Submitted on February 9, 2006
Accepted on May 16, 2006
In vivo oxygen imaging using green fluorescent protein
Eiji Takahashi1*, Tomohiro Takano1, Yasutomo Nomura2, Satoshi Okano3, Osamu Nakajima4, and Michihiko Sato5
1 Physiology, Yamagata University School of Medicine, Yamagata, Japan
2 Yamagata, Japan; Environmental Life Science, Graduate School of Medical Sciences, Yamagata University, Yamagata, Japan
3 Research Laboratory for Molecular Genetics, Yamagata University, Yamagata, Japan
4 Research Laboratory for Molecular Genetics, Yamagata University, Yamagata, Japan; Yamagata, Japan
5 CLRE, Yamagata University School of Medicine, Yamagata, Japan
* To whom correspondence should be addressed. E-mail: eiji{at}med.id.yamagata-u.ac.jp.
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 molecular imaging of oxygen in in vivo organs using green fluorescent protein (GFP). Green fluorescent protein expressing COS7 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 a 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 knocked-in mouse (green mouse) heart. Then, we tried molecular imaging of hypoxia in in vivo organs. Using the macroscopic optics, the red shift could be imaged in the ischemic liver and kidney in the green mouse 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 molecular imaging of oxygen in in vivo organs.
