Intracellular Calcium Imaging (Part III): Practical Methods and Concepts (II) for Mouse in vivo Ca²⁺ Imaging

Abstract

Intracellular calcium (Ca²⁺) imaging in living animals has emerged as a powerful approach for examining how cellular activity is integrated into organ-level and whole-body physiology. Unlike ex vivo preparations, in vivo imaging preserves native tissue architecture, blood flow, autonomic input, endocrine regulation, and other systemic influences that shape cellular Ca²⁺ signals. This is particularly important in peripheral organs, although imaging these tissues remains technically challenging due to motion artifacts and optical scattering, which often obscure subtle fluorescence changes. Recent advances in genetically encoded Ca²⁺ indicators (GECIs), genetic targeting strategies, and optical and computational technologies have greatly expanded the feasibility of these experiments. In particular, ratiometric GECIs reduce motion-dependent artifacts and improve measurement reliability in moving tissues. Combined with transgenic strategies, they enable high-level, cell type-specific expression suitable for quantitative in vivo analysis. In this article, we outline the technical background and conceptual basis of mouse in vivo Ca²⁺ imaging, with emphasis on peripheral organs. As an example, we present our approach to in vivo Ca²⁺ imaging of mouse hepatocytes. This application illustrates both the technical challenges of imaging in moving visceral organs and the unique physiological insights obtainable only under intact in vivo conditions.