Development of Multi-Scale X-ray Fluorescence Tomography for Examination of Nanocomposite-Treated Biological Samples

Abstract

Research in cancer nanotechnology is entering its third decade, and the need to study interactions between nanomaterials and cells remains urgent. Heterogeneity of nanoparticle uptake by different cells and subcellular compartments represent the greatest obstacles to a full understanding of the entire spectrum of nanomaterials’ effects. In this work, we used flow cytometry to evaluate changes in cell cycle associated with non-targeted nanocomposite uptake by individual cells and cell populations. Analogous single cell and cell population changes in nanocomposite uptake were explored by X-ray fluorescence microscopy (XFM). Very few nanoparticles are visible by optical imaging without labeling, but labeling increases nanoparticle complexity and the risk of modified cellular uptake. XFM can be used to evaluate heterogeneity of nanocomposite uptake by directly imaging the metal atoms present in the metal-oxide nanocomposites under investigation. While XFM mapping has been performed iteratively in 2D with the same sample at different resolutions, this study is the first example of serial tomographic imaging at two different resolutions. A cluster of cells exposed to non-targeted nanocomposites was imaged with a micron-sized beam in 3D. Next, the sample was sectioned for immunohistochemistry as well as a high resolution “zoomed in” X-ray fluorescence (XRF) tomography with 80 nm beam spot size. Multiscale XRF tomography will revolutionize our ability to explore cell-to-cell differences in nanomaterial uptake.

A reliable workflow for improving nanoscale X-ray fluorescence tomographic analysis on nanoparticle-treated HeLa cells

“…The overlay µ-XRF data, with Si, P, and Ti shown as red, green, and cyan colors, respectively, provide an initial assessment of nanocomposite distribution in HeLa cells. Congruent with the flow cytometry findings generated with the same samples, 14 nanoparticles are the most abundant in cells undergoing cell division. We captured eight cells in their division process, two of which had a high nanoparticle accumulation in their cytokinetic bridges, as shown in Supplementary material Fig.…”

“…In this work, we are interested in assessing the submicron distribution of non-targeted Fe 3 O 4 /TiO 2 nanocomposites taken up by the cells during cell division. 14 Incident photon energy of 10 keV was used for all the XRF measurements to excite the characteristic XRF signals from the nanocomposites and the native cellular elements, including, e.g., sulfur (S), phosphorous (P), and Zn.…”

“…While additional work will be necessary to establish details of the structure generated by interactions between cellular proteins and nanocomposites, it has been established that this type of nanocomposites generates complexes with baculoviral inhibitors of apoptosis repeat-containing 5 (BIRC5) and actin. 14 Both of these proteins participate in mitotic spindle structure.…”

“…The same samples used in the prior work 14 were imaged using the novel workflow detailed in this work. The nanocomposites made of an Fe 3 O 4 core and a TiO 2 shell were synthesized following the same preparation procedure as in the earlier studies.…”

Atomic spectrometry update: review of advances in X-ray fluorescence spectrometry and its special applications

“…In the field of cancer research , Chen et al 6 developed a multi-scale XRF-CT method for the examination of nanocomposite-treated biological samples to study interactions between NPs and cells. Flow cytometry was used to evaluate changes in cell cycles associated with non-targeted nanocomposite uptake by individual cells.…”