The role of intracellular magnesium ions is of high interest in the fields of pharmacology andcellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg
2+, there isa strong desire for the development of optimized Mg
2+ fluorescent probes. In this paper we describe thedesign, synthesis, and cellular application of the three novel Mg
2+ fluorescent probes KMG-101, -103, and-104. The compounds of this series feature a charged
-diketone as a binding site specific for Mg
2+ anda fluorescein residue as the fluorophore that can be excited with an Ar
+ laser such as is widely used inconfocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent responsetoward Mg
2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants(
Kd,Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ionconcentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg
2+ measurements. Forintracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfullyincorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporatedcells, the resulting increase in the free magnesium ion concentration could be followed over time. By usinga confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorilyobserved.