Organelle-specific blue-emitting two-photon probes for calcium ions: Combination with green-emitting two-photon probe for simultaneous detection of proton ions

Abstract

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca2+ (BCa-1, BCa-2mito, and BCa-3mem), with absorption maxima (λmax) at 350–358 nm, emission maxima (λfl) at 464–466 nm, and TP action cross-section (Φδmax) values of 55–70 × 10−50 cm4s/photon, in the presence of excess Ca2+ at 750 nm. Moreover, the probes had dissociation constants of 0.18, 2.7, and 100 μM, respectively, which are appropriate values for sensing Ca2+ in the cytoplasm, mitochondria, and plasma membrane, respectively. The measurements were conducted using a calcium calibration buffer (10 mM 3-[N-morpholino]propanesulfonic acid and 100 mM KCl) at pH 7.2. The TP microscopy results revealed that the probes could facilitate the real-time detection of Ca2+ in the cytoplasm, mitochondria, and plasma membranes of live cells and tissues. Additionally, we developed a green-emitting TP probe for H+ (FHEt-1lyso) with λmax = 359 nm, λfl = 571 nm, and Φδmax = 70 × 10−50 cm4s/photon at pH 4.3 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M tris[hydroxymethyl]aminomethane, and 0.1 M KCl); this probe could detect H+ in the lysosomes. Using BCa-1 and FHEt-1lyso, it was possible to simultaneously monitor the changes in cytosolic Ca2+ and lysosomal H+ concentrations in live cells and tissues using dual-color TP microscopy in real time. When used with TP probes emitting wavelengths of green light or longer, these blue-emitting Ca2+ probes can be used to investigate the physiological role of Ca2+ in cellular organelles as well as the crosstalk between Ca2+ and other metal ions in specific organelles.