High-resolution measurement of dissolved oxygen concentration in vivo using two-photon microscopy

Because oxygen is vital to the metabolic processes of all eukaryotic cells, a detailed understanding of its transport and consumption is of great interest to researchers. Existing methods of quantifying oxygen delivery and consumption are non-ideal for in vivo measurements. They either lack the three-dimensional spatial resolution needed, are invasive and disturb the local physiology, or they rely on hemoglobin spectroscopy, which is not a direct measure of the oxygen available to cells. Consequently, many fundamental physiology research questions remain unanswered. This dissertation presents our development of a novel in vivo oxygen measurement technique that seeks to address the shortcomings of existing methods. Specifically, we have combined two-photon microscopy with phosphorescence quenching oximetry to produce a system that is capable of performing depth-resolved, high-resolution dissolved oxygen concentration (PO2) measurements. Furthermore, the new technique allows for simultaneous visualization of the micro-vasculature and measurement of blood velocity. We demonstrate the technique by quantifying PO2 in rodent cortical vasculature under normal and pathophysiologic conditions. We also demonstrate the technique’s usefulness in examining the changes in oxygen transport that result from acute focal ischemia in rodent animal models.