Prifret: An ImageJ plugin for pixel based ratio imaging

A pixel based ratio imaging plugin for ImageJ is developed for image series using ratiometric FRET probes such as Cameleon. The use of a control image with red fluorescent protein (RFP) as described in Henquet et all 2016, is implemented. Furthermore, image series can be thresholded (automatic or manually) and normalized on a the first x images from the serie. Measured features include two types of ratio calculations: “sample” or whole image ratio and “sample image based” where the images are ratioed pixel by pixel. Furthermore, a standard deviation, standard error, image size (after thresholding) and peak area are included.

For cellular imaging the use of ratio metric FRET probes have become widely used. Typically, measurements result in series of CFP and YFP fluorescence emission images over a certain time frame. To track activation of a probe, the CFP and YFP fluorescence are typically ratioed against each other. Basically, a region of interest is determined and the average intensity over that region for YFP emission is divided over the CFP emission. However, this results in systematic under representation of the maximum ratio. In the prifret plugin we have designed, a pixel based ratio imaging that determines the ratio of each individual pixel before the pixels are averaged. This will result in higher and more significant ratio values. The pixel-based YFP/CFP ratio is calculated according to the following equation:


Where: f is the YFP/CFP ratio as a function of timestep t; y and c are the pixel values of the YFP and CFP images respectively. One of the images YFP or CFP, selected by the user, is thresholded, taking only values larger than q into account. The threshold value can be set by the user or calculated automatically, where it optimizes for a maximum f(t) ratio. The function is normalized by subtracting the average YFP/CFP ratio over the first n timesteps.
The combination with a control image makes this plugin particularly interesting for image series where the control cells are tagged with a red fluorescent protein. The plugin is able to filter a set of images against a control image and produce output data and charts for both sample and control within the same experiment. This provides unique opportunities to combine a control measurement direct with the sample measurement.

The plugin output provides the user with a graphical representation of the ratio obtained during the given time serie of images for both the sample (blue line) and control (red line) cells. If no control image is provided, only a sample graph and output will be generated. The output data set includes all parameters such as normalization and threshold settings as well at whole image and pixel based ratio outputs.


Availability - Software available soon

The software is available under a non-profit as well as under a commercial license. Details can be found in the license file after clicking the download button.