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

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.