Oxygen Mapping of Melanoma Spheroids using Small Molecule Platinum Probe and Phosphorescence Lifetime Imaging Microscopy (original) (raw)

Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application. The use of metal complexes as dyes and probes for emission-based cellular imaging has developed into a vibrant area of research over the past decade 1-3. This emerging class of probes offers photo-physical properties that differ to a range of commercially available organic probes, and is enabling a new range of technologies to be explored. Notably, recent advances in optics and electronics, combined with the long emission lifetimes typical for transition metal complexes (from hundreds of nanoseconds to microseconds), has resulted in the emergence of multiphoton microsecond lifetime mapping techniques, such as Phosphorescence Lifetime Imaging Microscopy (PLIM) 4, 5 and Time-Resolved Emission Imaging Microscopy (TREM) 4. Luminescent transition metal complexes typically emit from a triplet excited state. Although transitions between states of a different spin are formally forbidden, intersystem crossing to the triplet state and subsequent relaxation to the ground state via phosphorescence are facilitated in transition metal complexes by the high spin orbit coupling constant associated with the heavy metal atom. The forbidden nature of the phosphorescence transition, results in a slow rate of emission; hence phosphorescence lifetimes are typically the order of hundreds of nanoseconds to microseconds. It is well documented that molecular oxygen quenches such triplet emitters and that the rate of quenching is dependent on oxygen concentration 5-10. One application of phosphorescent emitters in biological imaging is in oxygen detection, where more sensitive, non-invasive methods are in demand. The combination of phosphorescence quenching and high-resolution lifetime imaging is a powerful approach for non-invasive, real-time hypoxia detection and oxygen quantification. Oxygen quantification in biological systems is primarily focused on the use of large platinum and palladium porphyrins. These compounds display long emission lifetimes (typically 40-60 µs), which typically decreases