Computational protocol: Quantitative assessment of angiogenesis and pericyte coverage in human cell-derived vascular sprouts

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Protocol publication

[…] We used an inverted microscope (Zeiss Axio Observer.Z1) with an automated stage to acquire multi-tile z-stacks (10-μm intervals) of stained sprouts, using a Zeiss 10×/NA0.45 objective lens. Both a Zeiss ApoTome system equipped with Colibri LED light source, as well as a Zeiss LSM 710 confocal microscope system were used for imaging. Images were acquired in grid positions and subsequently stitched using the Grid/Collection stitching plugin in Fiji [], resulting in a continuous image coverage of 70% of each well (3 × 3 mm). [...] Images were analyzed using the Fiji distribution of ImageJ [] at version ImageJ 2.0.0-rc-15/1.49k; Java 1.6.0_65 (available as a Life-Line Version from We developed a plugin for sprout analysis that is available from the Angiogenesis update site within ImageJ and is maintained to work with the newest version of Fiji (see for instructions how to install the plugin in Fiji). Documentation for our plugin is available at For each z-stack acquired, the maximum intensity projection (MIP) was created (see Additional file ) and processed using our plugin. For each 96-well plate, representative MIPs were used to adjust analysis parameters to improve image clarity across the entire dataset. Using the provided script (see Additional file ), the plugin was then run on every continuous image to consistently analyze the entire dataset. […]

Pipeline specifications

Software tools Stitching, ImageJ, Sprout Morphology
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Alzheimer Disease, Diabetes Mellitus