The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green(®) (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields of photosensitized singlet oxygen production, and can also lead to photooxygenation-dependent adverse effects in the system being investigated.

Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so-called "mini Singlet Oxygen Generator" (miniSOG), an FMN-containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (ϕΔ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron-transfer reactions (Type-I chemistry) at the expense of photosensitized singlet oxygen production (Type-II chemistry) and results in a comparatively poor yield of singlet oxygen (ϕΔ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well-characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.

Singlet oxygen, O(2)(a(1)Δ(g)), was produced upon pulsed-laser irradiation of an intracellular photosensitizer and detected by its 1275 nm O(2)(a(1)Δ(g)) → O(2)(X(3)Σ(g)(-)) phosphorescence in time-resolved experiments using (1) individual mammalian cells on the stage of a microscope and (2) suspensions of mammalian cells in a 1 cm cuvette. Data were recorded using hydrophilic and, independently, hydrophobic sensitizers. The microscope-based single cell results are consistent with a model in which the behavior of singlet oxygen reflects the environment in which it is produced; nevertheless, the data also indicate that a significant fraction of a given singlet oxygen population readily crosses barriers between phase-separated intracellular domains. The singlet oxygen phosphorescence signals reflect the effects of singlet-oxygen-mediated damage on cell components which, at the limit, mean that data were collected from dead cells and, in some cases, reflect contributions from both intracellular and extracellular populations of singlet oxygen. Despite the irradiation-induced changes in the environment to which singlet oxygen is exposed, the "inherent" intracellular lifetime of singlet oxygen does not appear to change appreciably as the cell progresses toward death. The results obtained from cell suspensions reflect key features that differentiate cell ensemble from single cell experiments (e.g., the ensemble experiment is more susceptible to the effects of sensitizer that has leaked out of the cell). Overall, the data clearly indicate that measuring the intracellular lifetime of singlet oxygen in a O(2)(a(1)Δ(g)) → O(2)(X(3)Σ(g)(-)) phosphorescence experiment is a challenging endeavor that involves working with a dynamic system that is perturbed during the measurement. The most important aspect of this study is that it establishes a useful framework through which future singlet oxygen data from cells can be interpreted.

Singlet oxygen, O2(a(1)Δg), the first excited electronic state of molecular oxygen, is an important reactive oxygen species. Its chemistry plays a role in processes ranging from polymer degradation to cell death. Although O2(a(1)Δg) is routinely produced through natural events, including photosensitized processes mediated by organic chromophores, the controlled and selective laboratory production of O2(a(1)Δg) remains a challenge, particularly in biological systems. Here we exploit the fact that ground-state oxygen, O2(X(3)Σg(-)), absorbs 765 nm light to selectively produce O2(b(1)Σg(+)) which, in turn, decays to O2(a(1)Δg). We have quantified this process in different solvents using the time-resolved 1275 nm O2(a(1)Δg) phosphorescence as an optical probe. Most importantly, 765 nm falls in the so-called "biological window", where endogenous chromophores minimally absorb. We show that femtosecond-laser-based, spatially resolved 765 nm irradiation of human tumor cells induces O2(a(1)Δg)-mediated cell death. We thus provide an accessible tool for the controlled sensitizer-free production and study of O2(a(1)Δg) in complex biological systems.

Singlet molecular oxygen, O(2)(a(1)Delta(g)), can be created in photosensitized experiments with sub-cellular spatial resolution in a single cell. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a(1)Delta(g)--> X(3)Sigma). Cellular responses to the creation of singlet oxygen can be monitored using viability assays. Time- and spatially-resolved optical measurements of both singlet oxygen and its precursor, the excited state sensitizer, reflect the complex and dynamic morphology of the cell. These experiments help elucidate photoinduced, oxygen-dependent events that compromise cell function and ultimately lead to cell death. In this perspective, recent work on the photosensitized production and detection of singlet oxygen in single cells is summarized, highlighting the advantages and current limitations of this unique experimental approach to study an old problem.