Synaptic localization of cadherin-9 at mossy fiber terminals with Colocalization Colormap

The association of cadherin-9 immunoreactivity with mossy fiber pathway was demonstrated with Colocalization Colormap (top panels). Moreover, single confocal plain of mouse brain section containing cadherin-9 immunoreactivity within mossy fiber boutons was analyzed with the plugin and nMDP distributions as well as Icorr value were calculated to assess the colocalization between cadherin-9 and mossy fiber terminal (bottom panels).

Simulated data

The proper algorithm implementation was tested on artificial images prepared to simulate complete colocalization, partial colocalization, and lack of colocalization. For that purpose, we have created an artificial spherical object based on a 3D intensity gradient (fig. 1a). The object was represented by a stack of images, that was first duplicated and then all the pixels in the duplicated stack were translated (fig. 1b). Complete colocalization was produced with translation by 0 pixels, whereas partial colocalization was produced with translation by 10, 20, or 30 pixels, and lack of colocalization (separation of the objects) was produced with translation by 40 pixels (fig. 1c, top panels). Reduced intensity correlation between pairs of corresponding pixels within generated images was observed proportionally to fewer overlap between objects (fig. 1c, middle panels). This set of data with dissimilarly colocalized spheres was then analyzed with Colocalization Colormap to calculate nMDPs distribution and Icorr values (fig. 1c, middle panels). The threshold value for the analysis of all translated objects was set as ‘118’ due to the fact that this was the minimum threshold value that assured complete separation of objects translated by 40 pixels (fig. 1c, bottom panels). As a result, we observed gradual decrease of Icorr values that was proportional to the decrease of colocalization between analyzed images. Icorr values were in range of 1 (for complete colocalization) to 0 (for lack of colocalization) (fig. 1c, bottom panels). Intermediate Icorr values for partial colocalization were as follows: 0.573 for 10-pixel translation, 0.132 for 20-pixel translation, and 0.055 for 30-pixl translation (fig. 1c, bottom panels). Decrease of colocalization between analyzed objects was also indicated by the loss of warm colors in corresponding colocalization maps due to reduced fraction of nMDP values with indexes above zero (fig. 1c, bottom panels).

In addition, the color map of duplicated stack was also inverted in order to generate pair of stacks containing negatively correlated intensities that simulate lack of colocalization while analyzed specimens overlap (exclusion of objects) (fig 1d). In this case we used the ‘autothreshold’ option for the analysis. Calculated Icorr value equaled 0 demonstrating lack of colocalization between objects. The negative global intensity correlation between stacks was reflected by color map of nMDPs distribution that did not contain any positively correlated pairs of corresponding pixel intensities (fig 1d, panels on the right).