Ratio Imaging Analyzer (RIA): A Lightweight, Standalone Python Tool for Portable Ratiometric Fluorescence Analysis

Published in Zenodo, 2026

Abstract

Ratiometric fluorescence imaging stands as a cornerstone technique in modern quantitative biology. By measuring the ratio of fluorescence intensities at two distinct wavelengths, this method renders measurements independent of sensor concentration, optical path length, and uneven illumination, making it the “gold standard” for quantifying dynamic intracellular events. Its application spans from monitoring ion dynamics (e.g., Calcium, pH) to tracking essential metabolites (e.g., Tryptophan, DA, and ATP) using an expanding toolkit of genetically encoded biosensors.

Ratio Imaging Analyzer (RIA) is a lightweight, open-source desktop application designed to streamline the processing of such ratiometric imaging data. Unlike complex image processing libraries that require scripting skills, RIA bridges the gap between raw data and biological insight through a user-friendly Graphical User Interface (GUI). It empowers researchers to perform motion correction, dynamic background subtraction, interactive thresholding, and real-time Region of Interest (ROI) analysis on standard personal computers, facilitating rapid hypothesis testing and data exploration.

Check out the orginal paper.
Check out the GitHub Repositories Epivitae/RatioImagingAnalyzer