Three-dimensional Hard X-ray Ptychographic Reflectometry Imaging on Extended Mesoscopic Surface Structures

Many nano and quantum devices, with their sizes often spanning from millimeters down to sub-nanometer, have intricate low-dimensional, non-uniform, or hierarchical structures on surfaces and interfaces. Since their functionalities are dependent on these structures, high-resolution surface-sensitive characterization becomes imperative to gain a comprehensive understanding of the function-structure relationship. We thus developed hard X-ray ptychographic reflectometry imaging, a new technique that merges the high-resolution two-dimensional imaging capabilities of hard X-ray ptychography for extended objects, with the high-resolution depth profiling capabilities of X-ray reflectivity for layered structures. The synergy of these two methods fully leverages both amplitude and phase information from ptychography reconstruction to not only reveal surface topography and localized structures such as shapes and electron densities, but also yields statistical details such as interfacial roughness that is not readily accessible through coherent imaging solely. The hard X-ray ptychographic reflectometry imaging is well-suited for three-dimensional imaging of mesoscopic samples, particularly those comprising planar or layered nanostructures on opaque supports, and could also offer a high-resolution surface metrology and defect analysis on semiconductor devices such as integrated nanocircuits and lithographic photomasks for microchip fabrications

Ultraviolet Germicidal Irradiation and Its Effects on Elemental Distributions in Mouse Embryonic Fibroblast Cells in X-Ray Fluorescence Microanalysis

<div><p>Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.</p></div

Representative elemental maps of frozen hydrated mouse embryonic fibroblast cells (S1 File).

<p>Cells previously cryofixed by plunge freezing were scanned using 10 keV X rays in the cryojet x-ray fluorescence microscope at APS beamline 2-ID-D at 100 K. These scans have 800 nm pixel size and a per-pixel dwell time of 1 second. Data are available in Supporting Information file <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s001" target="_blank">S1.zip</a>.</p

Effects of UVGI, cryo transfer and cryo-scanning on the elemental contents and distributions of frozen hydrated mouse embryonic fibroblast cells.

<p>Cryofixed cells were first imaged in the cryojet x-ray fluorescence microscope at 100 K without any prior ultraviolet exposure (UV0, the first scan). They were then removed while being maintained at cryogenic conditions, exposed to 10 minutes of UVGI while immersed in liquid nitrogen, and cryogenically imaged again (UV10, the second scan) before an additional 20 minutes of UV exposure were used and followed by a third scan (UV30 for 30 minutes accumulative exposure time). The x-ray scan parameters were identical to those used to produce the images of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.g002" target="_blank">Fig. 2</a> as described in Materials and Methods. The elemental image for the same element was scaled to the same maximum and minimum values among these scans for direct visual comparison. Data are available in Supporting Information files <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s002" target="_blank">S2.zip</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s003" target="_blank">S3.zip</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s004" target="_blank">S4.zip</a> for 0, 10, and 30 minutes of UV exposure, respectively.</p

The survival rate of T7 bacteriophage under prolonged UV irradiation.

<p>About 60 million T7 bacteriophages in 3 <i>μ</i>l solution were dropped on Si₃N₄ windows and frozen by liquid-nitrogen-cooled liquid ethane, and then subjected for 0, 1, 2, 5, 10 and 20 minutes of ultraviolet germicidal irradiation while immersed in 3 cm of liquid nitrogen bath. A standard plaque assay was carried out to calculate the survival rate under each exposure, with three independent experiments being averaged to arrive at each point.</p

Elemental content in frozen hydrated mouse embryonic fibroblast cells, based on the images shown in Fig. 3.

<p>Elemental content within entire cells (red crosses) and their nuclei (gray squares) are plotted as fractions against the content in the first, non-UV-irradiated scan. These measurements were made from four of the five cells shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.g003" target="_blank">Fig. 3</a>; the next-to-top cell was excluded due to the difficulty in outlining its area for analysis. The slight decrease observed for the measured content of lighter elements (P, S, K) levels is consistent with a possible explanation of ice or frost accretion on the specimen between the second and third scan, leading to increased self-absorption of these lower energy fluorescent x-rays. This was further supported by the nearly unchanged ratio of elemental contents in the cell’s nucleus versus the entire cell among these three scans.</p

Comparison of elemental distribution maps of four elements (S, K, Fe, Zn) in frozen hydrated cells (rows A and C) as well as freeze-dried counterparts (panels B and D), with (rows C and D) and without (rows A and B) 20 minutes of ultraviolet germicidal irradiation (UVGI) under liquid nitrogen prior to frozen hydrated imaging.

<p>Visible light micrographs (VLM) of frozen hydrated (rows A and C) and freeze dried (rows B and D) cells show that the cells suffered similar degree of shrinkage after x-ray cryo-scanning and freeze drying, whether or not UVGI was used. Please note the maximum and minimum concentration values for the light elements S and K in row A were scaled to be the same as that in row C, but different from that in rows B and D. Scan parameters were the same as used for Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.g002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.g004" target="_blank">4</a>. Data are available in Supporting Information as files <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s005" target="_blank">S5.zip</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s006" target="_blank">S6.zip</a> for dehydrated cells exposed to UVGI for times of 0 and 20 minutes, respectively, and as files <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s007" target="_blank">S7.zip</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117437#pone.0117437.s008" target="_blank">S8.zip</a> for frozen hydrated cells exposed to UVGI for times of 0 and 20 minutes, respectively.</p

Epidermal Growth Factor Receptor Targeted Nuclear Delivery and High-Resolution Whole Cell X‑ray Imaging of Fe₃O₄@TiO₂ Nanoparticles in Cancer Cells

Sequestration within the cytoplasm often limits the efficacy of therapeutic nanoparticles that have specific subcellular targets. To allow for both cellular and subcellular nanoparticle delivery, we have created epidermal growth factor receptor (EGFR)-targeted Fe₃O₄@TiO₂ nanoparticles that use the native intracellular trafficking of EGFR to improve internalization and nuclear translocation in EGFR-expressing HeLa cells. While bound to EGFR, these nanoparticles do not interfere with the interaction between EGFR and karyopherin-β, a protein that is critical for the translocation of ligand-bound EGFR to the nucleus. Thus, a portion of the EGFR-targeted nanoparticles taken up by the cells also reaches cell nuclei. We were able to track nanoparticle accumulation in cells by flow cytometry and nanoparticle subcellular distribution by confocal fluorescent microscopy indirectly, using fluorescently labeled nanoparticles. More importantly, we imaged and quantified intracellular nanoparticles directly, by their elemental signatures, using X-ray fluorescence microscopy at the Bionanoprobe, the first instrument of its kind in the world. The Bionanoprobe can focus hard X-rays down to a 30 nm spot size to map the positions of chemical elements tomographically within whole frozen-hydrated cells. Finally, we show that photoactivation of targeted nanoparticles in cell nuclei, dependent on successful EGFR nuclear accumulation, induces significantly more double-stranded DNA breaks than photoactivation of nanoparticles that remain exclusively in the cytoplasm

Epidermal Growth Factor Receptor Targeted Nuclear Delivery and High-Resolution Whole Cell X‑ray Imaging of Fe₃O₄@TiO₂ Nanoparticles in Cancer Cells

Sequestration within the cytoplasm often limits the efficacy of therapeutic nanoparticles that have specific subcellular targets. To allow for both cellular and subcellular nanoparticle delivery, we have created epidermal growth factor receptor (EGFR)-targeted Fe₃O₄@TiO₂ nanoparticles that use the native intracellular trafficking of EGFR to improve internalization and nuclear translocation in EGFR-expressing HeLa cells. While bound to EGFR, these nanoparticles do not interfere with the interaction between EGFR and karyopherin-β, a protein that is critical for the translocation of ligand-bound EGFR to the nucleus. Thus, a portion of the EGFR-targeted nanoparticles taken up by the cells also reaches cell nuclei. We were able to track nanoparticle accumulation in cells by flow cytometry and nanoparticle subcellular distribution by confocal fluorescent microscopy indirectly, using fluorescently labeled nanoparticles. More importantly, we imaged and quantified intracellular nanoparticles directly, by their elemental signatures, using X-ray fluorescence microscopy at the Bionanoprobe, the first instrument of its kind in the world. The Bionanoprobe can focus hard X-rays down to a 30 nm spot size to map the positions of chemical elements tomographically within whole frozen-hydrated cells. Finally, we show that photoactivation of targeted nanoparticles in cell nuclei, dependent on successful EGFR nuclear accumulation, induces significantly more double-stranded DNA breaks than photoactivation of nanoparticles that remain exclusively in the cytoplasm