Nanoparticles with non-centrosymmetric crystal structures exhibit second harmonic generation (SHG) of light when illuminated by a femtosecond pulsed laser. Such nanoparticles can be used as optical biomarkers to circumvent the drawbacks associated with fluorescent proteins and semiconductor quantum dots, such as photobleaching and fluorescent intermittency (blinking). Bulk barium titanate has a tetragonal crystal structure at room temperature however, reduction in particle sizes generally correlates with an increasing phase fraction of cubic material which does not exhibit SHG [1].

In this study we have produced barium titanate (BaTiO₃) and barium-strontium titanate (Ba_1-xSr_xTiO₃) nanoparticles by the hydrothermal method. These nanoparticles appear predominantly cubic by laboratory-XRD but Rietveld refinement on synchrotron X-Ray powder diffraction data suggests a mixture of tetragonal and cubic phases. Transmission electron microscopy (TEM) analysis techniques such as electron energy loss spectroscopy (EELS) and energy-dispersive X-ray (EDX) spectroscopy have been used to determine the inter- and intra-particle phase and composition of BaTiO₃ and Ba_1-xSr_xTiO₃ nanoparticles. Prior STEM-EELS work, suggests an intra-particle phase distribution of cubic and tetragonal phases [2]. STEM-EEL linescans by aberration corrected scanning transmission electron microscopy (SuperSTEM) confirm that these hydrothermal samples exhibit intra-particle phase distribution of a tetragonal core and a cubic shell (Figure 1 & 2).

Multi-photon microscopy correlated with SEM demonstrates the SHG signals from the BaTiO₃ and Ba_1-xSr_xTiO₃ nanoparticles [3]. The cellular uptake and biocompatibility of the BaTiO₃ and Ba_1-xSr_xTiO₃ nanoparticles have been determined by cell viability (MTT) and genotoxicity (Comet) assays. Uptake was confirmed by backscattered Z-contrast imaging by SEM and EDX (Figure 3), along with bright field TEM and HAADF-STEM of resin embedded cell sections. Direct correlation between electron microscopy (SEM & TEM) and multi-photon microscopy will be used to determine SHG characteristics at the individual particle level when taken up by cells.

[1]        E. Kim, A. Steinbrück, M. T. Buscaglia, V. Buscaglia, T. Pertsch, R. Grange, et al., Second-Harmonic Generation of Single BaTiO₃ Nanoparticles down to 22 nm Diameter, ACS Nano. 7 (2013) 5343–5349.

[2]      S M Moon, X Wang, N.H. Cho, Identification of Local Phase of Nanoscale BaTiO₃ Powders by High-Resolution Electron Energy Loss Spectroscopy, Microsc. Microanal. 19 (2013) 123.

[3]      O. Matar, O.M. Posada, N.S. Hondow, C. Wälti, M. Saunders, C.A. Murray, et al., Barium Titanate Nanoparticles for Biomarker Applications, J. Phys. Conf. Ser. 644 (2015). 012037

Figures:

Figure 1 - HAADF-STEM image of a BaTiO3 nanoparticle. EEL spectra extracted from the core (1) to the surface (2) are shown in Figure 2.

Figure 2 - EEL spectra from Area 1 & 2 in Figure 1. The separation of the Ti-L3,2 (t2g, eg) peaks show a variation in separation between the peaks suggesting a phase shift from a tetragonal core (1) to a cubic shell (2) [2].

Figure 3 - SEM backscattered electron image of an A549 cell following a 24 hour incubation with 100 μg/mL of BaTiO3 nanoparticles. Inset, a lower magnification secondary electron image showing the corresponding areas. The blue circle shows nanoparticles on the cell surface whereas the red circle highlights nanoparticles potentially internalised by the cell. (EDX confirms that these are barium titanate nanoparticles).

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EMC Abstracts - https://emc-proceedings.com/abstract/analytical-electron-microscopy-of-barium-titanate-and-barium-strontium-titanate-nanoparticles-for-second-harmonic-biomarkers/