Fig. 4.

The optically cleared spheroid is mounted onto the sample holder (the edge of the sample holder is indicated by the white dashed line). Following image acquisition, an average projection of the image stack is computed. At the center of the spheroid, the intensity profile along a line orthogonal to the direction of the light sheet (orange line) is measured in each channel (i.e.: α-tubulin, β-catenin and DAPI). The DAPI signal is regarded as the ideal dispersion of the fluorophores in the spheroid. The intensity profiles are smoothed to reduce morphological variations. Finally, the smoothed grey values of the antibodies are plotted against the smoothed DAPI values and a linear regression is calculated. The red circles show the location of the highlighted values in the different representation modes. The output of this analysis is the Pearson’s R value. One and zero indicates that the antibody profile matches, respectively does not match, the DAPI profile.