Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors

•A guide for selection of a fluorescent reporter for intravital microscopy.•Demonstration of adenoviral delivery of an intramolecular FRET biosensor.•A full protocol for an intravital skin flap imaging experiment in a xenograft.•Detailed protocols for FLIM-FRET imaging and analysis by intravital microscopy.•Microenvironmental imaging is demonstrated using oxygen-sensitive nanoparticles. Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays. Here we present a practical guide for the application of intravital microscopy, looking at the available fluorescent reporters and their respective expression systems and analysis considerations. Moving in vivo, we also discuss the microscopy set up and methods available for overlaying microenvironmental context to the experimental readouts. This enables a smooth transition into applying higher fidelity intravital imaging to improve the drug discovery process.