Cellular processes are regulated by the local concentrations of signaling molecules. To investigate these dynamic processes in detail, quantitative imaging of their absolute concentrations together with their spatiotemporal distributions is essential. Among fluorescence-based imaging methods, fluorescence lifetime imaging microscopy (FLIM) is particularly powerful since the fluorescence lifetime is a robust photophysical parameter. To exploit this advantage, FLIM-based biosensors that quantify molecular concentration through the alteration of the lifetime have been valuable. However, the development of FLIM biosensors remains technically challenging, particularly for those based on a single fluorescent protein (FP). Here, we present a platform for generating single-FP FLIM biosensor. Firstly, we engineered ATP-binding protein mutants inserted into Citrine, a yellow FP, using various linker designs. Screening for lifetime alterations led to the development of two green ATP biosensors: qMaLioffG (off-type) and qMaLionG (on-type), both showing a substantial fluorescence lifetime shift of more than 1.0 ns upon ATP binding. These biosensors enable quantitative ATP imaging across different cell types, including Drosophila brains. In a parallel effort, we developed FLIM biosensors for Ca 2+ , enabling quantification of its concentration in distinct subcellular and extracellular compartments such as the cytoplasm, mitochondria, endoplasmic reticulum, and extracellular milieu. Notably, in both ATP and Ca 2+ , biosensor developments, we extended this approach to generate different color variants, thereby facilitating multicolor FLIM imaging. In this presentation, we will highlight the applicability of our biosensors and discuss the molecular mechanisms underlying fluorescence lifetime alterations.
Arai et al. (Sun,) studied this question.