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Live-Cell PALM of Intracellular Proteins in Neurons

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Nanoscale Imaging of Synapses

Part of the book series: Neuromethods ((NM,volume 84))

Abstract

Synaptic communication is a tightly regulated process requiring the coordinated assembly and regulation of a multitude of scaffolding and signaling molecules critical to synaptic maintenance and plasticity. The small spatial scales of the synapse and the dendritic spines which house the postsynaptic machinery have limited the direct visualization of their structure in living cells. A number of imaging methods have evolved in recent years with the capability to resolve structures within living neurons at resolution far exceeding that achievable by confocal microscopy. We will focus in this chapter on this laboratory’s experience with Photoactivated Localization Microscopy (PALM) over the last several years and attempt to provide a useful and pragmatic guide to its utilization in the study of neuronal structures and their dynamic reorganization within living neurons. PALM offers several advantages over other imaging modalities. First, it permits the localization of single molecules with precision on the order of tens of nanometers and the rendering of neuronal structures at high resolution. As this technique utilizes expressed proteins tagged with photoconvertible fluorescent proteins, it permits time-resolved measurement of both the behavior of single molecules and the reshaping of protein networks within living neurons. We provide a framework to aid the reader through the initial process of designing or selecting an appropriate optical system, optimizing acquisition and hardware settings, and the analysis of single-molecule data.

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References

  1. Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011) Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8:527–528

    Article  CAS  PubMed  Google Scholar 

  2. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  CAS  PubMed  Google Scholar 

  3. Fischer RS, Wu Y, Kanchanawong P, Shroff H, Waterman CM (2011) Microscopy in 3D: a biologist's toolbox. Trends Cell Biol 21:682–691

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Frost NA, Kerr JM, Lu HE, Blanpied TA (2010) A network of networks: cytoskeletal control of compartmentalized function within dendritic spines. Curr Opin Neurobiol 20:578–587

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Frost NA, Lu HE, Blanpied TA (2012) Optimization of cell morphology measurement via single-molecule tracking PALM. PLoS One 7:e36751

    Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Gould TJ, Gunewardene MS, Gudheti MV, Verkhusha VV, Yin SR, Gosse JA, Hess ST (2008) Nanoscale imaging of molecular positions and anisotropies. Nat Methods 5:1027–1030

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Gould TJ, Verkhusha VV, Hess ST (2009) Imaging biological structures with fluorescence photoactivation localization microscopy. Nat Protoc 4:291–308

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Hedde PN, Fuchs J, Oswald F, Wiedenmann J, Nienhaus GU (2009) Online image analysis software for photoactivation localization microscopy. Nat Methods 6:689–690

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Huang B, Babcock H, Zhuang X (2010) Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143:1047–1058

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Izeddin I, Specht CG, Lelek M, Darzacq X, Triller A, Zimmer C, Dahan M (2011) Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe. PLoS One 6:e15611

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Jones SA, Shim SH, He J, Zhuang X (2011) Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8:499–508

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Kerr JM, Blanpied TA (2012) Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density. J Neurosci 32:658–673

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Lippincott-Schwartz J, Manley S (2009) Putting super-resolution fluorescence microscopy to work. Nat Methods 6:21–23

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. MacGillavry HD, Kerr JM, Blanpied TA (2011) Lateral organization of the postsynaptic density. Mol Cell Neurosci 48:321–331

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. MacGillavry HD, Song Y, Raghavachari S, Blanpied TA (2013) Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78(4):615–622

    Google Scholar 

  21. Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5:155–157

    Article  CAS  PubMed  Google Scholar 

  22. Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolkova K, Dlaskova A, Santorova J, Jezek P, Bewersdorf J (2011) Sample drift correction in 3D fluorescence photoactivation localization microscopy. Opt Express 19:15009–15019

    Article  PubMed  Google Scholar 

  23. Ober RJ, Ram S, Ward ES (2004) Localization accuracy in single-molecule microscopy. Biophys J 86:1185–1200

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Ram S, Prabhat P, Chao J, Ward ES, Ober RJ (2008) High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells. Biophys J 95:6025–6043

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Renner M, Domanov Y, Sandrin F, Izeddin I, Bassereau P, Triller A (2011) Lateral diffusion on tubular membranes: quantification of measurements bias. PLoS One 6:e25731

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Ritter JG, Veith R, Veenendaal A, Siebrasse JP, Kubitscheck U (2010) Light sheet microscopy for single molecule tracking in living tissue. PLoS One 5:e11639

    Article  PubMed Central  PubMed  Google Scholar 

  28. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Saxton MJ (2001) Anomalous subdiffusion in fluorescence photobleaching recovery: a Monte Carlo study. Biophys J 81:2226–2240

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Shao L, Kner P, Rego EH, Gustafsson MG (2011) Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods 8:1044–1046

    Article  CAS  PubMed  Google Scholar 

  31. Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5:417–423

    Article  CAS  PubMed  Google Scholar 

  32. Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, Olenych S, Davidson MW, Betzig E (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A 104:20308–20313

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Sugiyama Y, Kawabata I, Sobue K, Okabe S (2005) Determination of absolute protein numbers in single synapses by a GFP-based calibration technique. Nat Methods 2:677–684

    Article  CAS  PubMed  Google Scholar 

  35. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Triller A, Choquet D (2008) New concepts in synaptic biology derived from single-molecule imaging. Neuron 59:359–374

    Article  CAS  PubMed  Google Scholar 

  37. Urban NT, Willig KI, Hell SW, Nagerl UV (2011) STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys J 101:1277–1284

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6:991–1009

    Article  PubMed  Google Scholar 

  39. Vaziri A, Tang J, Shroff H, Shank CV (2008) Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci U S A 105:20221–20226

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Willig KI, Kellner RR, Medda R, Hein B, Jakobs S, Hell SW (2006) Nanoscale resolution in GFP-based microscopy. Nat Methods 3:721–723

    Article  CAS  PubMed  Google Scholar 

  41. Wolter S, Schuttpelz M, Tscherepanow M, Van De Linde S, Heilemann M, Sauer M (2010) Real-time computation of subdiffraction-resolution fluorescence images. J Microsc 237:12–22

    Article  CAS  PubMed  Google Scholar 

  42. Xu K, Babcock HP, Zhuang X (2012) Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods 9(2):185–188

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. York AG, Ghitani A, Vaziri A, Davidson MW, Shroff H (2011) Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat Methods 8:327–333

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, USA

    Nicholas A. Frost & Thomas A. Blanpied

  2. Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA

    Harold D. MacGillavry & Thomas A. Blanpied

  3. Program in Molecular Medicine, University of Maryland School of Medicine, Baltimore, MD, USA

    Hsiangmin E. Lu & Thomas A. Blanpied

Authors
  1. Nicholas A. Frost
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  2. Harold D. MacGillavry
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  3. Hsiangmin E. Lu
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  4. Thomas A. Blanpied
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Editor information

Editors and Affiliations

  1. Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France

    U. Valentin Nägerl

  2. Institute of Biology at Ecole Normale Supérieure, Ecole Normale Supérieure, Paris, France

    Antoine Triller

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Frost, N.A., MacGillavry, H.D., Lu, H.E., Blanpied, T.A. (2014). Live-Cell PALM of Intracellular Proteins in Neurons. In: Nägerl, U., Triller, A. (eds) Nanoscale Imaging of Synapses. Neuromethods, vol 84. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-9179-8_6

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  • DOI: https://doi.org/10.1007/978-1-4614-9179-8_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4614-9178-1

  • Online ISBN: 978-1-4614-9179-8

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