Abstract
Neuronal synapses are functional nodes in neural circuits. Their organization and activity define an individual’s level of intelligence, emotional state and mental health. Changes in the structure and efficacy of synapses are the biological basis of learning and memory. However, investigation of the molecular architecture of synapses has been impeded by the lack of efficient techniques with sufficient resolution. Recent developments in state-of-the-art nano-imaging techniques have opened up a new window for dissecting the molecular organization of neuronal synapses with unprecedented resolution. Here, we review recent technological advances in nano-imaging techniques as well as their applications to the study of synapses, emphasizing super-resolution light microscopy and 3-dimensional electron tomography.
Keywords: synaptic architecture, nano-imaging, super-resolution imaging, STED microscopy, STORM, PALM, cryoET
Footnotes
These authors contributed equally to this work.
References
- [1].Cajal SR. The Structure and Connexions of Neurons. Nobel Lecture, 1906. URL: http://nobelprize.org/medicine/laureates/1906/cajal-lecture.html.
- [2].DeFelipe J. From the connectome to the synaptome: An epic love story. Science. 2010;330:1198–1201. doi: 10.1126/science.1193378. [DOI] [PubMed] [Google Scholar]
- [3].Mayford M, Siegelbaum SA, Kandel ER. Synapses and memory storage. Cold Spring Harb Perspect Biol 2012, 4. doi: 10.1101/cshperspect.a005751. [DOI] [PMC free article] [PubMed]
- [4].Goto Y., Yang C.R., Otani S. Functional and dysfunctional synaptic plasticity in prefrontal cortex: roles in psychiatric disorders. Biol Psychiatry. 2010;67:199–207. doi: 10.1016/j.biopsych.2009.08.026. [DOI] [PubMed] [Google Scholar]
- [5].Llinas R.R. The contribution of Santiago Ramon y Cajal to functional neuroscience. Nat Rev Neurosci. 2003;4:77–80. doi: 10.1038/nrn1011. [DOI] [PubMed] [Google Scholar]
- [6].Sherrington C.S. The Integrative Action of the Nervous System. New York: Charles Scribner’s Sons; 1906. [Google Scholar]
- [7].Foster M., Sherrington C.S. A Textbook of Physiology. 7th ed. London: MacMillan & Co Ltd; 1897. [Google Scholar]
- [8].Lopez-Munoz F., Boya J., Alamo C. Neuron theory, the cornerstone of neuroscience, on the centenary of the Nobel Prize award to Santiago Ramon y Cajal. Brain Res Bull. 2006;70:391–405. doi: 10.1016/j.brainresbull.2006.07.010. [DOI] [PubMed] [Google Scholar]
- [9].Palay S.L. Synapses in the central nervous system. J Biophys Biochem Cytol. 1956;2:193–202. doi: 10.1083/jcb.2.4.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [10].Palay S.L. The morphology of synapses of the central nervous system. Exp Cell Res. 1958;14:275–293. [PubMed] [Google Scholar]
- [11].Gray E.G. Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J Anat. 1959;93:420–433. [PMC free article] [PubMed] [Google Scholar]
- [12].Gray E.G. Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex. Nature. 1959;183:1592–1593. doi: 10.1038/1831592a0. [DOI] [PubMed] [Google Scholar]
- [13].Heuser J.E., Reese T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at frog neuromuscular junction. J Cell Biol. 1973;57:315–344. doi: 10.1083/jcb.57.2.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Harris KM, Weinberg RJ. Ultrastructure of synapses in the mammalian brain. Cold Spring Harb Perspect Biol 2012, 4. doi: 10.1101/cshperspect.a005587. [DOI] [PMC free article] [PubMed]
- [15].Sheng M., Hoogenraad C.C. The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu Rev Biochem. 2007;76:823–847. doi: 10.1146/annurev.biochem.76.060805.160029. [DOI] [PubMed] [Google Scholar]
- [16].Wilt B.A., Burns L.D., Ho E.T.W., Ghosh K.K., Mukamel E.A., Schnitzer M.J. Advances in light microscopy for neuroscience. Annu Rev Neurosci. 2009;32:435–506. doi: 10.1146/annurev.neuro.051508.135540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [17].Giepmans B.N., Adams S.R., Ellisman M.H., Tsien R.Y. The fluorescent toolbox for assessing protein location and function. Science. 2006;312:217–224. doi: 10.1126/science.1124618. [DOI] [PubMed] [Google Scholar]
- [18].Bayes A., Grant S.G.N. Neuroproteomics: understanding the molecular organization and complexity of the brain. Nat Rev Neurosci. 2009;10:635–646. doi: 10.1038/nrn2701. [DOI] [PubMed] [Google Scholar]
- [19].Hell S.W. Microscopy and its focal switch. Nat Methods. 2009;6:24–32. doi: 10.1038/nmeth.1291. [DOI] [PubMed] [Google Scholar]
- [20].Hurbain I., Sachse M. The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biol Cell. 2011;103:405–420. doi: 10.1042/BC20110015. [DOI] [PubMed] [Google Scholar]
- [21].Hell S.W., Wichmann J. Breaking the diffraction resolution limit by stimulated emission:stimulated-emission-depletion fluorescence microscopy. Opt Lett. 1994;19:780–782. doi: 10.1364/OL.19.000780. [DOI] [PubMed] [Google Scholar]
- [22].Hell S.W. Far-field optical nanoscopy. Science. 2007;316:1153–1158. doi: 10.1126/science.1137395. [DOI] [PubMed] [Google Scholar]
- [23].Gustafsson M.G. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A. 2005;102:13081–13086. doi: 10.1073/pnas.0406877102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [24].Rust M.J., Bates M., Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) Nat Methods. 2006;3:793–795. doi: 10.1038/nmeth929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [25].Betzig E., Patterson G.H., Sougrat R., Lindwasser O.W., Olenych S., Bonifacino J.S., et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006;313:1642–1645. doi: 10.1126/science.1127344. [DOI] [PubMed] [Google Scholar]
- [26].Hess S.T., Girirajan T.P., Mason M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006;91:4258–4272. doi: 10.1529/biophysj.106.091116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [27].Lucic V., Forster F., Baumeister W. Structural studies by electron tomography: from cells to molecules. Annu Rev Biochem. 2005;74:833–865. doi: 10.1146/annurev.biochem.73.011303.074112. [DOI] [PubMed] [Google Scholar]
- [28].Dani A., Huang B. New resolving power for light microscopy: applications to neurobiology. Curr Opin Neurobiol. 2010;20:648–652. doi: 10.1016/j.conb.2010.07.006. [DOI] [PubMed] [Google Scholar]
- [29].Huang B., Babcock H., Zhuang X.W. Breaking the diffraction barrier: super-resolution imaging of cells. Cell. 2010;143:1047–1058. doi: 10.1016/j.cell.2010.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [30].Dyba M., Hell S.W. Focal spots of size λ/23 open up far-field florescence microscopy at 33 nm axial resolution. Phys Rev Lett. 2002;88:163901. doi: 10.1103/PhysRevLett.88.163901. [DOI] [PubMed] [Google Scholar]
- [31].Schmidt R., Wurm C.A., Jakobs S., Engelhardt J., Egner A., Hell S.W. Spherical nanosized focal spot unravels the interior of cells. Nat Methods. 2008;5:539–544. doi: 10.1038/nmeth.1214. [DOI] [PubMed] [Google Scholar]
- [32].Schmidt R., Wurm C.A., Punge A., Egner A., Jakobs S., Hell S.W. Mitochondrial cristae revealed with focused light. Nano Lett. 2009;9:2508–2510. doi: 10.1021/nl901398t. [DOI] [PubMed] [Google Scholar]
- [33].Hofmann M., Eggeling C., Jakobs S., Hell S.W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A. 2005;102:17565–17569. doi: 10.1073/pnas.0506010102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [34].Ding J.B., Takasaki K.T., Sabatini B.L. Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy. Neuron. 2009;63:429–437. doi: 10.1016/j.neuron.2009.07.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [35].Urban N.T., Willig K.I., Hell S.W., Nagerl U.V. STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys J. 2011;101:1277–1284. doi: 10.1016/j.bpj.2011.07.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [36].Berning S., Willig K.I., Sfeffens H., Dibaj P., Hell S.W. Nanoscopy in a living mouse brain. Science. 2012;335:551. doi: 10.1126/science.1215369. [DOI] [PubMed] [Google Scholar]
- [37].Liu K.S., Siebert M., Mertel S., Knoche E., Wegener S., Wichmann C., et al. RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science. 2011;334:1565–1569. doi: 10.1126/science.1212991. [DOI] [PubMed] [Google Scholar]
- [38].Hua Y., Sinha R., Thiel C.S., Schmidt R., Huve J., Martens H., et al. A readily retrievable pool of synaptic vesicles. Nat Neurosci. 2011;14:833–839. doi: 10.1038/nn.2838. [DOI] [PubMed] [Google Scholar]
- [39].Denker A., Krohnert K., Buckers J., Neher E., Rizzoli S.O. The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling. Proc Natl Acad Sci U S A. 2011;108:17183–17188. doi: 10.1073/pnas.1112690108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [40].Huang B., Bates M., Zhuang X. Super-resolution fluorescence microscopy. Annu Rev Biochem. 2009;78:993–1016. doi: 10.1146/annurev.biochem.77.061906.092014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [41].Xu K., Babcock H.P., Zhuang X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods. 2012;9:185–188. doi: 10.1038/nmeth.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [42].Bates M., Huang B., Dempsey G.T., Zhuang X. Multicolor superresolution imaging with photo-switchable fluorescent probes. Science. 2007;317:1749–1753. doi: 10.1126/science.1146598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [43].Shroff H., Galbraith C.G., Galbraith J.A., White H., Gillette J., Olenych S., et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A. 2007;104:20308–20313. doi: 10.1073/pnas.0710517105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [44].Bates M., Dempsey G.T., Chen K.H., Zhuang X. Multicolor superresolution fluorescence imaging via multi-parameter fluorophore detection. Chemphyschem. 2012;13:99–107. doi: 10.1002/cphc.201100735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [45].Westphal V., Rizzoli S.O., Lauterbach M.A., Kamin D., Jahn R., Hell S.W. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science. 2008;320:246–249. doi: 10.1126/science.1154228. [DOI] [PubMed] [Google Scholar]
- [46].Jones S.A., Shim S.H., He J., Zhuang X. Fast, three-dimensional super-resolution imaging of live cells. Nat Methods. 2011;8:499–508. doi: 10.1038/nmeth.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [47].Dani A., Huang B., Bergan J., Dulac C., Zhuang X. Superresolution imaging of chemical synapses in the brain. Neuron. 2010;68:843–856. doi: 10.1016/j.neuron.2010.11.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [48].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. 2010;67:86–99. doi: 10.1016/j.neuron.2010.05.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [49].Tardin C., Cognet L., Bats C., Lounis B., Choquet D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 2003;22:4656–4665. doi: 10.1093/emboj/cdg463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [50].Heine M., Groc L., Frischknecht R., Beique J.C., Lounis B., Rumbaugh G., et al. Surface mobility of postsynaptic AMPARs tunes synaptic transmission. Science. 2008;320:201–205. doi: 10.1126/science.1152089. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [51].Kerr J.M., Blanpied T.A. Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density. J Neurosci. 2012;32:658–673. doi: 10.1523/JNEUROSCI.2927-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [52].Derosier D.J., Klug A. Reconstruction of three dimensional structures from electron micrographs. Nature. 1968;217:130–134. doi: 10.1038/217130a0. [DOI] [PubMed] [Google Scholar]
- [53].Ruiz T., Erk I., Lepault J. Electron cryo-microscopy of vitrified biological specimens: towards high spatial and temporal resolution. Biol Cell. 1994;80:203–210. doi: 10.1111/j.1768-322x.1994.tb00931.x. [DOI] [PubMed] [Google Scholar]
- [54].Adrian M., Dubochet J., Lepault J., McDowall A.W. Cryo-electron microscopy of viruses. Nature. 1984;308:32–36. doi: 10.1038/308032a0. [DOI] [PubMed] [Google Scholar]
- [55].Erk I., Michel M., Lepault J. Electron cryo-microscopy of vitrified bulk biological specimens: ideal and real structures of water-lipid phases. J Microsc. 1996;182:15–23. doi: 10.1111/j.1365-2818.1996.tb04793.x. [DOI] [PubMed] [Google Scholar]
- [56].Kirschning E., Rutter G., Hohenberg H. High-pressure freezing and freeze-substitution of native rat brain: Suitability for preservation and immunoelectron microscopic localization of myelin glycolipids. J Neurosci Res. 1998;53:465–474. doi: 10.1002/(SICI)1097-4547(19980815)53:4<465::AID-JNR8>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
- [57].Vanhecke D., Asano S., Kochovski Z., Fernandez-Busnadiego R., Schrod N., Baumeister W., et al. Cryo-electron tomography: methodology, developments and biological applications. J Microsc. 2011;242:221–227. doi: 10.1111/j.1365-2818.2010.03478.x. [DOI] [PubMed] [Google Scholar]
- [58].Zuber B., Nikonenko I., Klauser P., Muller D., Dubochet J. The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes. Proc Natl Acad Sci U S A. 2005;102:19192–19197. doi: 10.1073/pnas.0509527102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [59].Chen X., Winters C., Azzam R., Li X., Galbraith J.A., Leapman R.D., et al. Organization of the core structure of the postsynaptic density. Proc Natl Acad Sci U S A. 2008;105:4453–4458. doi: 10.1073/pnas.0800897105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [60].Chen X., Nelson C.D., Li X., Winters C.A., Azzam R., Sousa A.A., et al. PSD-95 is required to sustain the molecular organization of the postsynaptic density. J Neurosci. 2011;31:6329–6338. doi: 10.1523/JNEUROSCI.5968-10.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [61].Liu J., Taylor D.W., Krementsova E.B., Trybus K.M., Taylor K.A. Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature. 2006;442:208–211. doi: 10.1038/nature04719. [DOI] [PubMed] [Google Scholar]
- [62].Grunewald K., Desai P., Winkler D.C., Heymann J.B., Belnap D.M., Baumeister W., et al. Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science. 2003;302:1396–1398. doi: 10.1126/science.1090284. [DOI] [PubMed] [Google Scholar]
- [63].Milne J.L., Subramaniam S. Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol. 2009;7:666–675. doi: 10.1038/nrmicro2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [64].Nicastro D., Frangakis A.S., Typke D., Baumeister W. Cryo-electron tomography of neurospora mitochondria. J Struct Biol. 2000;129:48–56. doi: 10.1006/jsbi.1999.4204. [DOI] [PubMed] [Google Scholar]
- [65].Fernandez-Busnadiego R., Schrod N., Kochovski Z., Asano S., Vanhecke D., Baumeister W., et al. Insights into the molecular organization of the neuron by cryo-electron tomography. J Electron Microsc (Tokyo) 2011;60(Suppl1):S137–148. doi: 10.1093/jmicro/dfr018. [DOI] [PubMed] [Google Scholar]
- [66].Fernandez-Busnadiego R., Zuber B., Maurer U.E., Cyrklaff M., Baumeister W., Lucic V. Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography. J Cell Biol. 2010;188:145–156. doi: 10.1083/jcb.200908082. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [67].Beck M., Lucic V., Forster F., Baumeister W., Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature. 2007;449:611–615. doi: 10.1038/nature06170. [DOI] [PubMed] [Google Scholar]
- [68].Alber F., Dokudovskaya S., Veenhoff L.M., Zhang W., Kipper J., Devos D., et al. Determining the architectures of macromolecular assemblies. Nature. 2007;450:683–694. doi: 10.1038/nature06404. [DOI] [PubMed] [Google Scholar]
- [69].Lucic V., Kossel A.H., Yang T., Bonhoeffer T., Baumeister W., Sartori A. Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography. J Struct Biol. 2007;160:146–156. doi: 10.1016/j.jsb.2007.08.014. [DOI] [PubMed] [Google Scholar]
- [70].Lucic V., Yang T., Schweikert G., Forster F., Baumeister W. Morphological characterization of molecular complexes present in the synaptic cleft. Structure. 2005;13:423–434. doi: 10.1016/j.str.2005.02.005. [DOI] [PubMed] [Google Scholar]
- [71].Sartori A., Gatz R., Beck F., Rigort A., Baumeister W., Plitzko J.M. Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol. 2007;160:135–145. doi: 10.1016/j.jsb.2007.07.011. [DOI] [PubMed] [Google Scholar]
- [72].Landis D.M., Hall A.K., Weinstein L.A., Reese T.S. The Organization of cytoplasm at the presynaptic active zone of a central nervoussystem synapse. Neuron. 1988;1:201–209. doi: 10.1016/0896-6273(88)90140-7. [DOI] [PubMed] [Google Scholar]
- [73].Siksou L., Rostaing P., Lechaire J.P., Boudier T., Ohtsuka T., Fejtova A., et al. Three-dimensional architecture of presynaptic terminal cytomatrix. J Neurosci. 2007;27:6868–6877. doi: 10.1523/JNEUROSCI.1773-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [74].Dalva M.B., McClelland A.C., Kayser M.S. Cell adhesion molecules: signalling functions at the synapse. Nat Rev Neurosci. 2007;8:206–220. doi: 10.1038/nrn2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [75].Gerrow K., El-Husseini A. Cell adhesion molecules at the synapse. Front Biosci. 2006;11:2400–2419. doi: 10.2741/1978. [DOI] [PubMed] [Google Scholar]
- [76].Kennedy M.B. Signal-processing machines at the postsynaptic density. Science. 2000;290:750–754. doi: 10.1126/science.290.5492.750. [DOI] [PubMed] [Google Scholar]
- [77].Takumi Y., Ramirez-Leon V., Laake P., Rinvik E., Ottersen O.P. Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Nat Neurosci. 1999;2:618–624. doi: 10.1038/10172. [DOI] [PubMed] [Google Scholar]
- [78].Kharazia V.N., Weinberg R.J. Tangential synaptic distribution of NMDA and AMPA receptors in rat neocortex. Neurosci Lett. 1997;238:41–44. doi: 10.1016/S0304-3940(97)00846-X. [DOI] [PubMed] [Google Scholar]
- [79].Ji N., Shroff H., Zhong H., Betzig E. Advances in the speed and resolution of light microscopy. Curr Opin Neurobiol. 2008;18:605–616. doi: 10.1016/j.conb.2009.03.009. [DOI] [PubMed] [Google Scholar]
- [80].Freitag B., Bischoff M., Mueller H., Hartel P., von Harrach H.S. Subnanometer resolution in field-free imaging using a Titan80-300 with Lorentz lens and image Cs-corrector at 300kV acceleration voltage. Microsc Microanal. 2009;15:184–185. doi: 10.1017/S143192760909429X. [DOI] [Google Scholar]
- [81].Fukuda Y., Nagayama K. Zernike phase contrast cryo-electron tomography of whole mounted frozen cells. J Struct Biol. 2012;177:484–489. doi: 10.1016/j.jsb.2011.11.018. [DOI] [PubMed] [Google Scholar]
- [82].Murata K., Liu X., Danev R., Jakana J., Schmid M.F., King J., et al. Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure. 2010;18:903–912. doi: 10.1016/j.str.2010.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [83].Jin L., Milazzo A.C., Kleinfelder S., Li S.D., Leblanc P., Duttweiler F., et al. Applications of direct detection device in transmission electron microscopy. J Struct Biol. 2008;161:352–358. doi: 10.1016/j.jsb.2007.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [84].Zanetti G., Riches J.D., Fuller S.D., Briggs J.A. Contrast transfer function correction applied to cryo-electron tomography and subtomogram averaging. J Struct Biol. 2009;168:305–312. doi: 10.1016/j.jsb.2009.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [85].Beck M., Malmstrom J.A., Lange V., Schmidt A., Deutsch E.W., Aebersold R. Visual proteomics of the human pathogen Leptospira interrogans. Nat Methods. 2009;6:817–823. doi: 10.1038/nmeth.1390. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [86].Shu X., Lev-Ram V., Deerinck T.J., Qi Y., Ramko E.B., Davidson M.W., et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 2011;9:e1001041. doi: 10.1371/journal.pbio.1001041. [DOI] [PMC free article] [PubMed] [Google Scholar]