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INNOVATIVE METHODOLOGY

PO₂ measurements in the microcirculation using phosphorescence quenching microscopy at high magnification

Department of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 16 November 2007 ; accepted in final form 25 March 2008

In phosphorescence quenching microscopy (PQM), the multiple excitation of a reference volume produces the integration of oxygen consumption artifacts caused by individual flashes. We analyzed the performance of two types of PQM instruments to explain reported data on PO₂ in the microcirculation. The combination of a large excitation area (LEA) and high flash rate produces a large oxygen photoconsumption artifact manifested differently in stationary and flowing fluids. A LEA instrument strongly depresses PO₂ in a motionless tissue, but less in flowing blood, creating an apparent transmural PO₂ drop in arterioles. The proposed model explains the mechanisms responsible for producing apparent transmural and longitudinal PO₂ gradients in arterioles, a PO₂ rise in venules, a hypothetical high respiration rate in the arteriolar wall and mesenteric tissue, a low PO₂ in lymphatic microvessels, and both low and uniform tissue PO₂. This alternative explanation for reported paradoxical results of PO₂ distribution in the microcirculation obviates the need to revise the dominant role of capillaries in oxygen transport to tissue. Finding a way to eliminate the photoconsumption artifact is crucial for accurate microscopic oxygen measurements in microvascular networks and tissue. The PQM technique that employs a small excitation area (SEA) together with a low flash rate was specially designed to avoid accumulated oxygen photoconsumption in flowing blood and lymph. The related scanning SEA instrument provides artifact-free PO₂ measurements in stationary tissue and motionless fluids. Thus the SEA technique significantly improves the accuracy of microscopic PO₂ measurements in the microcirculation using the PQM.

oxygen consumption; oxygen tension; oxygen gradient