Spatially resolved two-photon irradiation of an intracellular singlet oxygen photosensitizer: correlating cell response to the site of localized irradiation.

The response of HeLa cells to subcellular spatially localized two-photon irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. Upon irradiation under these conditions, a localized population of PpIX excited states can be produced with meaningful intracellular spatial resolution; the dimensions of the domain where the incident light flux is high enough for PpIX two-photon absorption are defined by the microscope optics and by the diffraction of light (spot diameter at beam waist of ˜0.5-1.0 μm). In turn, the dimensions of the intracellular domain containing cytotoxic PpIX-sensitized singlet oxygen will likewise be confined. Most importantly, cell response (e.g., morphological signs of cell death) correlates with the light dose delivered and the intracellular domain irradiated. Thus, controlling light delivery can complement other techniques used to impart intracellular spatial localization in mechanistic studies of photoinitiated reactive oxygen species. Such controlled light delivery is also expected to be a particularly useful tool to study the so-called bystander effect in which a selectively-perturbed cell can influence a neighboring cell through intercellular signaling mechanisms.