Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability

- Fluorescence imaging of Ca2+ is widely used for investigation of signaling pathways.
- Our red-fluorescent probe is highly membrane-permeable and practically useful.
- The probe could visualize cytoplasmic Ca2+ in living cells and brain slices.
- The probe will be applicable for multicolor imaging studies of biological mechanisms.

Fluorescence imaging of calcium ions (Ca2+) has become an essential technique for investigation of signaling pathways involving Ca2+ as a second messenger. But, Ca2+ signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca2+ and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca2+ that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca2+ concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca2+ red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.

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