Microvascular and mitochondrial PO₂ simultaneously measured by oxygen‐dependent delayed luminescence

Abstract

Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO(2) values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO(2) (μPO(2) ) and mitochondrial PO(2) (mitoPO(2) ) in rats. The μPO(2) measurements are based on oxygen-dependent quenching of phosphorescence of the near-infrared phosphor Oxyphor G2. The mitoPO(2) measurements are based on oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber-based time-domain setup consisting of a tunable pulsed laser, 2 red-sensitive gated photomultiplier tubes and a simultaneous sampling data-acquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous μPO(2) and mitoPO(2) measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances.

Cutaneous mitochondrial respirometry: non-invasive monitoring of mitochondrial function

“…In case of a non-homogenous oxygen distribution inside the measurement volume, a reliable estimation of the average PO 2 can be made by the rectangular distribution method (RDM) [25,26]:…”

Vascular KATP channels mitigate severe muscle O2 delivery-utilization mismatch during contractions in chronic heart failure rats

“…As described previously (Behnke et al, 2001), this technique applies the Stern-Volmer relationship (Rumsey et al, 1988), which describes the quantitative O 2 dependence of the phosphorescent probe G2 via the equation: $${\text{PO}}_2\text{mv}=[(\tau °∕\tau )-1]∕({\text{k}}_{\text{Q}}\cdot \tau °)$$ where k Q is the quenching constant and τ and τ° are the phosphorescence lifetimes at the ambient O 2 concentration and in the absence of O 2 , respectively. For the phosphorescent probe G2, k Q is 273 mmHg −1 s −1 and τ° is 251 µs (Bodmer et al, 2012; Dunphy et al, 2002). These characteristics do not change over the physiological range of pH (~7.4) and temperature (~38°C) in vivo and, therefore, the phosphorescence lifetime is affected solely by the O 2 partial pressure.…”

Oxygenation measurement by multi-wavelength oxygen-dependent phosphorescence and delayed fluorescence: catchment depth and application in intact heart

“…OPO‐355: Opolette 355‐I, FDS: Fiber Delivery System, FOA: Fiber Optic Attenuator, BFP: Bifurcated Reflection Probe, FFC: Fiber to Fiber Coupling, BFA: Bifurcated Fiber Assembly, PCL: Plano Convex Lens, FS1 & FS2: Filter Sets, PES: Protective Electronic Shutter, MCP‐PMT: Micro Channel Plate Photomultiplier Tube, TEC: Thermo Electric Cooling, PMT‐MOD: Photomultiplier Module, AMP: Amplifier, DAQ: Data Acquisition. As earlier published .…”

“…Phosphorescence and delayed fluorescence have much in common and are both a type of oxygen‐dependent luminescence. The technical feasibility to combine phosphorescence‐based µPO 2 measurements with delayed fluorescence‐based mitoPO 2 measurements, by means of a dual‐dye technique, has been demonstrated . A next logical step in the evolution of the technology would be the exploration of the usefulness of multi‐wavelength excitation in combination with this dual‐dye approach.…”