Botchway, S. W. et al. Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes. Proc. Natl Acad. Sci. USA 105, 16071-16076

Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Oxfordshire OX11 0QX, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2008; 105(42):16071-6. DOI: 10.1073/pnas.0804071105
Source: PubMed


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.

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    • "Here we will focus exclusively on the use of such probes for two-photon imaging. In 2008 the group of Williams reported on a simple phenyl-bipyridine cyclometalated platinum complex (complex 4 in Figure 1A), which showed intense phosphorescence (f em =0.75) along with a low two-photon absorption crosssection [15]. The complex was found to accumulate selectively in the cell nuclei, supposedly favored through binding with DNA. "
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    • "When applied in real biological samples, the fluorescent probes often suffer from the interference from autofluorescence of biosamples and scattered excitation light, reducing the signal-to-noise ratio (SNR). To avoid such an interference, one effective solution is to use time-gated luminescence imaging based on long-lifetime emissive materials as probes [9] [10]. The advantage of measuring the lifetime of photoluminescence emission is that this parameter is directly dependent upon excited-state reactions but independent of compound concentration, light intensity and photobleaching, so it is an ideal parameter for monitoring the local environment of the materials. "
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    • "For O 2 sensing, Ru(II) complexes show many advantages (Watts and Crosby 1971, Bacon and Demas 1987, Castellano and Lakowicz 1998). Lifetime measurements (Draaijer et al 1995, Periasamy et al 1996, Morris et al 2007) obviate or minimize some of the deficiencies of intensity determinations especially with respect to environmental influences (concentration of fluorophore, variation in excitation intensity, collection efficiency, propinquity to hydrophobic proteins or membranes, gradients of pH or ionic strength, etc (Zhong et al 2003, Botchway et al 2008)). Encapsulation in porous nanoparticles goes a further stage to making reliable intracellular assessments (Clark et al 1999, Buck et al 2004, Poulsen et al 2007, 2008a, 2008b). "
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