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1 Department of Physiology, Academic Medical Center, Amsterdam, The Netherlands
2 Laser Center, Academic Medical Center, Amsterdam, The Netherlands; Biomedical Technology Institute, University of Twente, Twente, The Netherlands
* To whom correspondence should be addressed. E-mail: c.ince{at}amc.uva.nl.
This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the non-linear two-photon effect.
Using a focused laser beam from a 1064 nm Q-switched laser, providing 10 ns pulses of 10 mJ, albumin bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime versus oxygen tension was performed. The obtained calibration constants were kq = 356 mmHgs-1 (quenching constant) and 
0 = 550 µs (lifetime at zero-oxygen conditions) at 37 °C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney.
It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g. non-invasive oxygen scanning in tissue at high spatial resolution. By our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen- dependent quenching of phosphorescence lifetime measurements.
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