Journal of Applied Physiology
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J Appl Physiol 97: 1962-1969, 2004. First published July 9, 2004; doi:10.1152/japplphysiol.01399.2003
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INNOVATIVE METHODOLOGY

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements

Egbert G. Mik,1 Ton G. van Leeuwen,2,3 Nicolaas J. Raat,1 and Can Ince1

1Department of Physiology and 2Laser Center, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam; and 3Biophysical Engineering, Biomedical Technology Institute, University of Twente, 7522 Enschede, The Netherlands

Submitted 29 December 2003 ; accepted in final form 7 July 2004

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 nonlinear two-photon effect. By using a focused laser beam from a 1,064-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 vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr–1·s–1 (quenching constant) and {tau}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., noninvasive oxygen scanning in tissue at high spatial resolution. To 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.

phosphorescence quenching; oxygen measurement; palladium porphyrin; tissue oxygenation


Address for reprint requests and other correspondence: C. Ince, Dept. of Physiology, Academic Medical Center, Univ. of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (E-mail: c.ince{at}amc.uva.nl).




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