We established an intravital microscopy-based system to study actin cytoskeleton dynamics in secretory granules during regulated exocytosis in salivary glands and exocrine pancreas. Using transgenic mouse models that express selected fluorescently labeled molecules, we discovered several key aspects of exocytosis that were not seen in ex vivo models. Here we present data suggesting that tropomyosins (Tpms), which form co-polymers along the length of actin filaments, modulate the actomyosin scaffold post-fusion, and thus control the membrane integration phase of exocytosis. We found that upon initiation of granule fusion Tpm4.2 and Tpm3.1 are recruited onto the fused secretory granules together with F-actin, but with different kinetics to that of bulk actin filaments. Genetic ablation of Tpm3.1 or exposure to an anti-Tm compound altered the kinetics of granule exocytosis, but did not prevent the completion of granule exocytosis. More recently, we developed an intracellular intravital microscopy approach to quantitate GLUT4 trafficking events in vivo to investigate the role of the actin cytoskeleton in GLUT4 exocytosis in skeletal muscle. We employed a novel dual colour GLUT4 probe comprised of pH-sensitive pHluorin inserted into the first exofacial loop of GLUT4 and tdTomato at the C-terminus. This allows the simultaneous tracking of GLUT4 fusion events (bursts of green pHluorin signal) and GLUT4 vesicle movement (red tdTomato signal). We electroporated mouse skeletal muscle with rGLUTpHluor and detected for the first time GLUT4 vesicle fusion events in live anaesthetised animals. We recorded GLUT4 fusion events both at the T-tubules and sarcolemma that increased in frequency after insulin stimulation. To our knowledge this is the first report to capture sub-diffraction-sized vesicle fusion events in a living mammal and may serve as a powerful approach to study exocytosis in mouse models.