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.

Two-photon photosensitized production of singlet oxygen.

Singlet molecular oxygen (a(1)Delta(g)) has been produced and optically detected upon two-photon nonlinear excitation of a sensitizer with a focused laser beam. The experiments were performed using toluene solutions with either a substituted difuranonaphthalene or a substituted distyryl benzene as the sensitizer. The data indicate that the two-photon absorption cross sections of the difuranonaphthalenes are comparatively large and depend significantly on the functional groups attached to the chromophore. The time-resolved 1270 nm phosphorescence signals used to characterize the production of singlet oxygen are limited in much the same way as signals from other two-photon spectroscopic studies (e.g., weak signals that can be masked by scattered radiation). Nevertheless, the two-photon singlet oxygen signals also reflect the unique advantages of this nonlinear optical technique (e.g., depth penetration in the sample afforded by irradiation in a spectral region void of the more dominant one-photon linear transitions and spatial resolution afforded by irradiation with a focused laser beam).

Time-resolved detection of singlet oxygen in a transmission microscope.

The time-resolved absorption spectrum of singlet oxygen [O2(a1 delta g)-->O2(b1 sigma g+)] has been recorded in the region approximately 5100-5300 cm-1 from air-saturated polystyrene samples using a microscope attached to a step-scan Fourier transform IR spectrometer. Singlet oxygen signals were observed with a time resolution of approximately 160 ns from sample volumes of approximately 20 nL using moderate data-acquisition times. These data indicate that it is reasonable and worthwhile to consider the further development of a transmission microscope as a viable tool to create singlet oxygen images of inhomogeneous samples including samples of biological importance.