ABSTRACT
This chapter discusses the application of two-photon laser-scanning microscopy to monitor biosensor activity in living animals. Eukaryotic cells have spatial and temporal responses to extracellular changes that can be missed by many conventional endpoint assays, including immunostaining or western blotting. To monitor cell-signaling events in real-time, researchers developed genetically encoded fluorescent reporter probes herein referred to as biosensors. The directed evolution of the fluorescent proteins has produced an extensive palette of fluorescent proteins that emit across the visible spectrum. Wide-field microscopy has been used for intravital studies for nearly 200 years and to collect fluorescence images of the internal organs of living animals for nearly 100 years. A unique challenge of intravital microscopy of biosensors results from the attenuation of signals when imaging at increasing depth in biological tissues. One of the most difficult challenges confronting an investigator in studies of living animals is the startling amount of tissue motion.
