Abstract
Super-resolution light microscopy including pointillist methods based on single molecule localization (e.g., PALM/STORM) allow to image protein structures much smaller than the diffraction limit (200–300 nm). However, commonly used labeling strategies such as antibodies or protein fusions have several important drawbacks, including the risk to alter the function or distribution of the imaged proteins. We recently demonstrated that pointillist imaging can be performed using the alternative labeling technique known as FlAsH, which better preserves protein function, is compatible with live cell imaging, and may help reach single nanometer resolution. We applied FlAsH-PALM to visualize HIV integrase in isolated virions or infected cells, allowing us to obtain sub-diffraction resolution images of this enzyme’s spatial distribution and analyze HIV morphology without altering viral replication. The technique should also prove useful to image delicate proteins in intracellular vesicles and organelles at high resolution. Here, we present a detailed protocol in order to facilitate the application of FLAsH-PALM to other proteins and biological structures.
Mickaël Lelek and Francesca Di Nunzio have contributed equally to this chapter.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hell SW (2009) Microscopy and its focal switch. Nat Methods 6:24–32
Huang B, Babcock H, Zhuang X (2010) Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143:1047–1058
Herbert S, Soares H, Zimmer C, Henriques R (2012) Single-molecule super-resolution microscopy: deeper and faster. Microscopy & Microanalysis 18:1419–1429
Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795
Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Sci Signal 312:217
Schnell U, Dijk F, Sjollema KA, Giepmans BN (2012) Immunolabeling artifacts and the need for live-cell imaging. Nat Methods 9:152–158
Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5:417–423
Enninga J, Mounier J, Sansonetti P, Van Nhieu GT (2005) Secretion of type III effectors into host cells in real time. Nat Methods 2:959–965
Müller B, Daecke J, Fackler OT, Dittmar MT, Zentgraf H, Kräusslich HG (2004) Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J Virol 78:10803–10813
Engelman A, Englund G, Orenstein JM, Martin MA, Craigie R (1995) Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol 69:2729–2736
Adams SR, Campbell RE, Gross LA et al (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc 124:6063–6076
Martin BR, Giepmans BNG, Adams SR, Tsien RY (2005) Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat Biotechnol 23:1308–1314
Arhel N, Genovesio A, Kim KA et al (2006) Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nat Methods 3:817–824
Andresen M, Schmitz-Salue R, Jakobs S (2004) Short tetracysteine tags to beta-tubulin demonstrate the significance of small labels for live cell imaging. Mol Biol Cell 15:5616–5622
Lelek M, Di Nunzio F, Henriques R et al (2012) Superresolution imaging of HIV in infected cells with FlAsH-PALM. Proc Natl Acad Sci U S A 109:8564–8569
Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI (1999) Assembly and analysis of conical models for the HIV-1 core. Science 283:80–83
Arhel N (2010) Revisiting HIV-1 uncoating. Retrovirology 7:96
Di Nunzio F (2013) New insights in the role of nucleoporins: a bridge leading to concerted steps from HIV nuclear entry until integration. Virus Res 178:187–196
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
Charneau P, Mirambeau G, Roux P, Paulous S, Buc H, Clavel F (1994) HIV-1 reverse transcription a termination step at the center of the genome. J Mol Biol 241:651–662
van de Linde S, Löschberger A, Klein T et al (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6:991–1009
Heilemann M, van de Linde S, Schüttpelz M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47:6172–6176
Sergé A, Bertaux N, Rigneault H, Marguet D (2008) Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods 5:687–694
Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T (1994) A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc Natl Acad Sci U S A 91:9564–9568
Henriques R, Griffiths C, Hesper Rego E, Mhlanga MM (2011) PALM and STORM: unlocking live-cell super-resolution. Biopolymers 95:322–331
Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272
Pertsinidis A, Zhang Y, Chu S (2010) Subnanometre single-molecule localization, registration and distance measurements. Nature 466:647–651
Geisler C et al (2012) Drift estimation for single marker switching based imaging schemes. Opt Express 20:7274–7289
Wolter S, Löschberger A, Holm T et al (2012) rapidSTORM: accurate, fast open-source software for localization microscopy. Nat Methods 9:1040–1041
Brede N, Lakadamyali M (2012) GraspJ: an open source, real-time analysis package for super-resolution imaging. Opt Nanoscopy 1:11
Gaietta GM, Deerinck TJ, Ellisman MH (2011) Labeling tetracysteine-tagged proteins with biarsenical dyes for live cell imaging. Cold Spring Harb Protoc 2011. doi: 10.1101/pdb.prot5547
Acknowledgements
We acknowledge funding by Institut Pasteur, Région Ile-de-France (DIM Malinf), Fondation pour la Recherche Médicale (Equipe FRM 2010), Sidaction and ANRS. We thank Philippe Souque for comments on the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lelek, M., Di Nunzio, F., Zimmer, C. (2014). FlAsH-PALM: Super-resolution Pointillist Imaging with FlAsH-Tetracysteine Labeling. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0944-5_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0943-8
Online ISBN: 978-1-4939-0944-5
eBook Packages: Springer Protocols