Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical biological process in development, tissue repair, and various diseases such as cancer. The tube formation assay is a widely used in vitro model to study angiogenesis, where endothelial cells reorganize to form capillary-like structures on extracellular matrix substrates like Matrigel®. Monitoring and quantifying these structures through image analysis is pivotal for understanding mechanisms regulating angiogenesis and for screening potential therapeutic agents.

Image analysis tools provide objective, reproducible, and quantitative evaluation of angiogenic behaviors. These tools analyze microscopy images to extract meaningful morphometric parameters characterizing the degree of tube formation and vascular network complexity.

Key Quantitative Parameters

Typical image analysis of tube formation assays includes quantification of several critical parameters:

• Total tube length: the cumulative length of all endothelial cords formed.
• Number of branching points: intersections where tubes bifurcate.
• Number of loops: closed circular structures formed by tubes.
• Cell-covered area: percentage area covered by endothelial cells forming the network.

These parameters reflect the extent and complexity of angiogenesis and are used to assess the effects of pro- or anti-angiogenic factors.

Robust image analysis tools are essential in angiogenesis research to objectively quantify endothelial tube formation and vascular network properties. A range of software solutions, from open-source plugins to commercial packages, serve diverse experimental needs, enabling detailed morphometric characterization and supporting high-throughput drug screening and mechanistic studies in vascular biology.