Mark Wefers Bettink

Chapter 3 46 oxygen tension and oxygen consumption at the subcellular level in the mitochondria. This paper is the first description of this CE-marked device (Photonics Healthcare, Utrecht, The Netherlands). It provides the technical background, the construction of the device, and its use together with two examples of measurements in human skin. 2. Methods 2.1 Background of PpIX-TSLT Protoporphyrin IX (PpIX) is the final precursor of heme in the heme biosynthetic pathway and is synthesized inside the mitochondria [13]. The conversion of PpIX to heme in the mitochondria is a rate-limiting step. Therefore, administration of the porphyrin precursor 5-aminolevulinic acid (ALA) enhances the mitochondrial PpIX concentration [18]. Administration of ALA does not only enhance PpIX to detectable levels, but it also ensures mitochondrial origin of the delayed fluorescence signal [3, 6, 7]. Delayed fluorescence can be observed after pulsed excitation of PpIX as delayed luminescence with the same spectrum as prompt fluorescence (red light). In contrast to prompt fluorescence delayed fluorescence has a lifetime of tens to hundreds of microseconds [3]. Delayed fluorescence is the result of photon emission due to spontaneous relaxation of the excited triplet state via bi-directional intersystem crossing. Oxygen is a very effective quencher of the excited triplet state. In the process of quenching, energy is transferred to oxygen and PpIX relaxes to the ground state without emission of a photon. This causes the lifetime of the triplet state, and thus the lifetime of the emitted delayed fluorescence, to be oxygen-dependent. The delayed fluorescence lifetime is inversely proportional to the amount of oxygen according to the Stern-Volmer equation [8, 19] . With the assumption of a homogenous distribution of oxygen this relationship can be used to calculate the mitochondrial oxygen tension: (1) Where τ is the measured delayed fluorescence lifetime, τ 0 is the delayed fluorescence lifetime in the absence of oxygen (i.e. the lifetime of spontaneous relaxation), and k q is the quenching constant.