Photoactivated Localization Microscopy (PALM) of Adhesion Complexes

Hari Shroff1, Helen White2, Eric Betzig2

1 National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, 2 Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.21
DOI:  10.1002/0471143030.cb0421s58
Online Posting Date:  March, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Key to understanding a protein's biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction‐limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (∼200 nm) have emerged. Here, both single‐ and dual‐color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein‐filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA‐FP) tags necessary to achieve PALM images at ∼20 nm resolution in 5 to 30 min in fixed cells. Curr. Protoc. Cell Biol. 58:4.21.1‐4.21.28. © 2013 by John Wiley & Sons, Inc.

Keywords: PALM; superresolution; adhesion complex; fluorescent proteins

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparing PALM Instrumentation
  • Basic Protocol 2: PALM‐Imaging tdEos/Paxillin Distributions in Fixed Cells
  • Basic Protocol 3: Dual‐Color PALM‐Imaging of tdEos/Vinculin and Dronpa α‐Actinin in Fixed Cells
  • Support Protocol 1: Preparing Clean Coverslips
  • Support Protocol 2: Transfection of tdEos/Paxillin into HFF‐1 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparing PALM Instrumentation

  Materials
  • Optical table (Technical Manufacturing Corporation)
  • Inverted fluorescence microscope (e.g., Olympus IX‐81) assembly, including:
    • Brightfield and DIC optics
    • Capability for TIRF illumination
    • High‐NA objective lenses (1.45 NA, 1.49 NA, or 1.65 NA) with matching coverslips, and immersion media
    • Internal magnification lens (optional, depending on objective magnification)
    • Additional magnification before camera (optional, depending on objective magnification)
  • Mercury or xenon lamp
  • Mechanically stable stage and sample mount
  • Appropriate excitation and activation lasers and optics (beam expanders, neutral density filters, half‐wave plates, and dichroic beamsplitters) for coupling lasers into the TIRF microscope path
  • Appropriate excitation, emission, and dichroic filters
  • Acousto‐optic tunable filter (AA Opto‐Electronic, AA‐AOTFnC‐VIS)
  • EMCCD camera (see step 12 for details)
  • Computer with appropriate control software (see step 14 for details)
  • Computer with appropriate analysis software (see step 16 for details)

Basic Protocol 2: PALM‐Imaging tdEos/Paxillin Distributions in Fixed Cells

  Materials
  • EM grade paraformaldehyde
  • Clean water (filtered through a Millipore system)
  • 10 N NaOH solution
  • 2× PHEM buffer (see recipe)
  • Cleaned coverslips ( protocol 4) plated with HFF‐1 cells transfected with tdEos/paxillin, in 35‐mm plastic dishes ( protocol 5)
  • Immersion oil
  • 100‐nm and 40‐nm Au particles (Microspheres‐Nanospheres, cat. nos. 790114‐010 and 790122‐010)
  • Chemical hood
  • 1‐liter glass beaker
  • Hotplate with magnetic stirring capability
  • Magnetic stir bar
  • 0.2‐µm filter
  • 37°C warm room or equivalent heating system
  • Fine steel forceps
  • Microscope set up (as described in protocol 1)
  • Lens paper
  • EMCCD camera (see protocol 1)
  • Benchtop vortexer/sonicator

Basic Protocol 3: Dual‐Color PALM‐Imaging of tdEos/Vinculin and Dronpa α‐Actinin in Fixed Cells

  Materials
  • HFF‐1 cells cotransfected with tdEos/vinculin and Dronpa/α‐actinin plasmid constructs (these plasmids are made in‐house by the authors, but can also be obtained from Mike Davidson, Florida State University)
  • 100‐nm and 40‐nm Au particles (Microspheres‐Nanospheres, cat. nos. 790114‐010 and 790122‐010)
  • Additional reagents and equipment for cleaning coverslips ( protocol 4), transfecting cells ( protocol 5), and PALM‐imaging both Eos ( protocol 2) and Dronpa

Support Protocol 1: Preparing Clean Coverslips

  Materials
  • Ammonium hydroxide
  • Hydrogen peroxide
  • Clean water (filtered through a Millipore system)
  • Methanol (spectroscopic grade)
  • Clean air supply (preferably filtered through a 0.2‐µm pore‐size filter)
  • RBS‐35 liquid detergent concentrate (Pierce, cat. no. 27950)
  • Acetone (spectroscopic grade)
  • Chemical fume hood
  • 100‐ml graduated cylinder
  • 250‐ml glass beaker
  • 25‐mm diameter glass coverslips, no. 1.5 (Warner Instruments, cat. no. 64‐0715; for use with 1.45 NA or 1.49 NA objectives)
  • High‐index 20‐mm diameter coverslips (Olympus, cat. no. APO100X‐CG; for use with 1.65 NA objective)
  • Hotplate with magnetic stirring capability
  • Magnetic stir bar
  • Corrosion‐resistant staining rack for holding coverslips (Thomas Scientific, cat. no. 8542E40)
  • Metal tongs for holding staining rack
  • Fine steel forceps
  • Compressed butane/natural gas, burner, and lighter

Support Protocol 2: Transfection of tdEos/Paxillin into HFF‐1 Cells

  Materials
  • 70% ethanol
  • Human plasma fibronectin diluted in 1× PBS (without divalent cations)
  • 1% (w/v) BSA/DMEM HG, heat‐inactivated (see recipe)
  • HFF‐1 growth medium (see recipe)
  • 0.05% (w/v) trypsin‐0.53mM EDTA
  • Cell Line 96‐well Nucleofector Kit SE (Amaxa, VHCA‐1001) containing:
    • SE solution
    • Supplement
  • Plasmid: tdEos/paxillin (∼0.5 to 1 µg/µl)
  • Normal human foreskin fibroblast (HFF‐1) cells (ATCC cat. no. SCRC‐1041) grown in 75‐cm2 cell culture flasks with 0.2‐µm vent caps for 2 to 3 days before transfection
  • Compressed butane/natural gas, burner, and lighter
  • Fine steel forceps
  • Cleaned coverslips (see protocol 4)
  • 35 × 10–mm cell culture dishes
  • 37°C water bath
  • 1.5‐ml microcentrifuge tube or 15‐ml conical centrifuge tubes
  • Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent)
  • Centrifuge rotor (Eppendorf 5810 A‐4‐62 or equivalent)
  • Rainin LTS pipets (0.1 to 200 µl)
  • Rainin LTS tips (RT‐L10F and RT‐L200F)
  • Nucleofector 96‐well shuttle system (Amaxa Biosystems)
  • Additional reagents and equipment for performing a viable cell count (unit 1.1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ando, R., Hama, H., Yamamoto‐Hino, M., Mizuno, H., and Miyawaki, A. 2002. An optical marker based on the UV‐induced green‐to‐red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99:12651‐12656.
   Ando, R., Mizuno, H., and Miyawaki, A. 2004. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370‐1373.
   Axelrod, D. 2001. Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764‐774.
   Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott‐Schwartz, J., and Hess, H.F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642‐1645.
   Cella Zanacchi, F., Lavagnino, Z., Perrone Donnorso, M., Del Bue, A., Furia, L., Faretta, M., and Diaspro, A. 2011. Live‐cell 3D super‐resolution imaging in thick biological samples. Nat. Methods 8:1047‐1049.
   Chen, I. and Ting, A.Y. 2005. Site‐specific labeling of proteins with small molecules in live cells. Curr. Opin. Biotechnol. 16:35‐40.
   Chudakov, D.M., Verkhusha, V.V., Staroverov, D.B., Souslova, E.A., Lukyanov, S., and Lukyanov, K.A. 2004. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22:1435‐1439.
   Chudakov, D.M., Lukyanov, S., and Lukyanov, K.A. 2007. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS‐CFP2 and Dendra2. Nat. Protocols 2:2024‐2032.
   Dempsey, G.T., Vaughan, J.C., Chen, K.H., Bates, M., and Zhuang, X. 2011. Evaluation of fluorophores for optimal performance in localization‐based super‐resolution imaging. Nat. Methods 8:1027‐1036.
   Egner, A., Geisler, C., Middendorff, C., Bock, H., Wenzel, D., Medda, R., Andresen, M., Stiel, A.C., Jakobs, S., Eggeling, C., Schönle, A., and Hell, S.W. 2007. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93:3285‐3290.
   Fölling, J., Belov, V., Kunetsky, R., Medda, R., Schönle, A., Egner, A., Eggeling, C., Bossi, M., and Hell, S.W. 2007. Photochromic rhodamines provide nanoscopy with optical sectioning. Angew. Chem. Int. Ed. 46:6266‐6270.
   Galbraith, C.G., Skalak, R., and Chien, S. 1998. Shear stress induces spatial reorganization of the endothelial cell cytoskeleton. Cell Motil. Cytoskeleton 40:317‐330.
   Giepmans, B.N.G., Adams, S.R., Ellisman, M.H., and Tsien, R.Y. 2006. The fluorescent toolbox for assessing protein location and function. Science 312:217‐224.
   Gustafsson, M.G.L. 2000. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198:82‐87.
   Gustafsson, M.G.L. 2005. Nonlinear structured‐illumination microscopy: Wide‐field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. U.S.A. 102:13081‐13086.
   Heilemann, M., van de Linde, S., Schüttpelz, M., Kasper, R., Seefeldt, B., Mukherjee, A., Tinnefeld, P., and Sauer, M. 2008. Subdiffraction‐resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47:6172‐6176.
   Hess, S.T., Girirajan, T.P.K., and Mason, M.D. 2006. Ultra‐high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91:4258‐4272.
   Huang, B., Jones, S.A., Brandenburg, B., and Zhuang, X. 2008. Whole‐cell 3D STORM reveals interactions between cellular structures with nanometer‐scale resolution. Nat. Methods 5:1047‐1052.
   Juette, M.F., Gould, T.J., Lessard, M.D., Mlodzianoski, M.J., Nagpure, B.S., Bennett, B.T., Hess, S.T., and Bewersdorf, J. 2008. Three‐dimensional sub‐100 nm resolution fluorescence microscopy on thick samples. Nat. Methods 5:527‐529.
   McKinney, S.A., Murphy, C.S., Hazelwood, K.L., Davidson, M.W., and Looger, L.L. 2009. A bright and photostable photoconvertible fluorescent protein. Nat. Methods 6:131‐133.
   Mitchison, T.J., Sawin, K.E., Theriot, J.A., Gee, K., and Mallavarapu, A. 1998. Caged fluorescent probes. Methods Enzymol. 291:63‐78.
   Otey, C.A. and Carpen, O. 2004. Alpha‐actinin revisited: A fresh look at an old player. Cell Mot. Cytoskel. 58:104‐111.
   Patterson, G.H. and Lippincott‐Schwartz, J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873‐1877.
   Rust, M.J., Bates, M., and Zhuang, X. 2006. Sub‐diffraction‐limit imaging by stochastic optical reconstruction microscopy (STORM) Nat. Methods 3:793‐796.
   Shannon, C.E. 1949. Communication in the presence of noise. Proc. IRE 37:10‐21.
   Shroff, H., Galbraith, C.G., Galbraith, J.A., White, H., Gillette, J., Olenych, S., Davidson, M.W., and 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.
   Shroff, H., Galbraith, C.G., Galbraith, J.A., and Betzig, E. 2008. Live‐cell photoactivated localization microscopy of nanoscale adhesion complexes. Nat. Methods 5:417‐423.
   Subach, F.V., Patterson, G.H., Manley, S., Gillette, J.M., Lippincott‐Schwartz, J., and Verkhusha, V.V. 2009. Photoactivatable mCherry for high‐resolution two‐color fluorescence microscopy. Nat. Methods 6:153‐159.
   Thompson, R.E., Larson, D.R., and Webb, W.W. 2002. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82:2775‐2783.
   Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., and Miywaki, A. 2005. Semi‐rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6:233‐238.
   Weiss, S. 1999. Fluorescence spectroscopy of single biomolecules. Science 283:1676‐1683.
   Wiedenmann, J. and Nienhaus, G.U. 2006. Live‐cell imaging with EosFP and other photoactivatable marker proteins of the GFP family. Expert Rev. Proteomics 3:361‐374.
   Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.‐D., and Nienhaus, G.U. 2004. EosFP, a fluorescent marker protein with UV‐inducible green‐to‐red fluorescence conversion. Proc. Natl. Acad. Sci. U.S.A. 101:15905‐15910.
   Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R., and Hell, S.W. 2006. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440:935‐939.
   Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E., and Selvin, P.R. 2003. Myosin V walks hand‐over‐hand: Single fluorophore imaging with 1.5‐nm localization. Science 300:2061‐2065.
   York, A.G., Ghitani, A., Vaziri, A., Davidson, M.W., and Shroff, H. 2011. Confined activation and subdiffractive localization enables whole‐cell PALM with genetically expressed probes. Nat. Methods 8:327‐333.
   Zaidel‐Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R., and Geiger, B. 2007. Functional atlas of the integrin adhesome. Nat. Cell Biol. 9:858‐867.
   Zamir, E. and Geiger, B. 2001. Molecular complexity and dynamics of cell‐matrix adhesions. J. Cell Sci. 114:3583‐3590.
   Ziegler, W.H., Liddington, R.C., and Critchley, D.R. 2006. The structure and regulation of vinculin. Trends Cell Biol. 16:453‐460.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library