Comparative and Practical Aspects of Localization‐Based Super‐Resolution Imaging

Gary S. Laevsky1, Christopher B. O'Connell1

1 Nikon Instruments, Melville, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.20
DOI:  10.1002/0471142956.cy0220s63
Online Posting Date:  January, 2013
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Abstract

Super‐resolution microscopy overcomes diffraction to generate images with superior resolution compared to conventional light microscopy. Localization‐based super‐resolution methods result in up to ten‐fold improvement in resolution by determining the positions of fluorescent molecules with sub‐pixel accuracy. This process critically depends on controlled emission at the level of individual fluorophores so that fluorescence is non‐overlapping, allowing for accurate centroid determination of diffraction‐limited spots by Gaussian fitting of the pixel intensities. The intrinsic photoswitching behavior of many fluorophores provides a convenient way to achieve emitter isolation. Here, we describe methods for label preparation and staining of cellular structures to obtain high‐quality images using localization super resolution. We also compare labeling strategies and dye characteristics relevant to all localization‐based techniques, such as STORM and PALM. Curr. Protoc. Cytom. 63:2.20.1‐2.20.11. © 2013 by John Wiley & Sons, Inc.

Keywords: super‐resolution microscopy; STORM; PALM; tandem dyes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Multi‐Channel Labeling of Microtubules and Mitochondria with STORM Tandem Dye Pairs
  • Support Protocol 1: Dye Preparation and Secondary Antibody Labeling
  • Basic Protocol 2: Buffer and Imaging Conditions for Synthetic Photoswitchable Dyes
  • Basic Protocol 3: Labeling Proteins via SNAP Tags for Live‐Cell Localization Super Resolution
  • Support Protocol 2: Buffer and Imaging Conditions for Live‐Cell Localization Super Resolution
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Multi‐Channel Labeling of Microtubules and Mitochondria with STORM Tandem Dye Pairs

  Materials
  • Cells grown in Lab‐Tek II chambered coverglass, 8 well (Thermo Scientific)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 20012‐027), 1×
  • Fixation solution: 3% paraformaldehyde (PFA) and 0.1% glutaraldehyde in PBS
  • 0.1% Sodium borohydride (NaBH 4) in PBS
  • Blocking buffer: 3% BSA, 0.2% Triton X‐100 in PBS
  • Primary antibodies: e.g., Rat anti‐beta tubulin (Abcam, cat. no. ab6160) or Rabbit anti‐Tom20 (Santa Cruz Biotechnologies, cat. no. 11315)
  • Washing buffer: 0.2% BSA and 0.05% Triton X‐100 in PBS
  • Alexa405‐Alexa647 and Cy3‐Alexa647 conjugated secondary antibodies (see protocol 2)
  • Rocking platform shaker

Support Protocol 1: Dye Preparation and Secondary Antibody Labeling

  Materials
  • Recommended reporter/activator dyes:
    • Alexa Fluor 647 carboxylic acid, succinimidyl ester, 1 mg (Invitrogen, cat. no. A20006)
    • Alexa Fluor 405 carboxylic acid, succinimidyl ester, 1 mg (Invitrogen, cat. no. A30000)
    • Cy2 bis‐Reactive Dye Pack 5 vials (GE Healthcare, cat. no. PA22000)
    • Cy3 Mono‐reactive Dye Pack 5 vials (GE Healthcare, cat. no. PA23001)
  • Dimethyl sulfoxide (DMSO), anhydrous
  • Secondary antibody: e.g., AffiniPure donkey anti‐rat IgG (H+L) (Jackson ImmunoResearch Europe, cat. no. 712‐005‐153) or AffiniPure donkey anti‐rabbit IgG (H+L) (Jackson ImmunoResearch Europe, cat. no. 711‐005‐152) or AffiniPure donkey anti‐mouse IgG (H+L) (Jackson ImmunoResearch Europe, cat. no. 712‐005‐151) or other secondary antibody of your preference
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 20012‐027)
  • NaHCO 3
  • Evaporator
  • Aluminum foil
  • Shaking platform
  • NAP‐5 columns (GE Healthcare, cat. no. 17‐0853‐02)
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • UV/visible absorption spectrophotometer

Basic Protocol 2: Buffer and Imaging Conditions for Synthetic Photoswitchable Dyes

  Materials
  • GLOX solution: 14 mg glucose oxidase from Aspergillus niger‐Type VII, lyophilized (Sigma‐Aldrich), 50 µl catalase from bovine liver, lyophilized (Sigma‐Aldrich), and 200 µl Buffer A (10 mM Tris⋅Cl, pH 8.0, 50 mM NaCl)
  • Buffer B: 50 mM Tris⋅Cl, pH 8.0, 10 mM NaCl, and 10% D‐glucose stock solution
  • 1 M MEA: 77 mg cytsteamine (MEA) in 0.25 N HCl
  • Sample
  • Centrifuge
  • 1.5‐ml centrifuge tubes

Basic Protocol 3: Labeling Proteins via SNAP Tags for Live‐Cell Localization Super Resolution

  Materials
  • BS‐C‐1 cells
  • Culture medium for BS‐C‐1 cells: Eagle's minimum essential medium (ATTC, cat. no. 30‐2003)
  • Cell Line Nucleofector Kit V (Lonza, cat. no. VCA‐1003) containing:
    • Nucleofector Solution V
    • Nuclefection cuvette
  • pSNAP‐Clathrin, or for added labeling density pSNAP‐Clathrin‐SNAP, plasmid (see Jones et al., )
  • Fetal bovine serum (FBS; Invitrogen, cat. no. 16000‐44)
  • Potassium hydroxide
  • Milli‐Q water
  • Phosphate‐buffered saline (PBS), 1×
  • Trypsin‐EDTA
  • SNAP‐Surface Alexa Fluor 647 fluorescent substrate (New England Biolabs, cat. no. S9136S)
  • Labeling solution: 4 mM solution of SNAP‐Surface Alexa Fluor 647 dissolved in dimethyl sulfoxide
  • Centrifuge
  • 25‐cm2 culture flask
  • 37°C incubator
  • Nucleofector (Lonza)
  • Sonicator
  • Cover glass for plating cells: recommended: Lab‐Tek II Chambered Coverglass (Thermo Scientific Nunc, cat. no. 155409)
  • UV light source

Support Protocol 2: Buffer and Imaging Conditions for Live‐Cell Localization Super Resolution

  • Live‐cell imaging buffer: Dulbecco's modified Eagle medium (DMEM), 75 mM HEPES (from a 1 M HEPES buffer solution, pH 8.0), and 2% glucose
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Figures

Videos

Literature Cited

Literature Cited
   Annibale, P., Scarselli, M., Kodiyan, A., and Radenovic, A. 2010. Photoactivatable fluorescent protein mEos2 displays repeated photoactivation after a long‐lived dark state in the red photoconverted form. J. Phys. Chem. Lett. 1:1506‐1510.
   Bates, M., Blosser, T.R., and Zhuang, X. 2005. Short‐range spectroscopic ruler based on a single‐molecule optical switch. Phys. Rev. Lett. 94:108101.
   Bates, M., Huang, B., Dempsey, G.T., and Zhuang, X. 2007. Multicolor super‐resolution imaging with photo‐switchable fluorescent probes. Science 317:1749‐1753.
   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.
   Chudakov, D.M., Matz, M.V., Lukyanov, S., and Lukyanov, K.A. 2010. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90:1103‐1163.
   Dempsey, G.T., Bates, M., Kowtoniuk, W.E., Liu, D.R., Tsien, R.Y., and Zhuang, X. 2009. Photoswitching mechanism of cyanine dyes. J. Am. Chem. Soc. 131:18192‐18193.
   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.
   Huang, B., Wang, W., Bates, M., and Zhuang, X. 2008. Three‐dimensional super‐resolution imaging by stochastic optical reconstruction microscopy. Science 319:810‐813.
   Jones, S.A., Shim, S.‐H., He, J., and Zhuang, X. 2011. Fast, three‐dimensional super‐resolution imaging of live cells. Nat. Methods 8:499‐508.
   Patterson, G.H. 2011. Highlights of the optical highlighter fluorescent proteins. J. Microsc. 243:1‐7.
   Rust, M.J., Bates, M., and Zhuang, X. 2006. Sub‐diffraction‐limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3:793‐795.
   Shroff, H., Galbraith, C.G., Galbraith, J.A., and Betzig, E. 2008. Live‐cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5:417‐423.
   van de Linde, S., Löschberger, A., Klein, T., Heidbreder, M., Wolter, S., Heilemann, M., and Sauer, M. 2011. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protocols 6:991‐1009.
   Veatch, S.L., Machta, B.B., Shelby, S.A., Chiang, E.N., Holowka, D.A., and Baird, B.A. 2012. Correlation functions quantify super‐resolution images and estimate apparent clustering due to over‐counting. PloS One 7:e31457.
   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.
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