WMIC 2015: WIMIN Scholar Award Winner 4

Phosphorescent probes for in vivo two-photon microscopy of oxygen

Tatiana Esipova, University of Pennsylvania

The ability to quantify oxygen in vivo in 3D with high spatial and temporal resolution is invaluable for understanding of oxygen metabolism, delivery and consumption in normal and diseased tissues. An optical method based on oxygen-dependent quenching of phosphorescence is being developed, that allows quantitative minimally invasive real-time imaging of partial pressure of oxygen (pO2) in tissue.

In the past, dendritically protected phosphorescent oxygen probes with controllable quenching parameters and defined bio-distributions have been developed [1], and more recently our probe design strategy has been expanded on two-photon excitable oxygen probes [2]. These latter molecules comprise FRET-based antenna-core constructs, which brought about first demonstrations of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen in vivo, providing new valuable information for neuroscience [3-6] and stem cell biology [7]. However, current two-photon oxygen probes suffer from a number of limitations, such as low brightness and high cost of synthesis, which dramatically reduce imaging performance and limit usability of the method.

Here we present an approach to new bright phosphorescent chromophores with internally enhanced two-photon absorption cross-sections, which allow construction of antenna-free probes for 2PLM. In addition to substantial increase in performance, the new probes can be synthesized by much more efficient methods, thereby greatly reducing the cost of synthesis.

1. Vinogradov, S. A., and Wilson, D. F. Porphyrin-dendrimers as biological oxygen sensors, In Designing Dendrimers (Capagna, S., and Ceroni, P., Eds.), Wiley, New York (2012)
2. Finikova, O. S. et al. Oxygen microscopy by two-photon-excited phosphorescence. ChemPhysChem 9, 1673 (2008).
3. Sakadzic, S. et al. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue. Nature Methods 7, 755 (2010).
4. Lecoq, J. et al. Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nature Medicine 17, 893 (2011).
5. Devor, A. et al. “Overshoot” of O2 is required to maintain baseline tissue oxygenation at locations distal to blood vessels. J. Neuroscience 31, 13676 (2011).
6. Parpaleix, A., Houssen, Y. G. & Charpak, S. Imaging local neuronal activity by monitoring pO2 transients in capillaries. Nature Medicine 19, 241 (2013).
7. Spencer, J. A. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269 (2014).

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