Quantitative Colocalization Analysis of Fluorescence Microscopy Images

Abstract 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.