RatioImg -- Tools for Ratiometric & Single-Wavelength Imaging Analysis

Ion ratio imaging is a relatively new technique for measuring either absolute or relative changes in ion concentration within living cells. This is accomplished by measuring the fluorescence emission of special dyes which have been designed to change their spectral properties or emission intensities upon binding to the ion of interest.

RatioImg is a suite of Igor Pro 3.13 procedures for processing and analyzing data from ratiometric imaging experiments (i.e., A and B wavelength measurements of multiple ROIs); it was originally developed for use with the sodium indicator dye SBFI. RatioImg can handle single-wavelength or already-ratioed data; it can perform background subtractions and can correct ratio values based on in situ fluorescence measurements using ionophores and known concentrations of the ion in the perfusion bath. For single-wavelength dyes, it can calculate and display F/Fo or delta_F/F. Data from each cell (ROI) can be browsed and displayed in a graph window, graphs can be copied to a notebook and annotated, and a variety of measurements can be performed.

Detailed on-line help is included along with complete documentation in pdf format. There are also two sample data files for use with the documentation's tutorial section and an experiment file showing examples of annotation and measurement.

The Macintosh or Windows versions of RatioImg can be downloaded using one of the following links:



We have tested RatioImg on a Mac PPC 6400/200 with OS 8.1, and on a Dell Optiplex GXM 5166 with Win95. There are slight differences between the Mac (.sit) and Windows (.zip) archives due to mostly cosmetic OS differences.


Developed by:

Howard Rodstein

Mark Rand
Dept. of Neurology
University of Washington, Seattle




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