Real-time imaging and quantification of adenosine triphosphate (ATP) fluctuation in cells are significant for understanding the relationship between energy metabolism and cell functions. However, few synthetic fluorescent probes have been reported to tackle this challenge due to lack of accurate fluorescence readout and suitable response concentration. Herein we designed and synthesized a ratiometric fluorescent probe (Rh6G–ACFPN) for quantitatively detecting the fluctuation of mitochondrial ATP in living cells. Rh6G–ACFPN selectively and reversibly responds to ATP with an ideal dissociation constant (K d) of 4.65 mM (3–10 mM: the range of mitochondrial ATP concentrations). Live-cell imaging allows us to directly monitor the dynamic changes of mitochondrial ATP in high temporal resolution. Moreover, for the first time, mitochondrial ATP in normal and cancer cells lines was successfully quantified and discriminated. These results demonstrate the versatility of Rh6G–ACFPN as a useful imaging tool to elucidate the function of mitochondrial ATP in living cells.

Two-photon imaging is an emerging tool for biomedical research and clinical diagnostics. Electron donor-acceptor (D-A) type molecules are the most widely employed two-photon scaffolds. However, current D-A type fluorophores suffer from solvatochromic quenching in aqueous biological samples. To address this issue, we devised a novel class of D-A type green fluorescent protein (GFP) chromophore analogues that form a hydrogen-bond network in water to improve the two-photon efficiency. Our design results in two-photon chalcone (TPC) dyes with 0.80 quantum yield and large two-photon action cross section (210 GM) in water. This strategy to form hydrogen bonds can be generalized to design two-photon materials with anti-solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed a sulfide probe based on TPC dyes and monitored endogenous H S generation and scavenging in the cirrhotic rat liver for the first time.

A simple and reversible fluorescent probe bearing 7-nitrobenz-2-oxa-1,3-diazole and a selenomorpholine fragment was designed and synthesized.

Tw o-photon imaging is an emerging tool for biomedical researcha nd clinical diagnostics.E lectron donoracceptor (D-A) type molecules are the most widely employed two-photon scaffolds.H owever,c urrent D-A type fluorophores suffer from solvatochromic quenching in aqueous biological samples.T oa ddress this issue,w ed evised an ovel class of D-A type green fluorescent protein (GFP) chromophore analogues that form ahydrogen-bond network in water to improve the two-photon efficiency.O ur design results in two-photon chalcone (TPC) dyes with 0.80 quantum yield and large two-photon action cross section (210 GM) in water.This strategy to form hydrogen bonds can be generalized to design two-photon materials with anti-solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed as ulfide probe based on TPC dyes and monitored endogenous H 2 Sg eneration and scavenging in the cirrhotic rat liver for the first time.