25 and syntaxin 1A in hippocampal neurons. mAbs 11, 305–321 (2019).

    CAS  PubMed  Google Scholar 

  86. 86.

    Pleiner, T., Bates, M. & Gorlich, D. A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies. J. Cell Biol. 217, 1143–1154 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Schlichthaerle, T., Ganji, M., Auer, A., Kimbu Wade, O. & Jungmann, R. Bacterially derived antibody binders as small adapters for DNA-PAINT microscopy. Chembiochem 20, 1032–1038 (2019).

    CAS  PubMed  Google Scholar 

  88. 88.

    Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89.

    Lukinavicius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).

    CAS  PubMed  Google Scholar 

  90. 90.

    Tamura, T. & Hamachi, I. Chemistry for covalent modification of endogenous/native proteins: from test tubes to complex biological systems. J. Am. Chem. Soc. 141, 2782–2799 (2019).

    CAS  PubMed  Google Scholar 

  91. 91.

    Wakayama, S. et al. Chemical labelling for visualizing native AMPA receptors in live neurons. Nat. Commun. 8, 14850 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Arttamangkul, S. et al. Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry. eLife https://doi.org/10.7554/eLife.49319 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  93. 93.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. 94.

    Ghosh, R. P. et al. A fluorogenic array for temporally unlimited single-molecule tracking. Nat. Chem. Biol. 15, 401–409 (2019).

    CAS  PubMed  Google Scholar 

  95. 95.

    Szent-Gyorgyi, C. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26, 235–240 (2008).

    CAS  PubMed  Google Scholar 

  96. 96.

    Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    CAS  PubMed  Google Scholar 

  97. 97.

    Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    CAS  PubMed  Google Scholar 

  98. 98.

    Erdmann, R. S. et al. Labeling strategies matter for super-resolution microscopy: a comparison between halotags and SNAP-tags. Cell Chem. Biol. 26, 584–592 e586 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. 99.

    Brizzard, B. Epitope tagging. Biotechniques 44, 693–695 (2008).

    CAS  PubMed  Google Scholar 

  100. 100.

    Vandemoortele, G., Eyckerman, S. & Gevaert, K. Pick a tag and explore the functions of your pet protein. Trends Biotechnol. 37, 1078–1090 (2019).

    CAS  PubMed  Google Scholar 

  101. 101.

    Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).

    CAS  Google Scholar 

  102. 102.

    Virant, D. et al. A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging. Nat. Commun. 9, 930 (2018).

    PubMed  PubMed Central  Google Scholar 

  103. 103.

    Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).

    PubMed  PubMed Central  Google Scholar 

  104. 104.

    Zhao, N. et al. A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo. Nat. Commun. 10, 2947 (2019).

    PubMed  PubMed Central  Google Scholar 

  105. 105.

    Wieneke, R. & Tampe, R. Multivalent chelators for in vivo protein labeling. Angew. Chem. Int. Ed. 58, 8278–8290 (2019).

    CAS  Google Scholar 

  106. 106.

    Howarth, M. & Ting, A. Y. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat. Protoc. 3, 534–545 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107.

    Krogager, T. P. et al. Labeling and identifying cell-specific proteomes in the mouse brain. Nat. Biotechnol. 36, 156–159 (2018).

    CAS  PubMed  Google Scholar 

  108. 108.

    Neubert, F. et al. Bioorthogonal click chemistry enables site-specific fluorescence labeling of functional NMDA receptors for super-resolution imaging. Angew. Chem. Int. Ed. 57, 16364–16369 (2018).

    CAS  Google Scholar 

  109. 109.

    Lang, K. & Chin, J. W. Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 16–20 (2014).

    CAS  PubMed  Google Scholar 

  110. 110.

    Beliu, G. et al. Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Commun. Biol. 2, 261 (2019).

    PubMed  PubMed Central  Google Scholar 

  111. 111.

    Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. 112.

    Nishiyama, J., Mikuni, T. & Yasuda, R. Virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain. Neuron 96, 755–768 e755 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113.

    Gao, Y. et al. Plug-and-play protein modification using homology-independent universal genome engineering. Neuron 103, 583–597 e588 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. 114.

    Willems, J. et al. ORANGE: a CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons. PLoS Biol. 18, e3000665 (2020).

    PubMed  PubMed Central  Google Scholar 

  115. 115.

    Thevathasan, J. V. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat. Methods 16, 1045–1053 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. 116.

    Harris, K. M. & Stevens, J. K. Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9, 2982–2997 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Choquet, D. The 2014 Nobel Prize in Chemistry: a large-scale prize for achievements on the nanoscale. Neuron 84, 1116–1119 (2014).

    CAS  PubMed  Google Scholar 

  118. 118.

    Sigal, Y. M., Zhou, R. & Zhuang, X. Visualizing and discovering cellular structures with super-resolution microscopy. Science 361, 880–887 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. 119.

    Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21, 72–84 (2019).

    CAS  PubMed  Google Scholar 

  120. 120.

    Inavalli, V. et al. A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Nat. Methods https://doi.org/10.1038/s41592-019-0611-8 (2019).

    Article  PubMed  Google Scholar 

  121. 121.

    Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).

    CAS  PubMed  Google Scholar 

  122. 122.

    Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).

    CAS  PubMed  Google Scholar 

  123. 123.

    Cnossen, J. et al. Localization microscopy at doubled precision with patterned illumination. Nat. Methods 17, 59–63 (2020).

    CAS  PubMed  Google Scholar 

  124. 124.

    Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. 125.

    Ober, R. J., Ram, S. & Ward, E. S. Localization accuracy in single-molecule microscopy. Biophys. J. 86, 1185–1200 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. 126.

    Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253–266 (2014).

    CAS  PubMed  Google Scholar 

  127. 127.

    Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    PubMed  PubMed Central  Google Scholar 

  128. 128.

    Kusumi, A., Sako, Y. & Yamamoto, M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65, 2021–2040 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. 129.

    Sibarita, J. B. High-density single-particle tracking: quantifying molecule organization and dynamics at the nanoscale. Histochem. Cell Biol. https://doi.org/10.1007/s00418-014-1214-1 (2014).

    Article  PubMed  Google Scholar 

  130. 130.

    Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    CAS  PubMed  Google Scholar 

  131. 131.

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. 132.

    Juette, M. F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    CAS  PubMed  Google Scholar 

  133. 133.

    Pavani, S. R. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl Acad. Sci. USA 106, 2995–2999 (2009).

    CAS  PubMed  Google Scholar 

  134. 134.

    Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).

    CAS  PubMed  Google Scholar 

  135. 135.

    Sage, D. et al. Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software. Nat. Methods https://doi.org/10.1038/s41592-019-0364-4 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  136. 136.

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006). This is one of the first two studies, with Hess et al. (2006), using photoactivation of fluorescent proteins for single-molecule localization-based super-resolution microscopy.

    CAS  Google Scholar 

  137. 137.

    Hess, S. T., Girirajan, T. P. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006). This is one of the first two studies, with Betzig et al. (2006), using photoactivation of fluorescent proteins for single-molecule localization-based super-resolution microscopy.

    CAS  PubMed  PubMed Central  Google Scholar 

  138. 138.

    Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006). This is the first study using photoactivation of organic dyes for single-molecule localization-based super-resolution microscopy.

    CAS  PubMed  PubMed Central  Google Scholar 

  139. 139.

    Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

    CAS  PubMed  Google Scholar 

  140. 140.

    Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010). This is the first study to use SRI to image a neuronal structure, the synapse.

    CAS  PubMed  PubMed Central  Google Scholar 

  141. 141.

    Nair, D. et al. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J. Neurosci. 33, 13204–13224 (2013). This is one of the two first studies, with MacGillavry et al. (2013), reporting the nanoscale organization of glutamate receptors in synapses.

    CAS  PubMed  PubMed Central  Google Scholar 

  142. 142.

    MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013). This is one of the two first studies, with Nair et al. (2013), reporting the nanoscale organization of glutamate receptors in synapses.

    CAS  PubMed  PubMed Central  Google Scholar 

  143. 143.

    Kellermayer, B. et al. Differential nanoscale topography and functional role of GluN2-NMDA receptor subtypes at glutamatergic synapses. Neuron 100, 106–119 e107 (2018).

    CAS  PubMed  Google Scholar 

  144. 144.

    Goncalves, J. et al. Nanoscale co-organization and coactivation of AMPAR, NMDAR, and mGluR at excitatory synapses. Proc. Natl Acad. Sci. USA 117, 14503–14511 (2020).

    CAS  PubMed  Google Scholar 

  145. 145.

    Ferreira, J. S. et al. Distance-dependent regulation of NMDAR nanoscale organization along hippocampal neuron dendrites. Proc. Natl Acad. Sci. USA 117, 24526–24533 (2020).

    CAS  PubMed  Google Scholar 

  146. 146.

    Specht, C. G. et al. Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites. Neuron 79, 308–321 (2013).

    CAS  PubMed  Google Scholar 

  147. 147.

    Bannai, H. et al. Bidirectional control of synaptic GABAAR clustering by glutamate and calcium. Cell Rep. 13, 2768–2780 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. 148.

    Jullie, D. et al. A discrete presynaptic vesicle cycle for neuromodulator receptors. Neuron 105, 663–677 e668 (2020).

    CAS  PubMed  Google Scholar 

  149. 149.

    Hannan, S., Gerrow, K., Triller, A. & Smart, T. G. Phospho-dependent accumulation of GABABRs at presynaptic terminals after NMDAR Activation. Cell Rep. 16, 1962–1973 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. 150.

    Siddig, S. et al. Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones. Sci. Adv. 6, eaay7193 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. 151.

    Schneider, R. et al. Mobility of calcium channels in the presynaptic membrane. Neuron 86, 672–679 (2015).

    CAS  PubMed  Google Scholar 

  152. 152.

    Bademosi, A. T. et al. In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters. Nat. Commun. 8, 13660 (2017).

    CAS  PubMed  Google Scholar 

  153. 153.

    Sinnen, B. L. et al. Optogenetic control of synaptic composition and function. Neuron 93, 646–660 e645 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. 154.

    Frost, N. A., Shroff, H., Kong, H., Betzig, E. & Blanpied, T. A. Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines. Neuron 67, 86–99 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. 155.

    Izeddin, I. et al. Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe. PLoS ONE 6, e15611 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. 156.

    Garcia, M. et al. Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones. Proc. Natl Acad. Sci. USA 112, 6997–7002 (2015).

    CAS  PubMed  Google Scholar 

  157. 157.

    Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat. Methods 9, 185–188 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  158. 158.

    Rahbek-Clemmensen, T. et al. Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains. Nat. Commun. 8, 740 (2017).

    PubMed  PubMed Central  Google Scholar 

  159. 159.

    Heller, J. P., Odii, T., Zheng, K. & Rusakov, D. A. Imaging tripartite synapses using super-resolution microscopy. Methods 174, 81–90 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  160. 160.

    Sigal, Y. M., Speer, C. M., Babcock, H. P. & Zhuang, X. Mapping synaptic input fields of neurons with super-resolution imaging. Cell 163, 493–505 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  161. 161.

    Levet, F. et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nat. Methods 12, 1065–1071 (2015). This article reports the development of a new method for quantification of single-molecule localization data.

    CAS  PubMed  Google Scholar 

  162. 162.

    Hoze, N. et al. Heterogeneity of AMPA receptor trafficking and molecular interactions revealed by superresolution analysis of live cell imaging. Proc. Natl Acad. Sci. USA 109, 17052–17057 (2012).

    CAS  PubMed  Google Scholar 

  163. 163.

    Masson, J. B. et al. Mapping the energy and diffusion landscapes of membrane proteins at the cell surface using high-density single-molecule imaging and bayesian inference: application to the multiscale dynamics of glycine receptors in the neuronal membrane. Biophys. J. 106, 74–83 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  164. 164.

    Jungmann, R. et al. Quantitative super-resolution imaging with qPAINT. Nat. Methods 13, 439–442 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  165. 165.

    Levet, F. et al. A tessellation-based colocalization analysis approach for single-molecule localization microscopy. Nat. Commun. 10, 2379 (2019).

    PubMed  PubMed Central  Google Scholar 

  166. 166.

    Dudok, B. et al. Cell-specific STORM super-resolution imaging reveals nanoscale organization of cannabinoid signaling. Nat. Neurosci. 18, 75–86 (2015).

    CAS  PubMed  Google Scholar 

  167. 167.

    Gustafsson, M. G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    CAS  PubMed  Google Scholar 

  168. 168.

    Wu, Y. & Shroff, H. Faster, sharper, and deeper: structured illumination microscopy for biological imaging. Nat. Methods 15, 1011–1019 (2018).

    CAS  PubMed  Google Scholar 

  169. 169.

    Gustafsson, M. G. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).

    CAS  PubMed  Google Scholar 

  170. 170.

    Nozumi, M., Nakatsu, F., Katoh, K. & Igarashi, M. Coordinated movement of vesicles and actin bundles during nerve growth revealed by superresolution microscopy. Cell Rep. 18, 2203–2216 (2017).

    CAS  PubMed  Google Scholar 

  171. 171.

    Wang, T. et al. Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability. J. Cell Biol. https://doi.org/10.1083/jcb.201902001 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  172. 172.

    Liu, C., Kershberg, L., Wang, J., Schneeberger, S. & Kaeser, P. S. Dopamine secretion is mediated by sparse active zone-like release sites. Cell 172, 706–718 e715 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  173. 173.

    Turcotte, R. et al. Dynamic super-resolution structured illumination imaging in the living brain. Proc. Natl Acad. Sci. USA 116, 9586–9591 (2019).

    CAS  PubMed  Google Scholar 

  174. 174.

    Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994). This article reports the invention of STED microscopy.

    CAS  PubMed  Google Scholar 

  175. 175.

    Vicidomini, G., Bianchini, P. & Diaspro, A. STED super-resolved microscopy. Nat. Methods 15, 173–182 (2018).

    CAS  PubMed  Google Scholar 

  176. 176.

    Kittel, R. J. et al. Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312, 1051–1054 (2006).

    CAS  PubMed  Google Scholar 

  177. 177.

    Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

    CAS  PubMed  Google Scholar 

  178. 178.

    Sieber, J. J. et al. Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317, 1072–1076 (2007).

    CAS  PubMed  Google Scholar 

  179. 179.

    Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).

    CAS  PubMed  Google Scholar 

  180. 180.

    Nagerl, U. V., Willig, K. I., Hein, B., Hell, S. W. & Bonhoeffer, T. Live-cell imaging of dendritic spines by STED microscopy. Proc. Natl Acad. Sci. USA 105, 18982–18987 (2008).

    CAS  PubMed  Google Scholar 

  181. 181.

    Tonnesen, J., Katona, G., Rozsa, B. & Nagerl, U. V. Spine neck plasticity regulates compartmentalization of synapses. Nat. Neurosci. 17, 678–685 (2014). This article reports the invention of SUSHI of brain extracellular space in living organotypic brain slices.

    CAS  PubMed  Google Scholar 

  182. 182.

    Berning, S., Willig, K. I., Steffens, H., Dibaj, P. & Hell, S. W. Nanoscopy in a living mouse brain. Science 335, 551 (2012).

    CAS  PubMed  Google Scholar 

  183. 183.

    Kilian, N. et al. Assessing photodamage in live-cell STED microscopy. Nat. Methods 15, 755–756 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  184. 184.

    Grotjohann, T. et al. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478, 204–208 (2011).

    CAS  PubMed  Google Scholar 

  185. 185.

    Dreier, J. et al. Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo. Nat. Commun. 10, 556 (2019).

    PubMed  PubMed Central  Google Scholar 

  186. 186.

    Tonnesen, J., Inavalli, V. & Nagerl, U. V. Super-resolution imaging of the extracellular space in living brain tissue. Cell 172, 1108–1121 e1115 (2018).

    PubMed  Google Scholar 

  187. 187.

    Chmyrov, A. et al. Nanoscopy with more than 100,000 ‘doughnuts’. Nat. Methods 10, 737–740 (2013).

    CAS  PubMed  Google Scholar 

  188. 188.

    Yang, B., Przybilla, F., Mestre, M., Trebbia, J. B. & Lounis, B. Large parallelization of STED nanoscopy using optical lattices. Opt. Express 22, 5581–5589 (2014).

    PubMed  Google Scholar 

  189. 189.

    Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  PubMed  Google Scholar 

  190. 190.

    Horton, N. G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat. Photonics https://doi.org/10.1038/nphoton.2012.336 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  191. 191.

    Ouzounov, D. G. et al. In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain. Nat. Methods 14, 388–390 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  192. 192.

    Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    CAS  PubMed  Google Scholar 

  193. 193.

    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).

    CAS  PubMed  Google Scholar 

  194. 194.

    Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat. Neurosci. 11, 713–720 (2008).

    CAS  PubMed  Google Scholar 

  195. 195.

    Grewe, B. F., Langer, D., Kasper, H., Kampa, B. M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399–405 (2010).

    CAS  PubMed  Google Scholar 

  196. 196.

    Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).

    CAS  PubMed  Google Scholar 

  197. 197.

    Papagiakoumou, E., Ronzitti, E. & Emiliani, V. Scanless two-photon excitation with temporal focusing. Nat. Methods https://doi.org/10.1038/s41592-020-0795-y (2020).

    Article  PubMed  Google Scholar 

  198. 198.

    Schrodel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat. Methods 10, 1013–1020 (2013).

    PubMed  Google Scholar 

  199. 199.

    Prevedel, R. et al. Fast volumetric calcium imaging across multiple cortical layers using sculpted light. Nat. Methods 13, 1021–1028 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  200. 200.

    Zong, W. et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat. Methods 14, 713–719 (2017).

    CAS  PubMed  Google Scholar 

  201. 201.

    Hillman, E. M. C., Voleti, V., Li, W. & Yu, H. Light-sheet microscopy in Neuroscience. Annu. Rev. Neurosci. 42, 295–313 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  202. 202.

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

    CAS  PubMed  Google Scholar 

  203. 203.

    Keller, P. J., Schmidt, A. D., Wittbrodt, J. & Stelzer, E. H. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

    CAS  PubMed  Google Scholar 

  204. 204.

    Keller, P. J. & Ahrens, M. B. Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy. Neuron 85, 462–483 (2015).

    CAS  PubMed  Google Scholar 

  205. 205.

    Dodt, H. U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

    CAS  PubMed  Google Scholar 

  206. 206.

    Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    CAS  PubMed  Google Scholar 

  207. 207.

    Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).

    CAS  PubMed  Google Scholar 

  208. 208.

    Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M. & Fraser, S. E. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat. Methods 8, 757–760 (2011).

    CAS  PubMed  Google Scholar 

  209. 209.

    Krzic, U., Gunther, S., Saunders, T. E., Streichan, S. J. & Hufnagel, L. Multiview light-sheet microscope for rapid in toto imaging. Nat. Methods 9, 730–733 (2012).

    CAS  PubMed  Google Scholar 

  210. 210.

    Wu, Y. et al. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  211. 211.

    Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 108, 17708–17713 (2011).

    CAS  PubMed  Google Scholar 

  212. 212.

    Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  213. 213.

    Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014). This article reports the invention of LLSM for high-resolution fast thick sample imaging.

    PubMed  PubMed Central  Google Scholar 

  214. 214.

    Chu, L. A. et al. Rapid single-wavelength lightsheet localization microscopy for clarified tissue. Nat. Commun. 10, 4762 (2019).

    PubMed  PubMed Central  Google Scholar 

  215. 215.

    Gao, R. et al. Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science https://doi.org/10.1126/science.aau8302 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  216. 216.

    Galland, R. et al. 3D high- and super-resolution imaging using single-objective SPIM. Nat. Methods 12, 641–644 (2015).

    CAS  PubMed  Google Scholar 

  217. 217.

    Kim, J. et al. Oblique-plane single-molecule localization microscopy for tissues and small intact animals. Nat. Methods 16, 853–857 (2019).

    CAS  PubMed  Google Scholar 

  218. 218.

    Gustavsson, A. K., Petrov, P. N., Lee, M. Y., Shechtman, Y. & Moerner, W. E. 3D single-molecule super-resolution microscopy with a tilted light sheet. Nat. Commun. 9, 123 (2018).

    PubMed  PubMed Central  Google Scholar 

  219. 219.

    Bouchard, M. B. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photonics 9, 113–119 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  220. 220.

    Chakraborty, T. et al. Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution. Nat. Methods 16, 1109–1113 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  221. 221.

    Voleti, V. et al. Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0. Nat. Methods 16, 1054–1062 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  222. 222.

    Gradinaru, V., Treweek, J., Overton, K. & Deisseroth, K. Hydrogel-tissue chemistry: principles and applications. Annu. Rev. Biophys. 47, 355–376 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  223. 223.

    Karagiannis, E. D. & Boyden, E. S. Expansion microscopy: development and neuroscience applications. Curr. Opin. Neurobiol. 50, 56–63 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  224. 224.

    Chen, F., Tillberg, P. W. & Boyden, E. S. Optical imaging. Expansion microscopy. Science 347, 543–548 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  225. 225.

    Chozinski, T. J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485–488 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  226. 226.

    Ku, T. et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat. Biotechnol. 34, 973–981 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  227. 227.

    Tillberg, P. W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987–992 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  228. 228.

    Zwettler, F. U. et al. Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Nat. Commun. 11, 3388 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  229. 229.

    Reddy-Alla, S. et al. Stable positioning of Unc13 restricts synaptic vesicle fusion to defined release sites to promote synchronous neurotransmission. Neuron 95, 1350–1364 e1312 (2017).

    CAS  PubMed  Google Scholar 

  230. 230.

    Liu, K. S. et al. RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science 334, 1565–1569 (2011).

    CAS  PubMed  Google Scholar 

  231. 231.

    Sakamoto, H. et al. Synaptic weight set by Munc13-1 supramolecular assemblies. Nat. Neurosci. 21, 41–49 (2018).

    CAS  PubMed  Google Scholar 

  232. 232.

    Bohme, M. A. et al. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling. Nat. Neurosci. 19, 1311–1320 (2016).

    PubMed  Google Scholar 

  233. 233.

    Padmanabhan, P. et al. Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites. Neuropharmacology 169, 107554 (2020).

    CAS  PubMed  Google Scholar 

  234. 234.

    Reshetniak, S. et al. A comparative analysis of the mobility of 45 proteins in the synaptic bouton. EMBO J. 39, e104596 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  235. 235.

    Bademosi, A. T. et al. Trapping of syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic. Cell Rep. 22, 427–440 (2018).

    CAS  PubMed  Google Scholar 

  236. 236.

    Groc, L. & Choquet, D. Linking glutamate receptor movements and synapse function. Science https://doi.org/10.1126/science.aay4631 (2020).

    Article  PubMed  Google Scholar 

  237. 237.

    Heine, M. et al. Surface mobility of postsynaptic AMPARs tunes synaptic transmission. Sci. 320, 201–205 (2008).

    CAS  Google Scholar 

  238. 238.

    Constals, A. et al. Glutamate-induced AMPA receptor desensitization increases their mobility and modulates short-term plasticity through unbinding from stargazin. Neuron 85, 787–803 (2015).

    CAS  PubMed  Google Scholar 

  239. 239.

    Polenghi, A. et al. Kainate receptor activation shapes short-term synaptic plasticity by controlling receptor lateral mobility at glutamatergic synapses. Cell Rep. 31, 107735 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  240. 240.

    Penn, A. C. et al. Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors. Nature 549, 384–388 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  241. 241.

    Zhong, G. et al. Developmental mechanism of the periodic membrane skeleton in axons. eLife https://doi.org/10.7554/eLife.04581 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  242. 242.

    D’Este, E., Kamin, D., Gottfert, F., El-Hady, A. & Hell, S. W. STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. Cell Rep. 10, 1246–1251 (2015).

    PubMed  Google Scholar 

  243. 243.

    Shen, P. S. The 2017 Nobel Prize in Chemistry: cryo-EM comes of age. Anal. Bioanal. Chem. 410, 2053–2057 (2018).

    CAS  PubMed  Google Scholar 

  244. 244.

    Hoffman, D. P. et al. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells. Science https://doi.org/10.1126/science.aaz5357 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  245. 245.

    Meijering, E. A bird’s-eye view of deep learning in bioimage analysis. Comput. Struct. Biotechnol. J. 18, 2312–2325 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  246. 246.

    von Chamier, L., Laine, R. F. & Henriques, R. Artificial intelligence for microscopy: what you should know. Biochem. Soc. Trans. 47, 1029–1040 (2019).

    Google Scholar 

  247. 247.

    Liu, S., Huh, H., Lee, S. H. & Huang, F. Three-dimensional single-molecule localization microscopy in whole-cell and tissue specimens. Annu. Rev. Biomed. Eng. https://doi.org/10.1146/annurev-bioeng-060418-052203 (2020).

    Article  PubMed  Google Scholar 

  248. 248.

    Holden, S. J., Uphoff, S. & Kapanidis, A. N. DAOSTORM: an algorithm for high- density super-resolution microscopy. Nat. Methods 8, 279–280 (2011).

    CAS  PubMed  Google Scholar 

  249. 249.

    Henriques, R. et al. QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat. Methods 7, 339–340 (2010).

    CAS  PubMed  Google Scholar 

  250. 250.

    Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).

    CAS  PubMed  Google Scholar 

  251. 251.

    Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z. & Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30, 2389–2390 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  252. 252.

    Serge, A., Bertaux, N., Rigneault, H. & Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat. Methods 5, 687–694 (2008).

    CAS  PubMed  Google Scholar 

  253. 253.

    Kechkar, A., Nair, D., Heilemann, M., Choquet, D. & Sibarita, J. B. Real-time analysis and visualization for single-molecule based super-resolution microscopy. PLoS ONE 8, e62918 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  254. 254.

    Ester, M., Kriegel, H. P., Sander, J. & Xu, X. A density-based algorithm for discovering clusters in large spatial databases with noise. in KDD-96: Proceedings of the Second International Conference on Knowledge Discovery and Data Mining, 226-231 (ACM Digital Library, 1996).

  255. 255.

    Malkusch, S. & Heilemann, M. Extracting quantitative information from single-molecule super-resolution imaging data with LAMA - LocAlization Microscopy Analyzer. Sci. Rep. 6, 34486 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  256. 256.

    Axelrod, D. Total internal reflection fluorescence microscopy. Methods Cell Biol. 30, 245–270 (1989).

    CAS  PubMed  Google Scholar 

  257. 257.

    Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    CAS  PubMed  Google Scholar 

  258. 258.

    Ashby, M. C., Ibaraki, K. & Henley, J. M. It’s green outside: tracking cell surface proteins with pH-sensitive GFP. Trends Neurosci. 27, 257–261 (2004).

    CAS  PubMed  Google Scholar 

  259. 259.

    Miyawaki, A. et al. Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997). This article reports the invention of a GFP-based genetically encoded calcium indicator.

    CAS  PubMed  Google Scholar 

  260. 260.

    O’Banion, C. P. & Yasuda, R. Fluorescent sensors for neuronal signaling. Curr. Opin. Neurobiol. 63, 31–41 (2020).

    PubMed  Google Scholar 

  261. 261.

    Leopold, A. V., Shcherbakova, D. M. & Verkhusha, V. V. Fluorescent biosensors for neurotransmission and neuromodulation: engineering and applications. Front. Cell. Neurosci. 13, 474 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  262. 262.

    Fan, L. Z. et al. All-optical electrophysiology reveals the role of lateral inhibition in sensory processing in cortical layer 1. Cell 180, 521–535 e518 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  263. 263.

    Adam, Y. et al. Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics. Nature 569, 413–417 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  264. 264.

    Sha, F., Abdelfattah, A. S., Patel, R. & Schreiter, E. R. Erasable labeling of neuronal activity using a reversible calcium marker. eLife https://doi.org/10.7554/eLife.57249 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  265. 265.

    Lee, D., Hyun, J. H., Jung, K., Hannan, P. & Kwon, H. B. A calcium- and light-gated switch to induce gene expression in activated neurons. Nat. Biotechnol. 35, 858–863 (2017).

    CAS  PubMed  Google Scholar 

  266. 266.

    Wang, W. et al. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nat. Biotechnol. 35, 864–871 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  267. 267.

    Montesinos, M. S., Satterfield, R. & Young, S. M. Jr. Helper-dependent adenoviral vectors and their use for neuroscience applications. Methods Mol. Biol. 1474, 73–90 (2016).

    CAS  PubMed  Google Scholar 

  268. 268.

    Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  269. 269.

    Brown, A. J. & James, D. C. Constructing strong cell type-specific promoters through informed design. Methods Mol. Biol. 1651, 131–145 (2017).

    CAS  PubMed  Google Scholar 

  270. 270.

    Kugler, S., Kilic, E. & Bahr, M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347 (2003).

    CAS  PubMed  Google Scholar 

  271. 271.

    Lukashchuk, V., Lewis, K. E., Coldicott, I., Grierson, A. J. & Azzouz, M. AAV9-mediated central nervous system-targeted gene delivery via cisterna magna route in mice. Mol. Therapy. Methods Clin. Dev. 3, 15055 (2016).

    Google Scholar 

  272. 272.

    Borgius, L., Restrepo, C. E., Leao, R. N., Saleh, N. & Kiehn, O. A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons. Mol. Cell. Neurosci. 45, 245–257 (2010).

    CAS  PubMed  Google Scholar 

  273. 273.

    Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 19, 1743–1749 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  274. 274.

    Suzuki, J., Kanemaru, K. & Iino, M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophys. J. 111, 1119–1131 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the critical suggestions of A. Getz, R. Galland and C. Butler with regard to the manuscript. They express their warmest thanks to the many outstanding members of their team and collaborators who participated in elaboration of the concepts in this Review. This work is currently supported by funding from the Ministère de l’Enseignement Supérieur et de la Recherche, Centre National de la Recherche Scientifique, European Research Council grant number 787340 Dyn-Syn-Mem, LabEx BRAIN ANR-10-LABX-43, ANR-10-IDEX-03-02, ANR-16-CE13-0018, ANR-16-CE16-0026-01 and the Conseil Régional de Nouvelle Aquitaine.

Author information

Affiliations

Authors

Contributions

All authors contributed equally to the manuscript.

Corresponding authors

Correspondence to Daniel Choquet or Matthieu Sainlos or Jean-Baptiste Sibarita.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information

Nature Reviews Neuroscience thanks F. Meunier, who co-reviewed with M. Joensuu; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Quantum dots

Semiconductor particles a few nanometres in size having optical and electronic properties that differ from those of larger particles due to quantum mechanics.

Nanodiamonds

Diamond nanoparticles smaller than 1 µm.

Photobleaching

The photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce.

Photoswitching

Modification of the structure of a compound by light, especially when accompanied by a change in function.

Chromophore maturation

The post-translational process through which the chromophore of fluorescent proteins is formed.

Phytochrome

A class of photoreceptors in plants, bacteria and fungi used to detect light.

Nanobodies

Single-domain antibody fragments consisting of a monomeric variable antibody domain.

Aptamers

Oligonucleotide or peptide molecules that bind to a specific target molecule.

Galvanometric mirror

An ammeter that indicates it has sensed an electric current by deflecting a light beam with a mirror and is used in laser scanning microscopy.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Choquet, D., Sainlos, M. & Sibarita, JB. Advanced imaging and labelling methods to decipher brain cell organization and function. Nat Rev Neurosci 22, 237–255 (2021). https://doi.org/10.1038/s41583-021-00441-z

Download citation

Search

Quick links

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