Singlet oxygen phosphorescence detection in living adherent cells in vitro.

Abstract

Objective: Optical detection of singlet oxygen in cells affects the investigated cells and potentially, photosensitization skews the observed results, since the generated singlet oxygen undergoes chemical reactions with cellular quenchers. For this reason, the illumination intensity has to be kept low during a measurement to avoid killing and destruction of the investigated cells. The scope of this paper is to show, that scanning detection can meet both, sufficient sensitivity and low illumination dose, for adherent cells in 6-well plates.

Material and methods: The individual illumination dose for cells under investigation is reduced by distributing it among a large number of adherent growing cells. Adherent cell lines were investigated with regard to singlet oxygen production by means of simultaneous fiber-coupled irradiation at 635 nm and singlet oxygen phosphorescence detection. Singlet oxygen kinetics could be measured in two different cell types incubated with different photosensitizers, by scanning a major part of the area of a confluent layer of cells in a well.

Result: In each case, singlet oxygen signals kinetics could be observed and analyzed. Incubation with increasing amounts of photosensitizer did correlate to the determined signal amplitude and photosensitizer triplet decay times.

Conclusion: Focused excitation and observation in combination with scanning across the whole well results in successful determination of singlet oxygen kinetics from incubated adherent cells without detaching them. Further development is required to reduce artifacts, likely originating from scattered excitation light.