Product Description
Singlet oxygen (¹O₂) is one of the Reactive Oxygen Species (ROS). ¹O₂ is known to be a cause of spots and wrinkles of the skin due to its very strong oxidizing potential. In the field of cancer research, ¹O₂ is of a particular importance because of its key role in photodynamic therapy (PDT), an emerging anticancer treatment using photoirradiation and photosensitizers. For the detection of ¹O₂, the existing singlet oxygen fluorescence detection reagent cannot be used in living cells because of its cell membrane impermeability.
Majima et al. synthesized a new far-red fluorescence probe composed of silicon-containing rhodamine and anthracene moieties, namely Si-DMA, 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⁽¹⁾,⁽²⁾. Among seven different ROS, Si-DMA is able to selectively detect the ¹O₂ (Fig. 3). In addition, Si-DMA is able to visualize the real-time generation of ¹O₂ from protoporphyrin IX in mitochondria with 5-aminolevulinic acid (5-ALA), a precursor of heme(Fig. 4).

No.	Sample	Instrument	Reference
1)	Cell(HeLa; RAW264.7)	Fluorescence microscope	S. Kim, T. Tachikawa, M. Fujitsuka, T. Majima, "Far-Red Fluorescence Probe for Monitoring Singlet Oxygen during Photodyanamic Therapy",　J. Am. Chem. Soc., 2014, 136 (33), 11707.
2)	Cell(CT26、TK6)	FCM	S. Bekeschus, A. Mueller, U. Gaipl, KD. Weltmann, "Physical plasma elicits immunogenic cancer cell death and mitochondrial singlet oxygen", TRPMS., 2017, 99, DOI:10.1109/TRPMS.2017.2766027.
3)	Cell(HeLa、HEK293)	Fluorescence microscope	Y. D. Riani, T. Matsuda, K. Takemoto and T. Nagai, "Green monomeric photosensitizing fluorescent protein for photo-inducible protein inactivation and cell ablation", BMC Biol, 2018, 16, 50.
4)	Cell(RIN-m5f)	Fluorescence microscope	T. Guo, T. Liu, Y. Sun, X. Liu, R. Xiong, H. Li, Z. Li, Z. Zhang, Z. Tian, and Y. Tian, "Sonodynamic therapy inhibits palmitateinduced beta cell dysfunction via PINK1/ Parkin-dependent mitophagy", Cell Death Dis., 2019, 10, 457.
5)	Cell(HepG2、NIH3T3)	Fluorescence microscope, FCM, Plate reader	K. Murotomi, A. Umeno, S, Sugino and Y. Yoshida. , "Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe", Sci Rep, 2020, 10, 10616.
6)	Cell (TIG-1 Cell)	Fluorescent Microscope	Y. Fujita, M. Iketani, M. Ito, I. Ohsawa, "Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts", 2022, doi:10.1016/j.exger.2022.111866.

Usage Example
Fluorescence microscopic detection of ¹O₂ in HeLa cells after added 5-aminolevulinic acid (5-ALA)

1. HeLa cells (2.4×10⁵ cells/ml, Hanks’ HEPES buffer 200 μl) were seeded on a μ-slide 8 well (Ibidi) and cultured at 37 ^oC in a 5%CO₂ incubator overnight.
2. Cells were washed with 200 μl Hanks’ HEPES buffer twice.
3. 150 μg/ml of 5-ALA in Hanks’ HEPES buffer was added to the μ-slide and cultured at 37 ^oC in a 5%CO₂ incubator for 4 hours.
4. Cells were washed with 200 μl Hanks’ HEPES buffer twice.
5. 200 μl of Si-DMA working solution (40 nmol/L) was added and cultured at 37 ^oC in a 5%CO₂ incubator for 45 minutes.
6. Cells were washed with 200 μl Hanks’ HEPES buffer twice.
7. 200 μl Hanks’ HEPES buffer was added and observe the cells under a fluorescence microscope.

Fluorescent of Si-DMA in 5-ALA-treated HeLa cells increased after 2.5 minutes irradiation. It was found that Si-DMA was able to visualize in real time the ¹O₂ generated from protoporphyrin IX in mitochondria.