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Am J Physiol Cell Physiol 291: C781-C787, 2006. First published May 31, 2006; doi:10.1152/ajpcell.00067.2006
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METHODS IN CELL PHYSIOLOGY

In vivo oxygen imaging using green fluorescent protein

Eiji Takahashi,1 Tomohiro Takano,1 Yasutomo Nomura,2 Satoshi Okano,3 Osamu Nakajima,3 and Michihiko Sato4

1Department of Physiology, Yamagata University School of Medicine; 2Department of Environmental Life Science, Graduate School of Medical Sciences, Yamagata University; 3Research Laboratory for Molecular Genetics, Yamagata University; and 4Central Laboratory for Research and Education, Yamagata University School of Medicine, Yamagata, Japan

Submitted 9 February 2006 ; accepted in final form 16 May 2006

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.

heart; ischemia; hypoxia; molecular imaging


Address for reprint requests and other correspondence: E. Takahashi, Dept. of Physiology, Yamagata University School of Medicine, Yamagata 990-9585, Japan (e-mail: eiji{at}med.id.yamagata-u.ac.jp)






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