Two-photon phosphorescence lifetime imaging of cells and tissues using a long-lived cyclometallated Npyridyl^Cphenyl^Npyridyl Pt(ii) complex

Abstract

Using a combination of multiphoton excitation, confocal scanning, TCSPC and beam blanking in conjunction with a cyclometallated N_pyridyl^C_phenyl^N_pyridyl Pt(II) complex (1) with a long luminescence lifetime, we demonstrate lifetime mapping of living cells and histological tissue sections over a time-frame of 50 microseconds, using a laser on/off “beam blanking” approach. This method of performing phosphorescence lifetime imaging microscopy (PLIM) represents an order of magnitude enhancement of the two-photon time-resolved emission imaging microscopy (TP-TREM) method, where in order to achieve a longer imaging window, the excitation laser repetition rate was reduced by cavity dumping [Chem. Sci., 2014, 5, 879]. The method complements and expands other existing imaging methodologies by enabling simultaneous PLIM and FLIM (fluorescence lifetime imaging microscopy – recorded between beam blanking), whilst maintaining essential sub-micron spatial resolution. We demonstrate how the Pt(II) complex can be used to distinguish between cell nuclei and matrix proteins on the basis of emission lifetime, in both structured and homogeneous tissue sections; whilst also revealing how the Pt(II) emission lifetime varies with tissue matrix composition. The proposed imaging approach can be used in conjunction with any biocompatible emissive probe with a long emission lifetime – exemplified here by (1) – and for an array of fluorescent/phosphorescent labels, where discrimination is lifetime-based.