Intracellular calcium ([Ca 2+ ]i) signaling regulates physiological functions in most cells. In secretory organs, such as the pancreas, salivary gland, and lacrimal gland (LG), [Ca 2+ ]i elevation in acinar cells triggers fluid secretion, which plays vital roles in the maintenance of functional health across the life- course. It is important to understand the secretory mechanism of secretory organs, but lack of analytic systems available for living animals limits the scope of research to gain

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... eper insights into the precise mechanism of secretion. We established an intravital imaging system for specific cell types of secretory organs to monitor the [Ca 2+ ]i changes using mouse line expressing Yellow Cameleon 3.60, a genetically encoded Ca 2+ indicator. Elevation of [Ca 2+ ]i in specific cell types of secretory organs could be monitored after cholinergic stimulation ex vivo and intravitally. We found that a marked attenuation of LG [Ca 2+ ] i response to cholinergic stimulation was induced under pathological conditions by postganglionic denervation. Intravital Ca 2+ imaging in secretory organs will broaden our understanding of the cellular mechanisms in animal models of secretory diseases. and photostability but the advantages of them are less sensitive to motion artifact and expression level differences because of rationing of two fluorescent proteins 13,14 . Previously, a transgenic mouse line conditionally expressing YC3.60 was established to monitor the long-term, spatiotemporal Ca 2+ signaling in living animals 13, 15 . This mouse line has been demonstrated to be useful in monitoring the Ca 2+ signaling in lymphoid tissues (spleen, Peyer's patches, and bone marrow) and intestinal gut epithelial cells 13, 15 . However, no studies have demonstrated intravital imaging of Ca 2+ signaling in specific secretory organs of YC3.60 transgenic mice. In this manuscript, we describe a useful and efficient visualization system to monitor the Ca 2+ signaling within secretory organs in a cell-type-specific manner using YC3.60 transgenic mice in combination with two-photon microscopy. This visualization system opens a wide range of new possibilities in the study of intravital secretory activities or behaviors. Results Localization of the YC3.60 probe in secretory organs isolated from YC3.60 transgenic mice. To determine the suitability of YC3.60 for Ca 2+ imaging in secretory organs, we examined the tissue morphology and distribution of YC3.60 expression in various secretory organs. There were no obvious differences in tissue morphology between YC3.60 and wild-type mice. (Fig. 1A left column) .