Fig. 3.

Configuration of the Sprout Analyzer plugin. a Dialog to configure output measurement parameters and to define input configuration, i.e., which image channel contains which staining information. b Example image showing a microcarrier bead with vascular sprouts stained for F-actin using phalloidin-AF488 (green) and nuclei using DAPI (blue). c Example image with EC/pericyte-derived sprouts stained for F-actin using phalloidin-AF488 (green), nuclei using DAPI (blue), EC nuclei using an anti-Erg-1/2/3 antibody (white), and pericytes using an anti-NG2 antibody (red). d Dialog to configure bead detection; when the preview checkbox is activated, a bead mask image (white) is overlaid onto the image (e and f for the original images in b and c, respectively). g Dialog to configure sprout detection; when the preview checkbox is activated, a sprout mask image (white) is overlaid onto the image (h, i). j Dialog to configure nucleus detection; when the preview checkbox is activated, a nucleus mask image (white) is overlaid onto the image (k, l). m Dialog to configure cell classification; when the preview checkbox is activated, a preview showing ECs (yellow) and pericytes (magenta) is overlaid onto the image (n). o Dialog to configure pericyte coverage measurement; when the preview checkbox is activated, a pericyte area mask image (white) is overlaid onto the image (p). For detailed instructions, see protocol in Additional file 3, Additional file 1: Video S1, and Additional file 2: Video S2