Type of presentation: Invited

ID-5-IN-2366 Characterizing bionanoparticles by STEM, EFTEM and electron tomography

Leapman R. D.¹, Aronova M. A.¹

¹National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA

leapmanr@mail.nih.gov

The ability to synthesize multicomponent hybrid nanocarriers with controlled architecture and chemical functionality offers great potential for developing in vivo and ex vivo medical diagnostics and therapeutics. Quantitative electron microscopy provides a tool to assess design strategies and to determine the degree of monodispersity, which are critical for controlling functionality and toxicity. Here, we illustrate that combining scanning transmission electron microscopy (STEM), energy-filtered transmission electron microscopy (EFTEM) and electron tomography (ET) gives elemental composition and 3D structure of hybrid nanocomplexes.

Fig. 1 shows how dark-field STEM tomography elucidates the structure of self-assembled, biodegradable, plasmonic gold nanovesicles that are designed for photoacoustic imaging and photothermal therapy [1]. Even at 300 kV it is not feasible to image these 200-nm diameter assemblies by bright-field TEM tomography because the strong scattering of gold gives a nonlinear signal when the nanoparticles overlap in the tilt series. However, the STEM tomogram clearly reveals a single shell of nanoparticles packed close to the vesicle membrane.

A combination of EFTEM and TEM tomography is used to characterize the flower-shaped optical nanosensor shown in Fig. 2. This nanocomplex consists of a central gold with surrounding iron oxide nanoparticles, which are attached to one end of a peptide substrate for matrix metalloproteinase enzyme, while the other end of the substrate is linked to a fluorescent dye molecule [2]. In the presence of the enzyme (expressed by cancer cells) the dye is cleaved and no longer quenched by the gold, resulting in a fluorescence signal. EFTEM (Fig. 2b) confirms that the petals of the nanosensor are iron oxide, and TEM tomography gives their 3D arrangement around the gold nanoparticle.

Fig. 3 demonstrates use of EFTEM to analyze a bionanoparticle developed for magnetic resonance imaging (MRI) of labeled cells. This nanocomplex consists of three FDA-approved drugs: heparin, protamine and ferumoxytol [3]. Each component can be mapped by its elemental composition: protamine contains nitrogen; heparin contains covalently bound sulfate groups; and fexumoxytol is a superparamagnetic iron oxide nanoparticle giving MRI contrast. The EFTEM elemental maps reveal a uniform distribution of protamine and heparin in the nanocomplex cores, whereas ferumoxytol is concentrated at the peripheries.

In summary, STEM, EFTEM and ET are valuable tools for optimizing the design and assessing the monodispersity of hybrid nanoparticles such as the ones illustrated here.

[1] P. Huang et al., Angew. Chem. Int. Ed. 52 (2013) 13958.

[2] X. Jie et al., ACS Nano 5 (2011) 3043.

[3] M.S. Thus et al., Nature Medicine 18 (2012) 463.

Research supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. The authors thank Drs. Xioayuan Chen, Peng Huang, Jin Xie, Joseph Frank and Henry Bryant for providing the nanoparticles used in this work.

Fig. 1: Self-assembled, plasmonic, biodegradable gold vesicle [1]: (a) annular dark-field STEM; (b) orthoslice through STEM tomographic reconstruction showing layer of nanoparticles next to membrane; (c) 3D visualization of the nanoassembly from the STEM tomogram.	Fig. 2: Hybrid Fe₃O₄/Au flower-shaped optical nanosensor [2]: (a) TEM; (b) EFTEM iron L_2,3 map superimposed on TEM bright-field image; (c) 3D visualization of nanosensor obtained by TEM tomography.
Fig. 3: EFTEM analysis of epon-embedded and sectioned nanocomplexes composed of heparin, protomaine and ferumoxytol [3]: (a) sulfur L_2,3 map; (b) nitrogen K map; (c) iron L_2,3 map; (d) overlay of the elemental distributions (S in green, N in blue, Fe in red).