A user-friendly ImageJ macro for ratio imaging analysis of fluorescence microscopy images

Authors

• N Khattar
• JM Reece

Abstract

Here we present a beta version of an ImageJ macro for ratio imaging analysis. Ratio imaging is a common analysis technique to evaluate intracellular ion concentrations, such as Ca2+ and pH, as well as biomolecular sensors based on FRET (Fluorescence Resonance Energy Transfer). Specifically, this technique makes use of a fluorescent probe (or tandem probes) with either two distinct excitation or emission wavelengths. Thus, two emission images are acquired, corresponding to either the pair of excitation or emission wavelengths, depending on the fluorophore(s) used. The benefits of ratio imaging are twofold: the ratio of the images normalizes for fluorophore concentration and reduces instrumentation artifacts. Moreover, mathematical analyses can be used to calculate quantitatively the (molecular) concentration over time from the ratio fluorescence intensities. Although ImageJ has had the capability for ROI analysis of ratio images since its inception, there are no known macros or plugins that make such analysis user-friendly and practical. This macro will guide users to preprocess a pair of images through background subtraction and shading correction prior to taking a ratio of the images. Furthermore, the mean fluorescence intensities of chosen regions of interest (ROIs) for the input image pair, as well as for the corresponding ratio image, and optionally the calibrated concentration values, will be evaluated and plotted for each time point. Thus, this macro will not only produce ratio images with reduced artifacts and even illumination, but can additionally facilitate the analysis of changes in calibrated concentration over time.

Scientific Focus Area: Cell Biology

This page was last updated on Friday, March 26, 2021