Background and objectives: Mitochondria require oxygen to generate energy for essential cellular processes. An in-vivo non-invasive technique to monitor mitochondrial oxygen tension (mitoPO2) is the protoporphyrin IX-triplet state lifetime technique. Protoporphyrin IX (PpIX), one of the key components of heme, is naturally present in the mitochondria. Topical administration of 5-aminolevulinic acid hydrochloride (ALA) increases PpIX concentration in the skin. After excitation with a laser pulse, PpIX emits red light with an oxygen-dependent delayed fluorescence lifetime. This signal is detected by a photomultiplier and the mitoPO2 can be derived from this. A clinical device has been developed using this technique. Although this device has been proven feasible, improvements in reliability and practicality are needed. This work aims to investigate possible improvements to the components of this device for better application in a clinical setting. The main areas of improvement are the excitation wavelength, the detection of the signal, and the light source.
Excitation wavelength: First, wavelengths from 415 nm to 695 nm are explored as possible excitation wavelengths. Second, four excitation wavelengths that exhibit low noise are investigated in more detail. For a better comparison, the pulse energy per wavelength is equated. The excitation wavelengths 415 nm and 515 nm showed low noise, no oversaturation, and accurate fits. These two excitation wavelengths are then validated on three test subjects with skin types II, IV, and V on the Fitzpatrick scale. Oxygen-dependent lifetimes are obtained in all test subjects with an excitation wavelength of 415 nm.
Detection: A new detector is explored to replace the large and expensive photomultiplier. The detector is tested in a fluorescent solution and on human skin. The first experiments resulted in oversaturation of the detector. To prevent this, several combinations of band-pass, long-pass, and absorption filters are tested. Even with these filters, the detector was oversaturated by the light pulse from the laser.
Light source: Two light sources are investigated as possible replacements for the currently used laser. These were first tested in a 10 μM PpIX solution. No delayed fluorescence was observed because the light sources and detector could not be placed close enough to each other. Therefore, for the measurements in human skin, the light source was coupled into a fiber. Both light sources showed oxygen-dependent lifetimes, suggesting that they are suitable for this application. As a final step, related parameters were optimized to reduce oversaturation and noise. These are described in the Confidential Appendix.
Conclusion: The potential of 415 nm as an excitation wavelength is demonstrated, but these results should be validated in more test subjects. The light sources showed the potential to replace the current laser, which could reduce maintenance and increase reliability. The next step should be to combine the 415 nm excitation wavelength, light source, and detector into a prototype to explore their combined potential.