Two‐Photon Microscopy and Spectroscopy of Lanthanide Bioprobes

Abstract

The linear and non-linear photophysical properties of tris-dipicolinate europium and terbium complexes (absorption, emission, lifetime, luminescence induced by two-photon absorption) are studied in the crystalline state as well as in protein derivative crystals and compared to those in solution. Upon laser irradiation at 532 nm, luminescence of terbium is induced by a two-photon antenna effect, whereas luminescence of europium results from one-photon absorption in forbidden f-f transitions. Finally, linear and two-photon microscopy imaging experiments on biological and bio-inspired crystals are performed. These first proof-of-concept experiments open the way for the development of time-resolved non-linear microscopy that should combine the advantages of lanthanide luminescence (long lifetime, sharp emission bands, insensitivity to oxygen) with those of confocal biphotonic excitation (near-IR excitation, 3D resolution and reduced photodamage).

Lanthanide Complexes for Nonlinear Optics: From Fundamental Aspects to Applications

“…[74] In spite of the very low TPA efficiency of this complex, the microscopy experiment was successful as a result of the high concentration of luminescent species in the crystal and under confocal irradiation at 532 nm, only a small glowing point was observed in the middle of one crystal plate (Figure 14). Inspired by the above-mentioned optimization study, we designed a new complex, [Na] 3 [EuDPA 2G 3 ], in which the ligand is functionalized by an alkoxyphenylethynyl system, and the hydrosolubility is ensured by 3,4,5-tris(triethyleneglycol)phenyl moieties and by the sodium countercations.…”

Interpretation of europium(III) spectra

“…Otherwise the one-photon absorption process will occur and not the two-photon absorption. This requirement restricts the use of two-photon absorption mainly to systems in the middle of the lanthanide series compounds is a very active research field at the moment, but for biological studies rather than theoretical studies [27,33,85,[579][580][581][582][583][584][585][586]. Europium(III) complexes are very often used for in vitro and in vivo studies of cells, but ultraviolet excitation light has a very limited penetration depth in tissues and it can also damage living cells.…”

Acridone‐Benzimidazole Ring‐Fused Ligands: A New Class of Sensitizers of Lanthanide Luminescence via Low‐Energy Excitation

“…[ (7). [56] ( 7) where c is the speed of light in cm s -1 , N A is Avogadro's number, J and J' are the quantum numbers for the ground and excited states, respectively, ͐ε(ν)dν is the integrated spectrum of the f-f transition, ν m is the barycenter of the transition and n is the refractive index. A solid sample of the [Yb(L AB2 ) 3 ] complex was dissolved in chloroform/methanol (9:1) for solubility reasons.…”

“…The first one relies on multi-photon excitation of lanthanide complexes absorbing in the UV or visible range and displaying important multi-photon absorption cross-sections. [3,[6][7][8][9][10] This technique presents the advantage of allowing excitation with near-infrared (NIR) light which is less absorbed by biological tissues and thus penetrates deeper into them; for instance, imaging depths of 500 µm are routinely obtained for brain tissues in this way. [11] On the other hand, powerful femto lasers are needed for achieving adequate excitation since multiphoton processes have very low probability.…”