Anne-Sophie Chauvin

New Opportunities for Lanthanide Luminescence

Trivalent lanthanide ions display fascinating optical properties. The discovery of the corresponding elements and their fist industrial uses were intimately linked to their optical properties. This relationship has been kept alive until today when many high-technology applications of lanthanide-containing materials such as energy -saving lighting devices, displays, optical fibers and amplifiers, lasers, responsive luminescent stains for biomedical analyses and in cellulo sensing and imaging heavily rely on the brilliant and pure-color emission of lanthanide ions. In this review we first outlined the basics of lanthanide luminescence with emphasis on f-f transitions, the sensitization mechanisms, and the assessment of the luminescence efficiency of lanthanide-containing emissive molecular edifices. Emphasis was then put on two fast developing aspects of lanthanide luminescence: materials for telecommunications and light emitting diodes, and biomedical imaging and sensing. Recent advances in NIR- emitting materials for plastic amplifiers and waveguides were described, together with the main solutions brought by researchers to minimize non-radiative deactivation of excited states. The demonstration in 1999 that erbium tris (8-hydroxyquinolinate) displayed a bright green emission suitable for orgenic light emitting diodes (OLEDs) was followed by realizing that in OLEDs, 25% of the excitation energy leads to singlet states and 75% to triplet states. Since lanthanide ions are good triplet quenchers, they now also play a key role in the development of these lighting devices. Luminescence analyses of biological molecules are among the most sensitive analytical techniques known. The long lifetime of the lanthanide excited states allows time- resolved spectroscopy to be used, suppressing the sample autofluorescence and reaching very low detection limits. Not only visible lanthanide sensors are now ubiquitously provided in medical diagnosis and in cell imaging, but the feasibility of using NIR emission of ions such as Ybm is now being tested because of deeper penetration in biological tissues.

Europium and Terbium tris(Dipicolinates) as Secondary Standards for Quantum Yield Determination

Abstract Aqueous solutions of europium(III) and terbium(III) tris(dipicolinates), around physiological pH are shown to be convenient secondary standards in the determination of quantum yields of lanthanide complexes containing these ions. Conditions for which a strict linearity is observed between the concentration of the solutions and the emission intensity are established. The speciation in these solutions, which contain a non‐negligible amount of bis species is presented and discussed on the basis of both stability constants and lifetime determinations of the Eu(5D0) level. The quantum yield Q L Eu displays a strong pH‐dependence: for a solution with an absorbance of 0.30, it increases sharply from about 2% at pH 2.5 to reach 11.5–12.5% in the pH range of 6–9. The proposed standard for EuIII is a solution of Cs3[Eu(dpa)3] 7.5 × 10−5 M in tris buffer 0.1 M (absorbance = 0.20) with a quantum yield of 13.5% ± 1.5% under excitation at 279 nm. For TbIII, we propose a standard solution of Cs3[Tb(dpa)3] 6.5 × 10−5 M in tris buffer 0.1 M (absorbance = 0.18) with a quantum yield of 26.5% ± 2% under excitation at 279 nm. Despite the speciation between bis and tris complexes, these two standard buffered solutions present a constant quantum yield within a reasonably large range of concentration and they are easy to handle, which makes them adequate for laboratory use.

Luminescent bimetallic lanthanide bioprobes for cellular imaging with excitation in the visible-light range.

A series of homoditopic ligands H(2)L(CX) (X=4-6) has been designed to self-assemble with lanthanide ions (Ln(III)), resulting in neutral bimetallic helicates of overall composition [Ln(2)(L(CX))(3)] with the aim of testing the influence of substituents on the photophysical properties, particularly the excitation wavelength. The complex species are thermodynamically stable in water (log beta(23) in the range 26-28 at pH 7.4) and display a metal-ion environment with pseudo-D(3) symmetry and devoid of coordinated water molecules. The emission of Eu(III), Tb(III), and Yb(III) is sensitised to various extents, depending on the properties of the ligand donor levels. The best helicate is [Eu(2)(L(C5))(3)] with excitation maxima at 350 and 365 nm and a quantum yield of 9 %. The viability of cervix cancer HeLa cells is unaffected when incubated with up to 500 mum of the chelate during 24 h. The helicate permeates into the cells by endocytosis and locates into lysosomes, which co-localise with the endoplasmatic reticulum, as demonstrated by counterstaining experiments. The relatively long excitation wavelength allows easy recording of bright luminescent images on a confocal microscope (lambda(exc)=405 nm). The new lanthanide bioprobe remains undissociated in the cell medium, and is amenable to facile derivatisation. Examination of data for seven Eu(III) and Tb(III) bimetallic helicates point to shortcomings in the phenomenological rules of thumb between the energy gap DeltaE((3)pipi*-(5)D(J)) and the sensitisation efficiency of the ligands.

Time-resolved luminescence microscopy of bimetallic lanthanide helicates in living cells

The cellular uptake mechanism and intracellular distribution of emissive lanthanide helicates have been elucidated by time-resolved luminescence microscopy (TRLM). The helicates are non-cytotoxic and taken up by normal (HaCat) and cancer (HeLa, MCF-7) cells by endocytosis and show a late endosomal-lysosomal cellular distribution. The lysosomes predominantly localize around the nucleus and co-localize with the endoplasmatic reticulum. The egress is slow and limited, around 30% after 24 h. The first bright luminescent images can be observed with an external concentration gradient of 5 microM of the Eu(III) helicate [Q = 0.21, tau = 2.43 ms], compared to >10 microM when using conventional luminescence microscopy. Furthermore, multiplex labeling could be achieved with the Tb(III) [Q = 0.11, tau = 0.65 ms], and Sm(III) [Q = 0.0038, tau = 0.030 ms] analogues.

Remarkable Tuning of the Photophysical Properties of Bifunctional Lanthanide tris(Dipicolinates) and its Consequence on the Design of Bioprobes

Derivatives of dipicolinic acid with a polyoxyethylene pendant arm at the pyridine 4-position have been functionalized for potential grafting with biological material. Four ligands with different terminal functions (alcohol, methoxy, phtalimide and amine) have been synthesized, which react with trivalent lanthanide ions Ln (III) to yield triple helical [Ln(L) 3] (3-) complexes, as shown by NMR and UV-vis titrations. The tris chelates display large thermodynamic stability with log beta 13 approximately 19-20 for all Eu (III) complexes for instance. Photophysical measurements reveal adequate sensitization of the metal-centered luminescence in the europium (eta sens = 33-72%) and terbium complexes, which is modulated by the nature of the terminal function. The lifetimes of the metal-centered excited states are long, up to 1.4 ms for [Eu(L) 3] (3-) and 1.6 ms for [Tb(L) 3] (3-) at room temperature, in line with hydration numbers essentially equal to zero. Quantum yields are as high as 29% for the [Eu( L ( NH2 )) 3] (3-) and 18% for the [Tb( L ( OH )) 3] (3-) tris chelates in water at physiological pH. These series of complexes demonstrate the extent of fine-tuning achievable for lanthanide luminescent probes and are simple models for investigating the effect of binding to biological molecules on the metal-centered luminescent properties.

Lanthanide Bimetallic Helicates forin VitroImaging and Sensing

As the need for targeting luminescent biolabels increases, for mapping selected analytes, imaging of cells and organs, and tracking in cellulo processes, lanthanide bimetallic helicates are emerging as versatile bioprobes. The wrapping of three ligand strands around two metallic centers by self‐assembly affords robust molecular edifices with tunable chemical and photophysical properties. In addition, heterometallic helical chelates can be assembled leading to bioprobes with inherent chiral properties. In this paper, we review the literature demonstrating that neutral [Ln2(LCX)3] (x = 1–3) helicates represent a viable alternative to existing chelating agents for bio‐analyses, while featuring specific enhanced properties. These bimetallic chelates self‐assemble in water, and at physiological pH the 2:3 (Ln:LCX) complex is by far the dominant species, conditional stability constants logβ23 being in the range 23–30. The metal ions are 9‐coordinate and lie in sites with slightly distorted D3 symmetry. Efficient protection from water interaction by the tightly wrapped ligand strands results in sizeable photophysical properties, with quantum yields up to 24% for EuIII and 11% for TbIII, while the luminescence of several other visible and/or near‐infrared emitting LnIII ions is also sensitized. Noncytotoxicity for all the helicates is established for several living cell lines including HeLa, HaCat, MCF‐7, 5D10, and Jurkat. We present new data pertaining to the live cell imaging ability of [Eu2(LC1)3] and compare the three systems with x = 1–3 with respect to thermodynamic stability, photophysics, cell‐permeation ability, and targeting capability for sensing in cellulo processes. Prospects of derivatization for characterizing specific biological interactions are discussed.

Luminescent lanthanide bimetallic triple-stranded helicates as potential cellular imaging probes

Water-soluble triple-stranded [Ln(2)(L)(3)] helicates have been successfully tested as imaging probes in human cervical adenocarcinoma cells (HeLa), the complex being not toxic and clearly staining their cytoplasm in a concentration-dependent manner.

Multiphoton-Excited Luminescent Lanthanide Bioprobes: Two- and Three-Photon Cross Sections of Dipicolinate Derivatives and Binuclear Helicates

Multiphoton excited luminescent properties of water-soluble Eu(III) and Tb(III) complexes with derivatives of dipicolinic acid functionalized with a polyoxyethylene pendant arm and terminal groups, [Eu(L(OMe))(3)](3-), [Eu(L(NH2))(3)](3-), and [Tb(L(OH))(3)](3-), as well as of binuclear helicates with overall composition [Ln(2)(L(CX))(3)] (X = 2, 5) are investigated. Characteristic emission from the (5)D(0) and (5)D(4) excited levels of Eu(III) and Tb(III), respectively, upon approximately 800 nm excitation results from three-photon absorption (3PA) for [Eu(L(OMe))(3)](3-), [Eu(L(NH2))(3)](3-), [Tb(L(OH))(3)](3-), and [Ln(2)(L(C2))(3)], while luminescence from [Eu(2)(L(C5))(3)] is induced by two-photon absorption (2PA) owing to its 1PA spectrum extending further into the visible. The 3PA cross sections have been determined and are the first ones reported for lanthanide complexes: (i) those of Eu(III) and Tb(III) bimetallic helicates [Ln(2)(L(C2))(3)] are 20 times larger compared to the corresponding values for tris(dipicolinates); (ii) derivatization of dipicolinic acid for Tb(III) complexes has almost no influence on the 3PA cross section; however, for Eu(III) complexes a approximately 2 times decrease is observed. The feasibility of [Eu(2)(L(C5))(3)] as multiphoton luminescence bioprobe is demonstrated by two-photon scanning microscopy imaging experiments on HeLa cells incubated with this bimetallic helicate.

Lanthanide 8-hydroxyquinoline-based podates with efficient emission in the NIR range

The novel hydroxyquinoline-containing tetrapodal ligand forms water soluble and stable chelates and is a good sensitizer of the NIR luminescence of its Nd(III) and Yb(III) complexes; its easy synthesis opens the way for potential biomedical applications.

Effect of the length of polyoxyethylene substituents on luminescent bimetallic lanthanide bioprobes

The new homoditopic ligand H2LC2′ self-assembles with lanthanide ions (LnIII) to yield neutral bimetallic helicates of overall composition [Ln2(LC2′)3]; it is fitted with two hexakis(oxyethylene) chains to test their effects on the thermodynamic, photophysical and biochemical properties of these complexes, with particular emphasis on their uptake by living cells. At physiological pH and under stoichiometric conditions, the conditional stability constants log β23 are around 28 resulting in the speciation of the EuIII helicate being >92% for a total ligand concentration of 1 mM. The ligand triplet state features adequate energy (0-phonon transition at ≈21 800 cm−1) for sensitising the luminescence of EuIII (Q = 19%) and TbIII (Q = 10%) in aerated water at pH 7.4. The Eu(5D0) emission spectrum and lifetime (2.43 ms) are characteristic of a species with pseudo-D3 symmetry and without bound water in the inner coordination sphere. The viability of HeLa cancerous cells is unaffected when incubated with up to 500 μM [Eu2(LC2′)3] during 24 h. The EuIII helicate permeates into the cytoplasm of these cells by endocytosis and remains essentially undissociated, despite a low intracellular concentration of 0.28 μM. In addition, the leakage of the EuIII helicate out of HeLa cells is very minimal over long periods of time. With respect to similar complexes with ligands bearing shorter tris(oxyethylene) chains, no substantial changes are observed, which opens the way for targeting experiments. This study also demonstrates that the [Ln2(LCX)3] helicates are fairly robust entities since their core is unaffected by the substitution in the pyridine 4-position.