Far-Red Fluorescence Probe for Monitoring Singlet Oxygen during Photodynamic Therapy

Singlet oxygen (1O2), molecular oxygen in the lowest excited state, has a critical role in the cell-killing mechanism of photodynamic therapy (PDT). Although 1O2 phosphorescence measurement has been mainly used to monitor 1O2 formation during PDT, its intensity is far insufficient to obtain two-dimensional images of intracellular 1O2 with the subcellular spatial resolution using the currently available near-IR detector. Here, we propose a new far-red fluorescence probe of 1O2, namely, Si-DMA, composed of silicon-containing rhodamine and anthracene moieties as a chromophore and a 1O2 reactive site, respectively. In the presence of 1O2, fluorescence of Si-DMA increases 17 times due to endoperoxide formation at the anthracene moiety. With the advantage of negligible self-oxidation by photoirradiation (ΦΔ < 0.02) and selective mitochondrial localization, Si-DMA is particularly suitable for imaging 1O2 during PDT. Among three different intracellular photosensitizers (Sens), Si-DMA could selectively detect the 1O2 that is generated by 5-amino­levulinic acid-derived protoporphyrin IX, colocalized with Si-DMA in mitochondria. On the other hand, mitochondria-targeted KillerRed and lysosomal porphyrins could not induce fluorescence change of Si-DMA. This surprising selectivity of Si-DMA response depending on the Sens localization and photosensitization mechanism is caused by a limited intracellular 1O2 diffusion distance (∼300 nm) and negligible generation of 1O2 by type-I Sens, respectively. For the first time, we successfully visualized 1O2 generated during PDT with a spatial resolution of a single mitochondrial tubule.