Here's a groundbreaking development in biosensor technology: a new green lifetime biosensor for calcium, named G-Ca-FLITS, has been engineered to maintain consistent brightness across its entire dynamic range. This innovation addresses a critical challenge in fluorescent biosensors, where one state is typically bright and the other dim, leading to significant differences in signal-to-noise ratios (SNR). The dim state's low SNR often compromises precise, quantitative measurements. G-Ca-FLITS, however, exhibits high brightness in both calcium-bound and unbound states, while still demonstrating a calcium-dependent lifetime change of over 1 ns. This sensor's brightness is comparable to the bright states of GCaMP3 and jGCaMP7c in mammalian cells, and its calcium-induced fluorescence intensity loss is minimal, at around 30%. Consequently, G-Ca-FLITS shows little variation in SNR when calcium levels change, making it an ideal tool for precise calcium measurements. But here's where it gets even more intriguing: G-Ca-FLITS also displays negligible sensitivity to pH within the physiological range, a trait inherited from its turquoise parent. Using fluorescence lifetime imaging (FLIM), researchers measured calcium concentrations with G-Ca-FLITS in various organelles, revealing transient and spatially heterogeneous calcium elevations in mitochondria of HeLa cells. And this is the part most people miss: the sensor's potential for two-photon FLIM in Drosophila brains was also evaluated, showcasing its versatility across different experimental systems. Controversially, while G-Ca-FLITS performs exceptionally in single-photon FLIM, its brightness under two-photon excitation in Drosophila neurons was surprisingly low, possibly due to lower expression or poor folding. This finding contrasts with its performance in other systems and raises questions about its optimal use in certain contexts. Thought-provoking question: Could the low brightness of G-Ca-FLITS in Drosophila brains be overcome by optimizing expression or folding, or is this a fundamental limitation of the sensor in this specific system? The discussion is open, and your insights are welcome in the comments!
