Intravital FLIM Pharmacodynamic Imaging Using FRET Biosensors

When studying the effectiveness of targeted anti-tumor therapy, measuring in vivo protein activity within the cancer cells in real-time is vital. This however presents challenges as post-translational modifications or binding of cofactors, and their dynamics, cannot be captured at the single cell level with traditional methods. The work of the last two decades led to the development of a broad range of genetically encoded FLIM-FRET biosensors that can be used to study protein activity, pharmacodynamics and -kinetics in real-time at the single cell level in the in vitro or in vivo setting. In this chapter, we will introduce FLIM-FRET biosensors for RAC1 and AKT activity, two key modulators of carcinogenesis. We will discuss their application in studying the effectiveness of anti-tumor therapy in vivo using intravital microscopy. Furthermore, we describe how to visualize RAC1 and AKT activity in vivo using these FLIM-FRET biosensors in combination with intravital imaging through imaging windows. Finally, we included a detailed description of the subsequent quantitative analysis of the readout of these biosensors in the in vivo context to dynamically evaluate the effectiveness of targeted anti-cancer therapies.