Quantitative Colocalization Analysis of Fluorescence Microscopy Images

Vadim Zinchuk1, Olga Grossenbacher‐Zinchuk2

1 Department of Neurobiology and Anatomy, Kochi University, Faculty of Medicine, 2 Institute of Anatomy, University of Bern, Bern
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.19
DOI:  10.1002/0471143030.cb0419s62
Online Posting Date:  March, 2014
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Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of fluorescence microscopy images with colocalization based on calculation of a number of specialized coefficients. Images of double‐stained sections are first subjected to background correction, and then various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of the results of calculations based on the use of linguistic variables are given. Success in colocalization studies depends on the quality of images analyzed, proper preparation of the images for coefficients calculations, and correct interpretation of results obtained. This protocol helps ensure reliability of colocalization coefficient calculations. Curr. Protoc. Cell Biol. 62:4.19.1‐4.19.14. © 2014 by John Wiley & Sons, Inc.

Keywords: quantitative colocalization; fluorescence microscopy; image analysis; data interpretation

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Quantitative Colocalization Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Quantitative Colocalization Analysis

  • 6‐ to 8‐µm‐thick cryostat sections of rat liver
  • Acetone
  • Blocking solution: 10% (v/v) goat serum in 0.1 M Tris‐buffered saline containing 0.1% Triton X‐100
  • Primary antibodies: anti‐Bsep (donated by Dr. Bruno Stieger, Department of Medicine, University of Zurich, Switzerland) and anti‐Mrp2 (Santa Cruz Biotechnology); primary antibodies should be raised in different species
  • Non‐immune IgG (control for specificity of immunostaining)
  • 0.1 M Tris‐buffered saline (TBS; appendix 2A)
  • Secondary antibodies with appropriate species specificity and labeled with fluorophores having different excitation spectra (e.g., Alexa 488 and Alexa 594; Molecular Probes); secondary antibodies should not cross‐react
  • Glycerol
  • Poly‐L‐lysine‐coated glass slides
  • Coverslips
  • Fluorescence microscope, including confocal (any brand) with appropriate oil‐immersion lens(es)
  • Argon‐krypton laser (any brand)
  • CoLocalizer Pro or CoLocalizer Express software (CoLocalization Research Software; http://www.colocalizer.com)
  • Additional reagents and equipment for immunofluorescence staining (unit 4.3) and fluorescence microscopy (units 4.2 & 4.5)
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Literature Cited

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  Yang, L., Lin, C., Liu, W., Zhang, J., Ohgi, K.A., Grinstein, J.D., Dorrestein, P.C., Rosenfeld, M.G. 2011. ncRNA‐ and Pc2 methylation‐dependent gene relocation between nuclear structures mediates gene activation programs. Cell 147:773‐788.
  Zinchuk, V. and Grossenbacher‐Zinchuk, O. 2009. Recent advances in quantitative colocalization analysis: Focus on neuroscience. Prog. Histochem. Cytochem. 44:125‐172.
  Zinchuk, V.S., Okada, T., Akimaru, K., and Seguchi, H. 2002. Asynchronous expression and colocalization of Bsep and Mrp2 during development of rat liver. Am. J. Physiol. Gastrointest. Liver Physiol. 282:G540‐G548.
  Zinchuk, O., Fukushima, A., Hangstefer, E., and Ueno, H. 2004. Dynamics of PAF‐induced conjunctivitis reveals differential expression of PAF receptor by macrophages and eosinophils in the rat. Cell Tissue Res. 317:265‐277.
  Zinchuk, O., Fukushima, A., Zinchuk, V., Fukata, K., and Ueno, H. 2005a. Direct action of platelet activating factor (PAF) induces eosinophil accumulation and enhances expression of PAF receptors in conjunctivitis. Mol. Vis. 11:114‐123.
  Zinchuk, V., Zinchuk, O., and Okada, T. 2005b. Experimental LPS‐induced cholestasis alters subcellular distribution and affects colocalization of Mrp2 and Bsep proteins: A quantitative colocalization study. Microsc. Res. Tech. 67:65‐70.
  Zinchuk, V., Wu, Y., Grossenbacher‐Zinchuk, O., and Stefani, E. 2011. Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations. Nat. Protoc. 6:1554‐1567.
  Zinchuk, V., Wu, Y., Grossenbacher‐Zinchuk, O. 2013. Bridging the gap between qualitative and quantitative colocalization results in fluorescence microscopy studies. Sci. Rep. 3:1365 doi:10.1038/srep01365.
Key References
  Manders et al., 1993. See above.
  First description of the correlation coefficient and examples of its use.
  Smallcombe, 2001. See above.
  Practical look at colocalization, some critical views, and advice for performing colocalization experiments.
  North, 2006. See above.
  Review with focus on proper interpretation of results from fluorescence microscopy studies, drawbacks, and limitations of biological imagery.
  Zinchuk and Grossenbacher‐Zinchuk, 2009. See above.
  Review describing applications of quantitative colocalization analysis in the field of neuroscience.
  Zinchuk et al., 2011. See above.
  A related protocol that also uses protein proximity index (PPI) to quantify colocalization.
  Zinchuk et al., 2013. See above.
  Interpretation of results from colocalization experiments based on the use of a fuzzy system model.
Internet Resources
  Website for CoLocalization Research Software, creators of CoLocalizer Pro and CoLocalizer Express software applications used for quantitative estimation of colocalization.
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