Nondestructive, serial in vivo imaging of a tissue-flap using a tissue adhesion barrier

Intravital Microscopy (IVM) is a powerful tool for imaging of dynamic events in living subjects and benefits from flexibility of various tissue preparation techniques. For example, a “tissue flap” (TF) approach initially affords high spatial resolution and physiological imaging with minimal tissue preparation, but serial TF imaging greatly increases the effects of pathological inflammation, resulting in postoperative adhesions and tissue injury. We took a materials science approach by implanting a commercially available, thin film, biopolymer tissue adhesion barrier (TAB) beneath the TF during serial imaging of the normal and developing breast in transgenic fluorescent mice, and with a fluorescent orthotopic mouse lymphoma model. We applied the TAB post-operatively beneath the TF to isolate the TF from the underlying peritoneum. When re-imaging the TF every 3–4 d, with a new TAB placed each time, we observed reduced hemorrhage, fibrous connective tissue and soft tissue damage. The presence of the TAB enabled sequential imaging of orthopically located EGFP+-lymphoma cells and associated vasculature at short intervals. In particular, it enabled visualization and tracking of the same individual fluorescent branches of the mammary gland in both adult and developing mice over time; likewise, it enabled tracking of lymph nodes. We conclude that this simple method affords great potential to serially track rare, microscopic, tissue-wide events in parenchyma or stroma. Potential applications include tracking proliferation and motility of transplanted cancer cells, stem cell-driven tissue growth, and tumor cell-stromal cell interactions at high spatial and temporal resolution.