Abstract

Summary Understanding of NAD + metabolism provides many critical insights into health and diseases, yet highly sensitive and specific detection of NAD + metabolism in live cells and in vivo remains difficult. Here, we present ratiometric, highly responsive genetically encoded fluorescent indicators, FiNad, for monitoring NAD + dynamics in living cells and animals. FiNad sensors cover physiologically relevant NAD + concentrations and sensitively respond to increases and decreases in NAD + . Utilizing FiNad, we performed a head-to-head comparison study of common NAD + precursors in various organisms and mapped their biochemical roles in enhancing NAD + levels. Moreover, we showed that increased NAD + synthesis controls morphofunctional changes of activated macrophages, and directly imaged NAD + declines during aging in situ . The broad utility of the FiNad sensors will expand our mechanistic understanding of numerous NAD + -associated physiological and pathological processes and facilitate screening for drug or gene candidates that affect uptake, efflux, and metabolism of this important cofactor.