Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new methodologies for detection. MCTS provides a useful model that mimic in vivo tumour microenvironment with varied metabolic gradient (oxygen, pH, glucose and ATP). Emission quenching of phosphorescence compounds by O2 is becoming a wide spread approach for sensing oxygen by optical method within biological model. The approach depends on the correlation of the lifetime of the phosphorescent probe with O2 pressure. The aim is to study the cell penetration and oxygen measurement potential of a novel phosphorescent PtLsCl probe in 2D and 3D biological models using a high resolution 1 and 2-photon emission Phosphorescence Lifetime Imaging Microscopy (PLIM). Quantitative analysis of fluorescence emission intensity and lifetime within cellular compartment showed preferred accumulation of PtLsCl in nucleoli of cells. Immunohistology experiments by Hypoxyprobe™ suggested hypoxia within MCTSs were dependent upon culture condition of MCTS (i.e. size and culture days). Thereafter, emission lifetime detected by 1 or 2-photon PLIM showed marked differences across the 3D MCTS and the variation in lifetime was dependent upon culture condition (size and culture days) of MCTS, suggesting varied oxygen concentration. The distribution of emission lifetime of PtLsCl in whole spheroids ranged from 0 to 12 microseconds with phosphorescence lifetime imaging revealing three distinct lifetime-related oxygen areas. Thereafter, emission lifetime of PtLsCl in the whole melanoma tissue engineered model was analysed. Distribution emission ranged from 0 to 13 microseconds, with phosphorescence lifetime imaging revealing three distinct areas – 1) a normal stromal region of 0 to 4.0 μsec; 2) a spheroid border 4.0 to 6.0 μsec and 3) an inner core of MCTS 6.0 to 13.0 μsec. A marked deviation in lifetime of PtLsCl across the melanoma tissue engineered model clearly demarcated tumour area within normal stroma. It is proposed that the depletion of O2 is known to increase the emission lifetime of the PtLsCl label described herein, and PtLsCl incorporated with 1 or 2 PE-PLIM system demonstrated an excellent potential for high-resolution mapping of oxygen concentration in multi-cellular tissue models. Furthermore, both 1 and 2P-PLIM of a highly sensitive PtLsCl label has shown the potential to detect changes in partial O2 pressure and related response (e.g. necrosis).