Abstract
Recent advances in fluorescence microscopy have provided researchers with powerful new tools to visualize cellular processes occurring in real time, giving researchers an unprecedented opportunity to address many biological questions that were previously inaccessible. With respect to neurobiology, these real-time imaging techniques have deepened our understanding of molecular and cellular processes, including the movement and dynamics of single proteins and organelles in living cells. In this review, we summarize recent advances in the field of real-time imaging of single synaptic vesicles in live neurons.
Similar content being viewed by others
References
Alabi A A, Tsien R W (2012). Synaptic vesicle pools and dynamics. Cold Spring HarbPerspectBiol, 4(8): a013680
Alabi A A, Tsien R W (2013). Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu Rev Physiol, 75: 393–422
Andreae L C, Fredj N B, Burrone J (2012). Independent vesicle pools underlie different modes of release during neuronal development. J Neurosci, 32(5): 1867–1874
Aravanis A M, Pyle J L, Tsien R W (2003). Single synaptic vesicles fusing transiently and successively without loss of identity. Nature, 423(6940): 643–647
Atasoy D, Ertunc M, Moulder K L, Blackwell J, Chung C, Su J, Kavalali E T (2008). Spontaneous and evoked glutamate release activates two populations of NMDA receptors with limited overlap. J Neurosci, 28(40): 10151–10166
Axelrod D, Thompson N L, Burghardt T P (1983). Total internal inflection fluorescent microscopy. J Microsc, 129(Pt 1): 19–28
Baba K, Nishida K (2012). Single-molecule tracking in living cells using single quantum dot applications. Theranostics, 2(7): 655–667
Balaji J, Ryan T A (2007). Single-vesicle imaging reveals that synaptic vesicle exocytosis and endocytosis are coupled by a single stochastic mode. Proc Natl Acad Sci U S A, 104(51): 20576–20581
Barroso M M (2011). Quantum Dots in Cell Biology. J Histochem Cytochem, 59: 237–251
Betzig E, Patterson G H, Sougrat R, Lindwasser O W, Olenych S, Bonifacino J S, Davidson M W, Lippincott-Schwartz J, Hess H F (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science, 313(5793): 1642–1645
Bianchini P, Peres C, Oneto M, Galiani S, Vicidomini G, Diaspro A (2015). STED nanoscopy: a glimpse into the future. Cell Tissue Res, 360(1): 143–150
Blum C, Meixner A J, Subramaniam V (2004). Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants. Biophys J, 87(6): 4172–4179
Bottrill M, Green M (2011). Some aspects of quantum dot toxicity. Chem Commun (Camb), 47(25): 7039–7050
Buxbaum A R, Yoon Y J, Singer R H, Park H Y (2015). Singlemolecule insights into mRNA dynamics in neurons. Trends Cell Biol, 25(8): 468–475
Chang Y P, Pinaud F, Antelman J, Weiss S (2008). Tracking biomolecules in live cells using quantum dots. J Biophotonics, 1(4): 287–298
Chater T E, Goda Y (2014). The role of AMPA receptors in postsynaptic mechanisms of synaptic plasticity. Front Cell Neurosci, 8: 401
Chéreau R, Tønnesen J, Nägerl U V (2015). STED microscopy for nanoscale imaging in living brain slices. Methods, 88: 57–66
Choquet D, Triller A (2013). The dynamic synapse. Neuron, 80(3): 691–703
Chung C, Barylko B, Leitz J, Liu X, Kavalali E T (2010). Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission. J Neurosci, 30(4): 1363–1376
Coelho M, Maghelli N, Tolic-Nørrelykke I M (2013). Single-molecule imaging in vivo: the dancing building blocks of the cell. Integr Biol (Camb), 5(5): 748–758
Dahan M, Lévi S, Luccardini C, Rostaing P, Riveau B, Triller A (2003). Diffusion dynamics of glycine receptors revealed by singlequantum dot tracking. Science, 302(5644): 442–445
Darcy K J, Staras K, Collinson L M, Goda Y (2006). Constitutive sharing of recycling synaptic vesicles between presynaptic boutons. Nat Neurosci, 9(3): 315–321
Deniz A A, Mukhopadhyay S, Lemke E A (2008). Single-molecule biophysics: at the interface of biology, physics and chemistry. J R Soc Interface, 5(18): 15–45
DePina A S, Wöllert T, Langford G M (2007). Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts. Cell Motil Cytoskeleton, 64(10): 739–755
Dreosti E, Lagnado L (2011). Optical reporters of synaptic activity in neural circuits. Exp Physiol, 96(1): 4–12
Duzdevich D, Greene E C (2013). Towards physiological complexity with in vitro single-molecule biophysics. Philos Trans R SocLond B BiolSci, 368(1611): 20120271
Fernandez-Alfonso T, Ryan T A (2008). A heterogeneous “resting” pool of synaptic vesicles that is dynamically interchanged across boutons in mammalian CNS synapses. Brain Cell Biol, 36(1-4): 87–100
Fernández-Suárez M, Ting A Y (2008). Fluorescent probes for superresolution imaging in living cells. Nat Rev Mol Cell Biol, 9(12): 929–943
Fioravante D, Regehr W G (2011). Short-term forms of presynaptic plasticity. Curr Opin Neurobiol, 21(2): 269–274
Gandhi S P, Stevens C F (2003). Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature, 423(6940): 607–613
Giepmans B N, Adams S R, Ellisman M H, Tsien R Y (2006). The fluorescent toolbox for assessing protein location and function. Science, 312(5771): 217–224
Groc L, Choquet D (2006). AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse. Cell Tissue Res, 326(2): 423–438
Gu H, Lazarenko R M, Koktysh D, Iacovitti L, Zhang Q (2015). A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses. Stem Cells Transl Med, 4(8): 887–893
Gust A, Zander A, Gietl A, Holzmeister P, Schulz S, Lalkens B, Tinnefeld P, Grohmann D (2014). A starting point for fluorescencebased single-molecule measurements in biomolecular research. Molecules, 19(10): 15824–15865
Haas B L, Matson J S, Di Rita V J, Biteen J S (2014). Imaging live cells at the nanometer-scale with single-molecule microscopy: obstacles and achievements in experiment optimization for microbiology. Molecules, 19(8): 12116–12149
Harke B, Keller J, Ullal C K, Westphal V, Schönle A, Hell S W (2008). Resolution scaling in STED microscopy. Opt Express, 16(6): 4154–4162
Hell SW, Wichmann J (1994). Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett, 19(11): 780–782
Herzog E, Nadrigny F, Silm K, Biesemann C, Helling I, Bersot T, Steffens H, Schwartzmann R, Nägerl U V, El Mestikawy S, Rhee J, Kirchhoff F, Brose N (2011). In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice. J Neurosci, 31(43): 15544–15559
Howarth M, Liu W, Puthenveetil S, Zheng Y, Marshall L F, Schmidt M M, Wittrup K D, Bawendi M G, Ting A Y (2008). Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat Methods, 5(5): 397–399
Hua Y, Sinha R, Martineau M, Kahms M, Klingauf J (2010). A common origin of synaptic vesicles undergoing evoked and spontaneous fusion. Nat Neurosci, 13(12): 1451–1453
Ifrim MF, Williams K R, Bassell G J (2015). Single-molecule imaging of PSD-95 mRNA translation in dendrites and its dysregulation in a mouse model of fragile X syndrome. J Neurosci, 35(18): 7116–7130
Jahn R, Fasshauer D (2012). Molecular machines governing exocytosis of synaptic vesicles. Nature, 490(7419): 201–207
Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T (2008).Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem, 77: 51–76
Kamin D, Lauterbach M A, Westphal V, Keller J, Schönle A, Hell S W, Rizzoli S O (2010). High- and low-mobility stages in the synaptic vesicle cycle. Biophys J, 99(2): 675–684
Kavalali E T (2015). The mechanisms and functions of spontaneous neurotransmitter release. Nat Rev Neurosci, 16(1): 5–16
Kavalali E T, Jorgensen E M (2014). Visualizing presynaptic function. Nat Neurosci, 17(1): 10–16
Kharazia V N, Weinberg R J (1997). Tangential synaptic distribution of NMDA and AMPA receptors in rat neocortex. NeurosciLett, 238(1-2): 41–44
Kural C, Kim H, Syed S, Goshima G, Gelfand V I, Selvin P R (2005). Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement? Science, 308(5727): 1469–1472
Kusumi A, Tsunoyama T A, Hirosawa K M, Kasai R S, Fujiwara T K (2014). Tracking single molecules at work in living cells. Nat ChemBiol, 10(7): 524–532
Kwakowsky A, Potapov D, Abrahám I M (2013). Tracking of single receptor molecule mobility in neuronal membranes: a quick theoretical and practical guide. J Neuroendocrinol, 25(11): 1231–1237
Lavis L D, Raines R T (2014). Bright building blocks for chemical biology. ACS ChemBiol, 9(4): 855–866
Lee S, Jung K J, Jung H S, Chang S (2012). Dynamics of multiple trafficking behaviors of individual synaptic vesicles revealed by quantum-dot based presynaptic probe. PLoS One, 7(5): e38045
Leitz J, Kavalali E T (2011). Ca²? influx slows single synaptic vesicle endocytosis. J Neurosci, 31(45): 16318–16326
Leitz J, Kavalali E T (2014). Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. Elife, 3: e03658
Levi V, Gratton E (2007). Exploring dynamics in living cells by tracking single particles. Cell Biochem Biophys, 48(1): 1–15
Liu G (2003). Presynaptic control of quantal size: kinetic mechanisms and implications for synaptic transmission and plasticity. Curr Opin Neurobiol, 13(3): 324–331
Liu Z, Lavis L D, Betzig E (2015). Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell, 58(4): 644–659
Loy K, Welzel O, Kornhuber J, Groemer T W (2014). Common strength and localization of spontaneous and evoked synaptic vesicle release sites. Mol Brain, 7: 23
Mahler B, Spinicelli P, Buil S, Quelin X, Hermier J P, Dubertret B (2008). Towards non-blinking colloidal quantum dots. Nat Mater, 7 (8): 659–664
Makino H, Malinow R (2009). AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron, 64(3): 381–390
Manzo C, Garcia-Parajo M F (2015). A review of progress in single particle tracking: from methods to biophysical insights. Rep Prog Phys, 78(12): 124601
Maschi D, Klyachko V A (2015).A nanoscale resolution view on synaptic vesicle dynamics. Synapse, 69(5): 256–267
Mattoussi H, Palui G, Na H B (2012). Luminescent quantum dots as platforms for probing in vitro and in vivo biological processes. Adv Drug Deliv Rev, 64(2): 138–166
Maysinger D, Ji J, Hutter E, Cooper E (2015). Nanoparticle-based and bioengineered probes and sensors to detect physiological and pathological biomarkers in neural cells. Front Neurosci, 9: 480
Medintz I L, Uyeda H T, Goldman E R, Mattoussi H (2005). Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater, 4 (6): 435–446
Michalet X, Colyer R A, Scalia G, Ingargiola A, Lin R, Millaud J E, Weiss S, Siegmund O H, Tremsin A S, Vallerga J V, Cheng A, Levi M, Aharoni D, Arisaka K, Villa F, Guerrieri F, Panzeri F, Rech I, Gulinatti A, Zappa F, Ghioni M, Cova S (2013). Development of new photon-counting detectors for single-molecule fluorescence microscopy. Philos Trans R SocLond B Biol Sci, 368(1611): 20120035
Michalet X, Pinaud F F, Bentolila L A, Tsay J M, Doose S, Li J J, Sundaresan G, Wu A M, Gambhir S S, Weiss S (2005). Quantum dots for live cells, in vivo imaging, and diagnostics. Science, 307 (5709): 538–544
Midorikawa M, Sakaba T (2015). Imaging exocytosis of single synaptic vesicles at a fast CNS presynaptic terminal. Neuron, 88(3): 492–498
Miesenböck G, De Angelis D A, Rothman J E (1998). Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature, 394(6689): 192–195
Mochida S (2011). Activity-dependent regulation of synaptic vesicle exocytosis and presynaptic short-term plasticity. Neurosci Res, 70 (1): 16–23
Monico C, Capitanio M, Belcastro G, Vanzi F, Pavone F S (2013). Optical methods to study protein-DNA interactions in vitro and in living cells at the single-molecule level. Int J Mol Sci, 14(2): 3961–3992
Müller T, Schumann C, Kraegeloh A (2012). STED microscopy and its applications: new insights into cellular processes on the nanoscale. Chemphyschem, 13(8): 1986–2000
Murthy V N, De Camilli P (2003). Cell biology of the presynaptic terminal. Annu Rev Neurosci, 26: 701–728
Nan X, Sims P A, Chen P, Xie X S (2005). Observation of individual microtubule motor steps in living cells with endocytosed quantum dots. J Phys Chem B, 109(51): 24220–24224
Neupane B, Ligler F S, Wang G (2014). Review of recent developments in stimulated emission depletion microscopy: applications on cell imaging. J Biomed Opt, 19(8): 080901
Opazo P, Sainlos M, Choquet D (2012). Regulation of AMPA receptor surface diffusion by PSD-95 slots. Curr Opin Neurobiol, 22(3): 453–460
Park H, Hanson G T, Duff S R, Selvin P R (2004). Nanometre localization of single ReAsH molecules. J Microsc, 216(Pt 3): 199–205
Park H, Li Y, Tsien R W (2012). Influence of synaptic vesicle position on release probability and exocytotic fusion mode. Science, 335 (6074): 1362–1366
Park H, Toprak E, Selvin P R (2007). Single-molecule fluorescence to study molecular motors. Q Rev Biophys, 40(1): 87–111
Pechstein A, Shupliakov O (2010). Taking a back seat: synaptic vesicle clustering in presynaptic terminals. Front Synaptic Neurosci, 2: 143
Peng A, Rotman Z, Deng P Y, Klyachko V A (2012). Differential motion dynamics of synaptic vesicles undergoing spontaneous and activity-evoked endocytosis. Neuron, 73(6): 1108–1115
Ramirez D M, Kavalali E T (2011). Differential regulation of spontaneous and evoked neurotransmitter release at central synapses. Curr Opin Neurobiol, 21(2): 275–282
Ratnayaka A, Marra V, Branco T, Staras K (2011). Extrasynaptic vesicle recycling in mature hippocampal neurons. Nat Commun, 2: 531
Regehr W G (2012). Short-term presynaptic plasticity. Cold Spring Harb Perspect Biol, 4(7): a005702
Rust MJ, Bates M, Zhuang X (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods, 3(10): 793–795
Sakaba T (2006). Roles of the fast-releasing and the slowly releasing vesicles in synaptic transmission at the calyx of Held. J Neurosci, 26 (22): 5863–5871
Sara Y, Bal M, Adachi M, Monteggia L M, Kavalali E T (2011). Usedependent AMPA receptor block reveals segregation of spontaneous and evoked glutamatergic neurotransmission. J Neurosci, 31(14): 5378–5382
Smith A M, Nie S (2010). Semiconductor nanocrystals: structure, properties, and band gap engineering. Acc Chem Res, 43(2): 190–200
Staras K, Branco T, Burden J J, Pozo K, Darcy K, Marra V, Ratnayaka A, Goda Y (2010). A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons. Neuron, 66(1): 37–44
Steyer J A, Almers W (2001). A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol, 2(4): 268–275
Südhof T C (2004). The synaptic vesicle cycle. Annu Rev Neurosci, 27: 509–547
Südhof T C (2008). Neurotransmitter release. Handb Exp Pharmacol, 184: 1–21
Takamori S, Holt M, Stenius K, Lemke E A, Grønborg M, Riedel D, Urlaub H, Schenck S, Brügger B, Ringler P, Møller S A, Rammner B, Gräter F, Hub J S, De Groot B L, Mieskes G, Moriyama Y, Klingauf J, Grubmüller H, Heuser J, Wieland F, Jahn R (2006). Molecular anatomy of a trafficking organelle. Cell, 127(4): 831–846
Tardin C, Cognet L, Bats C, Lounis B, Choquet D (2003). Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J, 22(18): 4656–4665
Tatavarty V, Ifrim MF, Levin M, Korza G, Barbarese E, Yu J, Carson J H (2012). Single-molecule imaging of translational output from individual RNA granules in neurons. MolBiol Cell, 23(5): 918–929
Thompson R E, Larson D R, Webb W W (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophys J, 82 (5): 2775–2783
Triller A, Choquet D (2008). New concepts in synaptic biology derived from single-molecule imaging. Neuron, 59(3): 359–374
Warshaw D M, Kennedy G G, Work S S, Krementsova E B, Beck S, Trybus K M (2005). Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity. Biophys J, 88(5): L30–L32
Westphal V, Rizzoli S O, Lauterbach M A, Kamin D, Jahn R, Hell S W (2008). Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science, 320(5873): 246–249
Wilhelm B G, Groemer T W, Rizzoli S O (2010). The same synaptic vesicles drive active and spontaneous release. Nat Neurosci, 13(12): 1454–1456
Willig K I, Rizzoli S O, Westphal V, Jahn R, Hell S W (2006). STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature, 440(7086): 935–939
Wu Y, Yeh F L, Mao F, Chapman E R (2009). Biophysical characterization of styryl dye-membrane interactions. Biophys J, 97 (1): 101–109
Xia T, Li N, Fang X (2013). Single-molecule fluorescence imaging in living cells. Annu Rev PhysChem, 64: 459–480
Xie X S, Trautman J K (1998). Optical studies of single molecules at room temperature. Annu Rev PhysChem, 49: 441–480
Yang Y, Calakos N (2013). Presynaptic long-term plasticity. Front Synaptic Neurosci, 5: 8
Yildiz A, Forkey J N, McKinney S A, Ha T, Goldman Y E, Selvin P R (2003). Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science, 300(5628): 2061–2065
Yildiz A, Selvin P R (2005). Fluorescence imaging with one nanometer accuracy: application to molecular motors. Acc Chem Res, 38(7): 574–582
Zenisek D, Steyer J A, Almers W (2000). Transport, capture and exocytosis of single synaptic vesicles at active zones. Nature, 406 (6798): 849–854
Zhang Q, Cao Y Q, Tsien R W (2007). Quantum dots provide an optical signal specific to full collapse fusion of synaptic vesicles. Proc Natl Acad Sci U S A, 104(45): 17843–17848
Zhang Q, Li Y, Tsien RW (2009). The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science, 323 (5920): 1448–1453
Zhang R, Rothenberg E, Fruhwirth G, Simonson P D, Ye F, Golding I, Ng T, Lopes W, Selvin P R (2011). Two-photon 3D FIONA of individual quantum dots in an aqueous environment. Nano Lett, 11 (10):4074–4078
Zhou X, Wang L (2010). Uses of single-particle tracking in living cells. Drug Discov Ther, 4(2): 62–69
Zhu Y, Xu J, Heinemann S F (2009). Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging. Neuron, 61(3): 397–411
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yu, C., Zhang, M., Qin, X. et al. Real-time imaging of single synaptic vesicles in live neurons. Front. Biol. 11, 109–118 (2016). https://doi.org/10.1007/s11515-016-1397-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11515-016-1397-z