Chapter 24: Overview of Detection Methods

The investigation of the involvement of singlet oxygen (¹O₂) in many important processes in Biology, Materials Science, Chemistry and Medicine demands its efficient and specific detection and quantification. Due to its highly reactive nature, ¹O₂ has only one direct method of determination, namely the detection of its phosphorescence emission at 1270 nm. As discussed in Chapters 25 and 26, time-resolved phosphorescence detection (TRPD) of ¹O₂ not only allows the confirmation of its presence, but also the study of its formation and decay kinetics. The determination of kinetic parameters of ¹O₂ with time-resolved studies is indeed crucial to characterize its reactivity. In turn, these studies also provide information about the precursors of ¹O₂.

However, the phosphorescence emission of ¹O₂ is extremely weak, which requires very sensitive NIR detectors that are not always available in all laboratories. Therefore, indirect methods that typically rely on a ¹O₂ trap have been developed and extensively used, even if there are some drawbacks regarding their specificity and crossreactivity with other ROS. Chapter 29 discusses the most relevant characteristics of classical ¹O₂ chemical traps. Many of these compounds constitute the building blocks to create more complex reporters such as those based on fluorescence (Chapter 30) and spin detection (Chapter 31). The analysis of specific reaction products with biomolecules (e.g. lipids and nucleic acids) is also a useful method to ascertain the participation of ¹O₂ in a given process, which can be aided by isotopic labeling (Chapter 32).

A conceptually different indirect method relies on the detection of the ¹O₂-sensitized delayed fluorescence (SOSDF) of the photosensitizer (Chapter 28). While this technique is rather complex and not yet mainstream, it does allow determining kinetic information, even with spatial resolution in a time-resolved fluorescence imaging experiment. Indeed, imaging the production of ¹O₂ is a goal that has long been pursued, either by detecting the weak ¹O₂ phosphorescence or by the use of fluorescent reporters (Chapter 27). Increasing the sensitivity, specificity, spatial and temporal resolution of ¹O₂ imaging in biological systems is probably one of the major challenges in the field.

Another challenge is the quantification of absolute doses of ¹O₂, which is especially relevant in photodynamic therapy. The importance of dosimetry as well as the difficulties associated with the different techniques are discussed in Chapter 33.

The choice of technique for detecting ¹O₂ will depend on a number of factors. For example, the lack of biological compatibility of some chemical traps and fluorescent probes rules out these methods for their use in (living) cells. On the other hand, fluorescent probes may be useful when the amounts of ¹O₂ are very small, as the fluorescent product can accumulate and photodetectors are very sensitive in the visible range. If time resolution is required, only TRPD and SOSDF are suitable.

The editors are aware that other techniques have been used for studying ¹O₂ processes that are not covered in this book. Among them, photothermal techniques, particularly laser-induced optoacoustic spectroscopy¹  and photothermal lensing²  have been used in different laboratories but their lack of specificity and the specialized equipment needed makes them less-popular options. The interested reader may find useful information in the references quoted at the end of this chapter. In conclusion, Chapters 25–33 provide the technical details, a critical evaluation of strengths and weaknesses, and some examples of applications that will help the readers to choose the most suitable ¹O₂ detection method for their particular sample.