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3 September 2012

Singlet-Oxygen-Mediated Cell Death Using Spatially-Localized Two- Photon Excitation of an Extracellular Sensitizer

Authors: Frederico M. Pimenta,† Rasmus L. Jensen,† Lotte Holmegaard,† Tatiana V. Esipova,‡ Michael Westberg,† Thomas Breitenbach,† and Peter R. Ogilby*,†


Controlling and quantifying the photosensitized production of singlet oxygen are key aspects in mechanistic studies of oxygendependent photoinitiated cell death. In this regard, the commonly accepted practice of using intracellular photosensitizers is, unfortunately, plagued by problems that include the inability to accurately (1) quantify the sensitizer concentration in the irradiated domain and (2) control the local environment that influences light delivery and sensitizer photophysics. However, capitalizing on the fact that singlet oxygen produced outside a cell is also cytotoxic, many of these problems can be avoided with the use of an extracellular sensitizer. For the present study, a hydrophilic dendrimer-encased membrane-impermeable sensitizer was used to generate an extracellular population of singlet oxygen upon spatially localized two-photon irradiation. Through the use of this sensitizer and this approach, it is now possible to better control the singlet oxygen dose in microscope-based time- and space-resolved single cell experiments. Thus, we provide a solution to a limiting problem in mechanistic studies of singlet-oxygen-mediated cell death.