We have created unique near-infrared (NIR)-emitting nanoscale metal-organic frameworks (nano-MOFs) incorporating a high density of Yb 3+ lanthanide cations and sensitizers derived from phenylene. We establish here that these nano-MOFs can be incorporated into living cells for NIR imaging. Specifically, we introduce bulk and nano-Yb-phenylenevinylenedicarboxylate-3 (nano-Yb-PVDC-3), a unique MOF based on a PVDC sensitizer-ligand and Yb 3+ NIRemitting lanthanide cations. This material has been structurally characterized, its stability in various media has been assessed, and its luminescent properties have been studied. We demonstrate that it is stable in certain specific biological media, does not photobleach, and has an IC 50 of 100 μg/mL, which is sufficient to allow live cell imaging. Confocal microscopy and inductively coupled plasma measurements reveal that nano-Yb-PVDC-3 can be internalized by cells with a cytoplasmic localization. Despite its relatively low quantum yield, nano-Yb-PVDC-3 emits a sufficient number of photons per unit volume to serve as a NIR-emitting reporter for imaging living HeLa and NIH 3T3 cells. NIR microscopy allows for highly efficient discrimination between the nano-MOF emission signal and the cellular autofluorescence arising from biological material. This work represents a demonstration of the possibility of using NIR lanthanide emission for biological imaging applications in living cells with single-photon excitation.uminescent reporters emitting in the near-infrared (NIR) region of the electromagnetic spectrum are highly advantageous for biological imaging applications for several reasons. Biological material has low autofluorescence in the NIR window, which allows facile discrimination between the desired signal of the reporter and the background, leading to an enhanced signalto-noise ratio and improved detection sensitivity (1). Additionally, NIR light scatters less than visible light, and therefore results in increased optical imaging resolution (2, 3). Finally, NIR photons interact less with biological material compared with visible photons, thus decreasing the risk of disturbing or damaging the biological systems being observed.NIR reporters, such as cyanine dyes (4, 5) and quantum dots (6), have previously been shown to be useful for biological imaging applications. However, these materials have broad emission bands that limit their ability to be easily discriminated from the background fluorescence. Additionally, cyanine dyes exhibit limited photostability and quantum dots can display blinking emission, making it difficult to conduct repeated or long-term experiments for such purposes as tracking a moiety or monitoring a process.Several lanthanide cations emit in the NIR and have some advantages with respect to organic fluorophores and semiconductor nanocrystals. Lanthanide cations have narrower emission bandwidths than organic fluorophores and semiconductor nanocrystals. Their emission wavelengths are not affected by the environment, allowing them to be used in a broad ra...

We report two prototype Ln(3+) complexes that address requirements for both MRI and luminescence imaging and we demonstrate that the presence of two H(2)O molecules bound to the Ln(3+), beneficial for MRI applications of the Gd(3+) analogue, is not a major limitation for the development of NIR luminescent agents.

In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology.

A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL)=17-19, with a high selectivity for lanthanides over Ca(2+), Cu(2+), and Zn(2+). The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex)(298)=7.7-9.3×10(6) s(-1)) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠)=7.2-8.8 cm(3) mol(-1)). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.

Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies.

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.

Transient absorption spectroscopy is a powerful tool for studying biological electron-transfer chains, provided that their members give rise to distinct changes of their absorption spectra. There are, however, chains that contain identical molecules, so that electron transfer between them does not change net absorption. An example is the chain flavin adenine dinucleotide (FAD)-W382-W359-W306 in DNA photolyase from E. coli. Upon absorption of a photon, the excited state of FADH* (neutral FAD radical) abstracts an electron from the tryptophan residue W382 in approximately 30 ps (monitored by transient absorption). The cation radical W382*+ is presumably reduced by W359 and W359*+ by W306. The latter two reactions could not be monitored directly so far because the absorption changes of the partners compensate in each step. To overcome this difficulty, we used linearly polarized flashes for excitation of FADH*, thus inducing a preferential axis in the a priori unoriented sample (photoselection). Because W359 and W306 are very differently oriented within the protein, detection with polarized light should allow us to distinguish them. To demonstrate this, W306 was mutated to redox-inert phenylalanine. We show that the resulting anisotropy spectrum of the initial absorption changes (measured at 10 ns time resolution) is in line with W359 being oxidized. The corresponding spectrum in wildtype photolyase is clearly different and identifies W306 as the oxidized species. These findings set an upper limit of 10 ns for electron transfer from W306 to W359*+ in wildtype DNA photolyase, consistent with previous, more indirect evidence [Aubert, C.; Vos, M. H.; Mathis, P.; Eker, A. P. M.; Brettel, K. Nature 2000, 405, 586-590].

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees . The catalytically potent state FADH- can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees , in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH degrees * <-- W382 <-- W359 <-- W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH degrees and leads to the formation of FADH- and a deprotonated tryptophanyl radical, most likely W359 degrees. These photoproducts are formed in less than 10 ns and recombine to the dark state in approximately 1 micros. These results support the electron hopping mechanism.