Single‐Molecule Tracking Photoactivated Localization Microscopy to Map Nano‐Scale Structure and Dynamics in Living Spines

Harold D. MacGillavry1, Thomas A. Blanpied1

1 University of Maryland School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.20
DOI:  10.1002/0471142301.ns0220s65
Online Posting Date:  October, 2013
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Abstract

Super‐resolution microscopy has rapidly become an indispensable tool in cell biology and neuroscience by enabling measurement in live cells of structures smaller than the classical limit imposed by diffraction. The most widely applied super‐resolution method currently is localization microscopy, which takes advantage of the ability to determine the position of individual fluorescent molecules with nanometer accuracy even in cells. By iteratively measuring sparse subsets of photoactivatable fluorescent proteins, protein distribution in macromolecular structures can be accurately reconstructed. Moreover, the motion trajectories of individual molecules within cells can be measured, providing a unique ability to measure transport kinetics, exchange rates, and binding affinities of even small subsets of molecules with high temporal resolution and great spatial specificity. This unit describes protocols to measure and quantify the distribution of scaffold proteins within single synapses of cultured hippocampal neurons, and to track and measure the diffusion of intracellular constituents of the neuronal plasma membrane. Curr. Protoc. Neurosci. 65:2.20.1‐2.20.19. © 2013 by John Wiley & Sons, Inc.

Keywords: single‐molecule tracking; photoactivated localization microscopy; PALM; STORM; live‐cell imaging; super‐resolution microscopy; neuron; dendritic spine; synapse; postsynaptic density; hippocampal cultures

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Single‐Molecule PALM in Living Neurons
  • Support Protocol 1: Dissociated Hippocampal Culture Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Single‐Molecule PALM in Living Neurons

  Materials
  • Hippocampal cultures (14 to 21 DIV; see Support Protocol) in 12‐well plates
  • Purified expression plasmid expressing protein of interest tagged with photoconvertible protein
  • Opti‐MEM I–reduced serum medium (Invitrogen)
  • Lipofectamine 2000 reagent (Invitrogen)
  • Extracellular imaging buffer (see recipe)
  • Inverted microscope (e.g., Olympus IX81)
  • Epifluorescence light source (e.g., Arc lamp)
  • Oil immersion objective suitable for TIRF (e.g., 100×/1.49 NA)
  • Lasers:
    • 405‐nm activation laser (e.g., Coherent Cube, 50 mW)
    • 561‐nm excitation laser (e.g., Cobolt Jive; ≥200 mW)
  • Acousto‐optical tunable filter (AOTF, e.g., Neos)
  • Filter cubes in the microscope
  • iXon+ 897 EM‐CCD camera (Andor Technology)
  • Stimulator or timing circuit (e.g., AMPI Master‐8)
  • iQ software version 2.4 or higher (Andor Technology)
  • MATLAB software (with Image Processing toolbox, MathWorks)

Support Protocol 1: Dissociated Hippocampal Culture Preparation

  Materials
  • Sterile‐filtered water
  • Ammonium hydroxide ( appendix 2A)
  • Hydrogen peroxide
  • 96% ethanol
  • Methanol
  • Poly‐L‐lysine solution (see recipe)
  • Plating medium (see recipe)
  • Dissection medium (see recipe), ice cold
  • E18 timed‐pregnant rat
  • CO 2 source
  • 0.25% Trypsin‐EDTA
  • DNase
  • Trypsin inhibitor, filter‐sterilized, 37°C
  • 0.4% Trypan Blue stain
  • Plating medium (see recipe)
  • Feeding medium (see recipe)
  • No. 1.5 glass coverslips (Warner instruments)
  • Heat‐resistant coverslip rack
  • 1000‐ml glass beaker
  • Magnetic stir plate and bar
  • 12‐well cell culture plates
  • 37°C cell culture incubator
  • Microsurgical tools:
    • Large scissors
    • 10‐cm scissors
    • 10‐cm Graefe forceps
    • Micro‐spatula
    • 11‐cm fine shanks forceps
    • 45‐tip forceps
    • 8‐cm spring scissors
  • 60‐ and 100‐mm dishes
  • 15‐ml tubes
  • Centrifuge
  • Pasteur pipets
  • Binocular dissecting microscope
  • Hemacytometer
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Figures

Videos

Literature Cited

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Internet Resources
  http://physics.georgetown.edu/matlab/
  Single‐molecule tracking software source.
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