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
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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

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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
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Basic Protocol 1: Preparing PALM Instrumentation

  • 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

  • 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

  • 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

  • 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

  • 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)
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