The Bionanoprobe
a TiO 2
Figure 6 : Elemental X-ray maps of the cell in Figure 4 acquired using a 50 nm step size. These maps show the distri- bution of the nanocomposites (identifi ed by the Fe and Ti signals) with respect to cellular structures. Reproduced with permission of the International Union of Crystallography from [ 3 ].
Figure 7 : Three-dimensional reconstruction (side view on left; cross section on right) of a frozen-hydrated HeLa cancer cell treated with Fe 3 O 4 @TiO 2 nanocomposites for 30 min showing that some of the nanocomposites (identifi ed by the Ti signals) were successfully delivered into the nuclear region (identifi ed by elevated Zn signals). The 3D volume reconstruction was based on 53 projections with an angular coverage from -78° to 78° and an angular step size of 3°. Each projection was acquired using a 250 nm step size. This fi gure was adapted from Figure 6 of [ 10 ].
damage to cryogenic samples has been observed at spatial resolutions of 100 nm and above. The quality of tomogram reconstruction is improved by projection image alignment, rotation axis correction, and background subtraction. Recently, XRF tomography has been developed and used to study 3D elemental distributions in biological samples on a sub-cellular level [ 7 , 8 ].
Results T e BNP has been employed to study nuclear DNA targeting using photoactive nanocomposites. Diff erent approaches have been developed to deliver nanoparticles composed of photoactivatable materials (such as TiO 2 ) to the nuclei of cancer cells where these nanoparticles can cleave nuclear DNA under photoactivation. We have synthe- sized 6–7 nm nanoparticles consisting of a Fe 3 O 4 core and
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shell (Fe 3 O 4 @TiO 2 ) and surface-conjugated these with several diff erent peptides to facilitate nuclear delivery [ 9 ]. Cultured HeLa cervical cancer cells were used for this study. T is cell line is the most understood and the most commonly used human cell line in scientifi c research. We then used the BNP to examine the nanocomposite-treated HeLa cell and determined the distribution of these nanocomposites. A particular question to answer is whether the nanocomposites would reach the nucleus aſt er cellular internalization. We chose incident photon energy of 10 keV, which allows excitation of Kα X-ray fl uorescence in elements with atomic numbers up to 30 (Zn). Distributions of both natural cellular elements (S and Zn, for example) and the elements introduced with the nanocomposites (Fe and Ti) were detected within a HeLa cell of about 20 µm in diameter. In a low- magnifi cation survey map ( Figure 4 ), the S fl uorescence signal was used to locate cells of interest because S is present in the amino acids methionine and cysteine and is therefore distributed throughout the cell [ 9 ]. Figure 5 shows X-ray spectra from a cell (white box in Figure 4 ) and a background region of the same area (yellow box in Figure 4 ). T ese spectra show that cells exhibit elevated levels of S, Ca, Ti, and Fe compared a sample region devoid of cells (background spectrum). Figure 6 shows the colocalization of Fe, Ti, and S signals of the cell in higher magnifi cation maps acquired using a 50 nm step size. T ese elemental maps demonstrate that some of the
nanocomposites were possibly taken into the cell by endocytosis. Multiple cells were examined using the same method. To confi rm that nanocomposites were located within the nucleus rather than on the cell outer surface, a 3D tomographic reconstruction was produced ( Figure 7 ). T e elevated Zn fl uorescence signal, probably from Zn fi nger proteins, is an indicator of the nuclear region [ 9 ]. T is 3D rendering shows the Ti hotspots inside the Zn-rich volume and therefore confi rms the success of nuclear delivery of the nanocomposites. Results from several of these X-ray 2D maps and 3D renderings indicate that about 20% of the nanocomposites were translocated into the nucleus [ 9 ].
Conclusion T e BNP is a new facility at the Advanced Photon Source of Argonne National Laboratory. T is beam-line setup allows fl uorescence X-ray mapping of elements in biological organisms
www.microscopy-today.com • 2015 May
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