Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes

Abstract

This work explores time-resolved emission imaging microscopy (TREM) for noninvasive imaging and mapping of live cells on a hitherto uncharted microsecond time scale. Simple robust molecules for this purpose have long been sought. We have developed highly emissive, synthetically versatile, and photostable platinum(II) complexes that make TREM a practicable reality. [PtLCl], {HL = 1,3-di(2-pyridyl)benzene and derivatives}, are charge-neutral, small molecules that have low cytotoxicity and accumulate intracellularly within a remarkably short incubation time of 5 min, apparently under diffusion control. Their microsecond lifetimes and emission quantum yields of up to 70% are exceptionally high for transition metal complexes and permit the application of TREM to be demonstrated in a range of live cell types—normal human dermal fibroblast, neoplastic C8161 and CHO cells. [PtLCl] are thus likely to be suitable emission labels for any eukaryotic cell types. The high photostability of [PtLCl] under intense prolonged irradiation has allowed the development of tissue-friendly NIR two-photon excitation (TPE) in conjunction with transition metal complexes in live cells. A combination of confocal one-photon excitation, nonlinear TPE, and microsecond time-resolved imaging has revealed ( i ) preferential localization of the complexes to intracellular nucleic acid structures, in particular the nucleoli and ( ii ) the possibility of measuring intracellular emission lifetimes in the microsecond range. The combination of TREM, TPE, and Pt(II) complexes will be a powerful tool for investigating intracellular processes in vivo , because the long lifetimes allow discrimination from autofluorescence and open up the use of commonplace technology.