Figure 10. The Fraction Mapper plugin.

Starting from a stack of two channels, Fraction Mapper calculates the fraction of the intensity in the first channel for every pixel (ch1/ch1+ch2), which can be obtained as a fraction map. A 2D plot of the intensities in each of the channels can also be generated. The plugin then colour-codes the numbers corresponding to the fraction according to a chosen LUT, and gives the pixels the light intensity of the sum of the two channels. The LUT used was based on six discrete colours from blue to red (LUT ‘6 colours 2,3,3,3,3,2’ available for download as Supplementary File 2). Bottom row: Starting from the 440nm and 490 nm channels, classically used for calculating GP, the picture obtained is much less informative. Of note, GP values are closely related to fraction values we use in this study: the GP can be obtained simply by multiplying the fraction by two and subtracting 1. Owen et al. have produced and published a ‘GP Calculator’ plugin ( Owen et al., 2011) that provides very similar results to those obtained with Fraction Mapper, albeit by a more indirect approach. The 10× magnifications correspond to the 52×52 pixels regions indicated by white squares in the adjacent image; each pixel is 100×100 nm and the whole pictures cover 52×52 microns.