Singlet Oxygen Generation by the Genetically Encoded Tag miniSOG

The genetically encodable fluorescent tag miniSOG is expected to revolutionize correlative light- and electron microscopy due to its ability to produce singlet oxygen upon light irradiation. The quantum yield of this process was reported as ΦΔ = 0.47 ± 0.05, as derived from miniSOG's ability to photooxidize the fluorescent probe anthracene dipropionic acid (ADPA). In this report, a significantly smaller value of ΦΔ = 0.03 ± 0.01 is obtained by two methods: direct measurement of its phosphorescence at 1275 nm and chemical trapping using uric acid as an alternative probe. We present insight into the photochemistry of miniSOG and ascertain the reasons for the discrepancy in ΦΔ values. We find that miniSOG oxidizes ADPA by both singlet oxygen-dependent and -independent processes. We also find that cumulative irradiation of miniSOG increases its ΦΔ value ∼10-fold due to a photoinduced transformation of the protein. This may be the reason why miniSOG outperforms other fluorescent proteins reported to date as singlet oxygen generators.