Ratiometric Imaging for Quantification of Elevated Ca in Neurons Using Synthetic Low-Affinity Fluorescent Probe.

The availability of various calcium ion (Ca) fluorescent probes has contributed to revealing physiological events related to intracellular Ca. However, conventional probes face challenges for quantitatively and selectively visualizing high Ca concentrations in cells induced by any stimuli, including biomolecules or electrical signal that disrupt Ca homeostasis. In this report, we designed and synthesized a low-affinity ratiometric Ca probe, , utilizing -aminophenol--diacetate--methylene-methylphosphinate (APDAP) as a ligand, for which we recently demonstrated the suitability as a new low-affinity ligand for Ca. showed a blue shift in excitation wavelength with increasing Ca concentration based on the intramolecular charge transfer (ICT). Its affinity for Ca is lower than commercially available conventional Ca probes. Furthermore, the selectivity for Ca and the fluorescence intensity were considered sufficient to accurately detect Ca. The corresponding acetoxymethyl ester, , was synthesized for intracellular imaging and applied to Ca quantification in neurons. enabled quantitative ratiometric monitoring of the two-step Ca concentration increase induced by glutamate stimulation. While this two-step response was not clearly observed with a commercially available low-affinity ratiometric Ca probe, Fura-FF, has demonstrated the potential to quantitatively visualize the behavior of high Ca concentrations. The ratiometric low-affinity Ca probe, , is expected to be a powerful tool for discovering new functions of Ca in neurons.