In Vivo Imaging of Near-Membrane Calcium Ions with Two-Photon Probes

We report a two-photon (TP) probe (ACaLN) for near-membrane Ca2+ that shows a 13-fold TP excited fluorescence (TPEF) enhancement in response to Ca2+, dissociation constants (K-d(TP)) of (1.9 +/- 0.2) mu m, pH-insensitivity at the biologically relevant pH, and can detect near-membrane Ca2+ in live cells for more than 1500 s and in living tissues at 120 mu m depth without interference from other metal ions. Comparison with existing probes provides a useful strategy for the design of efficient TP probes for the near-membrane metal ions.