Unit

UNIT 4.27 Image Correlation Spectroscopy for Measurements of Particle Densities and Colocalization

  1. Benjamin Rappaz1,
  2. Paul W. Wiseman1,2

Published Online: 1 JUN 2013

DOI: 10.1002/0471143030.cb0427s59

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Rappaz, B. and Wiseman, P. W. 2013. Image Correlation Spectroscopy for Measurements of Particle Densities and Colocalization. Current Protocols in Cell Biology. 59:4.27:4.27.1–4.27.15.

Author Information

  1. 1

    Department of Physics, McGill University, Montreal, Quebec, Canada

  2. 2

    Department of Chemistry, McGill University, Montreal, Quebec, Canada

Publication History

  1. Published Online: 1 JUN 2013
  2. Published Print: JUN 2013

Abstract

Cells interact with their environment through receptor proteins expressed at their plasma membrane, and protein-protein interactions govern the transduction of signals across the membrane into the cell. Therefore, the ability to measure receptor densities and protein colocalization within the membrane of intact cells is of paramount importance. This unit describes a technique to extract these parameters from fluorescence microscopy images obtained using a commercial confocal laser scanning microscope (CLSM) and other similar types of microscopes. It is based on the analysis of spatial fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state via calculation of a spatial autocorrelation function, or used to measure particle colocalization via calculation of a spatial cross-correlation function from dual-color images of proteins tagged with two different fluorophores and imaged in two detection channels. These parameters offer key insights on the interaction of the cell with its environment. Curr. Protoc. Cell Biol. 59:4.27.1-4.27.15. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • fluorescence fluctuations;
  • image correlation spectroscopy;
  • particle density;
  • colocalization;
  • aggregation state;
  • cross-correlation