Hong, Seung Taek, Kim, Mun Seok, Kim, Bo Ra, Lee, Eun Jeong, Yoon, Yeo Uk, Paik, Kyu Cheol, Han, Man So, Kim, Eun Sun, and Cho, Bong Rae
In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca2+ (BCa-1 , BCa-2 mito , and BCa-3 mem), with absorption maxima (λ max) at 350–358 nm, emission maxima (λ fl) at 464–466 nm, and TP action cross-section (Φδ max) 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-1 lyso) with λ max = 359 nm, λ fl = 571 nm, and Φδ max = 70 × 10−50 cm4s/photon at pH 4.3 in a universal buffer (0.1 M citric acid, 0.1 M KH 2 PO 4 , 0.1 M Na 2 B 4 O 7 , 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 lyso , 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. [Display omitted] • We developed organelle-specific blue-emitting two-photon Ca2+ probes. • They can detect Ca2+ in organelles of living cells and tissues in real-time. • They have appropriate K d values for Ca2+ for each organelle. • They show high sensitivity, low pH dependency, and negligible cytotoxicity. • They can monitor the crosstalk among metal ions using multicolor TPM imaging. [ABSTRACT FROM AUTHOR]