We report an acedan-derived two-photon (TP) probe (1-AM) that can accurately measure the cytosolic magnesium in a living Hep3B cell. To determine intracellular magnesium concentration, a variety of membrane-permeable one-photon fluorescence (OPF) probes has been developed and some of them are commercially available. One of the major drawbacks of such probes is that the excitation wavelengths are in the range of 350-560 nm, which can cause damage to the substrates. To avoid such problem, two-photon microscopy (TPM) employing two NIR photons for the excitation has been developed. The advantages of TPM include reduced photodamage and photobleaching, and enhanced resolution due to the improved penetration depth, small absorption coefficient of light in tissue, and lower tissue auto-fluorescence. Recently, TP probes sensitive to the metal ions, pH, and fluoride ion have been developed. However, they have been studied in organic solvents or model membranes and membrane-permeable TP probe that is useful for TPM is rare.

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