RESUMO
Image contrast is often limited by background autofluorescence in steady-state bioimaging microscopy. Upconversion bioimaging can overcome this by shifting the emission lifetime and wavelength beyond the autofluorescence window. Here we demonstrate the first example of triplet-triplet annihilation upconversion (TTA-UC) based lifetime imaging microscopy. A new class of ultra-small nanoparticle (NP) probes based on TTA-UC chromophores encapsulated in an organic-inorganic host has been synthesised. The NPs exhibit bright UC emission (400-500â nm) in aerated aqueous media with a UC lifetime of ≈1â µs, excellent colloidal stability and little cytotoxicity. Proof-of-concept demonstration of TTA-UC lifetime imaging using these NPs shows that the long-lived anti-Stokes emission is easily discriminable from typical autofluorescence. Moreover, fluctuations in the UC lifetime can be used to map local oxygen diffusion across the subcellular structure. Our TTA-UC NPs are highly promising stains for lifetime imaging microscopy, affording excellent image contrast and potential for oxygen mapping that is ripe for further exploitation.
RESUMO
The DNA binding and cellular uptake of the lambda enantiomer of two bis-tetraazaphenanthrene (TAP) Ru(II) polypyridyl complexes containing either a linear dppn (1) or a hooked bdppz (2) benzodipyridophenazine ligand are reported, and the role of different charge-transfer states of the structural isomers in the photo-oxidation of guanine is explored. Both complexes possess characteristic metal-to-ligand charge-transfer (MLCT) bands between 400 and 500 nm and emission at ca. 630 nm in an aerated aqueous solution. Transient visible absorption (TrA) spectroscopy reveals that 400 nm excitation of 1 yields a dppn-based metal-to-ligand charge-transfer (MLCT) state, which in turn populates a dppn intraligand (3IL) state. In contrast, photoexcitation of 2 results in an MLCT state on the TAP ligand and not the intercalating bdppz ligand. Both 1 and 2 bind strongly to double-stranded guanine-rich DNA with a loss of emission. Combined TrA and time-resolved infrared (TRIR) spectroscopy confirms formation of the guanine radical cation when 2 is bound to the d(G5C5)2 duplex, which is not the case when 1 is bound to the same duplex and indicates a different mechanism of action in DNA. Utilizing the long-lived triplet excited lifetime, we show good uptake and localization of 2 in live cells as well as isolated chromosomes. The observed shortening of the excited-state lifetime of 2 when internalized in cell chromosomes is consistent with DNA binding and luminescent quenching due to guanine photo-oxidation.