Analysis of the individual steps in metastasis is crucial if insights at the molecular level are to be linked to the cell biology of cancer. A technical hurdle to achieving the analysis of the individual steps of metastasis is the fact that, at the gross level, tumors are heterogeneous in both animal models and patients. Human primary tumors show extensive variation in all properties ranging from growth and morphology of the tumor through tumor-cell density in the blood and formation and growth of metastases. Methods capable of the direct visualization and analysis of tumor-cell behavior at single-cell resolution in vivo have become crucial in advancing the understanding of mechanisms of metastasis, the definition of microenvironment, and the markers related to both. High-resolution multiphoton imaging of tumors in vivo is a valuable tool in this regard. Because tumor cells have been found to be attracted to blood vessels, the in vivo invasion assay was developed to analyze which factors may stimulate invasion of these cells into the vessels. This protocol describes the preparation and handling of collection needles for the assay. A set of 33-gauge needles is used to create artificial or surrogate blood vessels that are injected into tumors, using a special holding device attached to a micromanipulator to stabilize the needle positions during and after insertion into the anesthetized animal. The needles are filled with Matrigel and various growth factors to determine which of these factors may influence the invading tumor cells.