Rare Earths-Doped and Ceria-Coated Strontium Aluminate PlateletsVersatile Luminescent Platforms for Correlated Lifetime Imaging by Multiphoton FLIM and PLIM

Abstract

We report our recent advances in the design and synthesis of functional and hybrid composite nanomaterials with properties geared toward life sciences assays and as platforms for biomedical imaging applications. Using a stepwise reverse micelle procedure, we synthesized hybrid platelets comprising rare earth-doped strontium aluminate cores labeled Eu,Dy:SrAlO, where the phase nominally denoted as Sr₀.₉₅Eu₀.₀₂Dy₀.₀₃Al₂O₄ dominates the nature of the composite, as demonstrated by extensive X-ray diffraction investigations. These were coated with a biocompatible cerium oxide shell, giving rise to the hierarchical hybrids denoted CeO₂@Eu,Dy:SrAlO. Such Eu/Dy codoped strontium aluminates exhibit broad luminescent emissions with high optical sensitivity. The CeO₂ shell further imparts biocompatibility and water dispersibility, resulting in kinetically stable nanoplatelets which can translocate into living cells in lifetime imaging protocols that were optimized for imaging across nano- and microscales. Multiphoton fluorescence lifetime imaging microscopy (MP FLIM) confirmed the luminescent properties in thin films and living cellular environments. These nanohybrids represent a significant step forward in the development of functional molecules and materials, leveraging directed and self-assembly strategies for their synthesis. Their luminescence (detectable by fluorescence as well as phosphorescence emission intensity correlated with emission lifetime), negligible toxicity on the time scale of imaging assays and up to 72 h, and biocompatibility with cellular milieu enabled their tracing with living cells. Their cellular activity was estimated by standard MTT assays in PC-3 and provided a further insight into their behavior in biological environments. The inclusion of heavy cerium and strontium atoms enhanced X-ray attenuation, supporting multimodal imaging by integrating optical and X-ray-based methods, which paves the way for potential applications in computed tomography correlated to confocal microscopy coupled with fluorescence lifetime imaging. These findings highlight the versatility of these luminescent hybrids for bioimaging and as synthetic scaffolds toward nanomedicine applications, bridging advanced imaging modalities with functional materials design.

1. Schematic representation of the synthetic protocol applied hereby. The process was monitored via TEM and the evaluation of the dispersed nanoparticles scaffolds was performed using DLS particle size measurements at 25 °C in water (conc. 0.5 mg/mL). Note: A combination of phases consisting of SrAl₂O₄ (Monoclinic), Sr_1.88Eu_0.12Al₂₄O₃₈ (Hexagonal), Sr₃Al₂O₆ (Cubic), and minor traces of Sr₁₀Al₆O₁₉ (Monoclinic) occurred from the synthesis, therefore the material obtained is denoted hereby using the abbreviation Eu,Dy:SrAlO

1

(a) Powder X-ray diffractogram and (b) DLS of the strontium aluminate codoped luminescent particles as obtained at 1300 °C. (c) Powder X-ray diffractogram and (d) DLS of the CeO₂ shell. (e) Powder X-ray diffractogram and (f) DLS of the CeO₂@Eu,Dy:SrAlO (* peaks corresponding to the CeO₂ phase ICCC: 00-034-0394).

2

Representative TEM micrographs of the core luminescent particles involved in this study, (a–g) imaged at different magnifications (Eu,Dy:SrAlO) and (h) corresponding energy-dispersive X-ray (EDX) analysis of the strontium aluminate codoped luminescent particles as obtained at 1300 °C.

3

Spectroscopic characterization: solid state UV–vis spectrum (a), fluorescence emission spectrum (λ_exc = 360 nm) of the luminescent particles (Eu,Dy:SrAlO) in solid state (b), fluorescence emission spectra (λ_exc = 360 nm) of the luminescent particles (Eu,Dy:SrAlO) and core–shell composite (CeO₂@Eu,Dy:SrAlO) in H₂O dispersed phase (1 mg/mL) (c), two photon fluorescence emission spectrum (λexc = 800 nm) of the luminescent particles (Eu,Dy:SrAlO) suspension in water at 1 mg/mL concentration (d), representative fluorescence lifetime data in dispersed phase (e) and in thin films (in representative pixel-by-pixel spot) for the nanomaterials investigated further details are given in Table and Supporting Information (f).

4

Solid state UV/visible absorption spectrum of the CeO₂ shell (a) and core–shell composite (b).

5

Representative TEM micrographs showing the platelet-like nature of particles investigated, on a range of magnifications (a–e) and EDX analysis of the core–shell composite CeO₂@Eu,Dy:SrAlO. (f). Alternative images are given in the Supporting Information.

6

Correlated imaging: single-photon laser confocal fluorescence (λ_ex = 400 nm and λ_em = 570–750 nm) for Eu,Dy:SrAlO (a) and CeO₂@Eu,Dy:SrAlO (e). Correlated two-photon fluorescence microscopy measurements (scale-bar 20 μm, laser power: 2.0 mW at 800 nm 2P excitation) of particle films showing: fluorescence intensity (b,f, respectively), fluorescence lifetime maps (c,g) and associated profile distribution for (a–d) uncoated core particles Eu,Dy:SrAlO and (e–h) ceria-coated composites, CeO₂@Eu,Dy:SrAlO. Rainbow colors mapping provides a direct correlation between the lifetime in field of view (c,g) and the fluorescence lifetime distribution histograms (d,h).

7

Correlated PLIM microscopy measurements of particles in thin films (400 nm excitation). Fluorescence emission intensity channel (λ_ex = 400 nm and λ_em = 570–750 nm) for Eu,Dy:SrAlO (a) and CeO₂@Eu,Dy:SrAlO (e). Correlated PLIM measurements of particle films showing: phosphorescence emission intensity (b,f, respectively), phosphorescence lifetime maps (c,g), and associated profile distribution for (a–d) uncoated core particles Eu,Dy:SrAlO and (e–h) core–shell composites CeO2@Eu,Dy:SrAlO. Rainbow colors mapping provides a direct correlation between the phosphorescence lifetime in field of view (c,g) and the phosphorescence lifetime distribution histograms (d,h).

8

In vitro two-photon fluorescence microscopy of CHO cells. Correlated imaging (scalebar 10 μm) with micrographs showing DIC channel, intensity map, lifetime distribution maps, and associated profiles for lifetime distribution for (a–d) luminescent particles Eu,Dy:SrAlO, (e–h) core–shell particles CeO₂@Eu,Dy:SrAlO, (i–l) luminescent particles Eu,Dy:SrAlO plus NucBlue CHO cells were treated with 1 mg/mL of the particles. Additional experiments are given in the Supporting Information. In vitro two-photon fluorescence microscopy of PC-3 cells. DIC channel, intensity map, lifetime map, and associated profile distribution for (m–p) core–shell particles CeO₂@Eu,Dy:SrAlO in PC-3 (37 °C, 15 min incubation). Colors provide a direct correlation between the lifetime maps and the lifetime histograms. Laser power: 2.0 mW at 800 nm wavelength (multiphoton excitation). PC-3 cells were treated with 1 mg/mL of the particles. Additional micrographs are given in the Supporting Information.

9

(a) Comparison of 2P fluorescence lifetime decays of Eu,Dy:SrAlO and CeO₂@Eu,Dy:SrAlO in CHO cells (800 nm, TCSPC measurements, CHO cells were treated with 1 mg/mL of the particles) and (b) comparison of 2P fluorescence lifetime decays of CeO₂@Eu,Dy:SrAlO in PC-3 cells (800 nm, TCSPC measurements, PC-3 cells were treated with 1 mg/mL of the particles).