Quantitative Analysis of Histological Staining and Fluorescence Using ImageJ

Abstract

An important objective for scientists is to statistically compare staining intensity (Fig. 1) or fluorescence (Fig. 2) for a particular marker between treatments or groups.Simply "eyeballing" an image and stating that a particular treatment or group is more densely stained or brightly fluorescent than another treatment or group is insufficient for scientific publications.Systems are available for image analysis in immunohistochemistry.However, many of these systems require expensive software and hardware attachments for acquisition, analysis, and storage of images.Therefore, an inexpensive and reliable alternative for image analysis is desirable.The cost effective answer for quantitative immunohistochemical analysis is ImageJ, developed by Wayne Rasband (http://imagej.nih.gov/ij/docs/index.html).ImageJ is a Java image processing and analysis program based on NIH Image for the Macintosh.ImageJ is available in the public domain (i.e., free).ImageJ runs on any computer that is a Java 1.5 or later virtual machine.Java runtime environments are available for free from Sun Microsystems or bundled with platform-specific installations of ImageJ (rsb.info.nih.gov/ij).Downloadable copies are available for Windows, Linux, and Mac OSX.For the evaluation of immunohistochemistry slides using ImageJ, images are captured onto the hard drive of the workstation computer.Thereafter, captured images are opened in NIH Image/ ImageJ for evaluating indices of positivity on immunohistochemistry slides, as well as fluorescence images.