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

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca (BCa-1, BCa-2, and BCa-3), with absorption maxima (λ) at 350-358 nm, emission maxima (λ) at 464-466 nm, and TP action cross-section (Φδ) values of 55-70 × 10 cms/photon, in the presence of excess Ca at 750 nm. Moreover, the probes had dissociation constants of 0.18, 2.7, and 100 μM, respectively, which are appropriate values for sensing Ca 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 Ca in the cytoplasm, mitochondria, and plasma membranes of live cells and tissues. Additionally, we developed a green-emitting TP probe for H (FHEt-1) with λ = 359 nm, λ = 571 nm, and Φδ = 70 × 10 cms/photon at pH 4.3 in a universal buffer (0.1 M citric acid, 0.1 M KHPO, 0.1 M NaBO, 0.1 M tris[hydroxymethyl]aminomethane, and 0.1 M KCl); this probe could detect H in the lysosomes. Using BCa-1 and FHEt-1, it was possible to simultaneously monitor the changes in cytosolic Ca 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 Ca probes can be used to investigate the physiological role of Ca in cellular organelles as well as the crosstalk between Ca and other metal ions in specific organelles.