Comparative hard x-ray tomography for virtual histology of zebrafish larva, human tooth cementum, and porcine nerve.

Purpose: Synchrotron radiation-based tomography yields microanatomical features in human and animal tissues without physical slicing. Recent advances in instrumentation have made laboratory-based phase tomography feasible. We compared the performance of three cutting-edge laboratory systems benchmarked by synchrotron radiation-based tomography for three specimens. As an additional criterion, the user-friendliness of the three microtomography systems was considered. Approach: The three tomography systems-SkyScan 2214 (Bruker-microCT, Kontich, Belgium), Exciscope prototype (Stockholm, Sweden), and Xradia 620 Versa (Zeiss, Oberkochen, Germany)-were given 36 h to measure three medically relevant specimens, namely, zebrafish larva, archaeological human tooth, and porcine nerve. The obtained datasets were registered to the benchmark synchrotron radiation-based tomography from the same specimens and selected ones to the SkyScan 1275 and phoenix nanotom m® laboratory systems to characterize development over the last decade. Results: Next-generation laboratory-based microtomography almost reached the quality achieved by synchrotron-radiation facilities with respect to spatial and density resolution, as indicated by the visualization of the medically relevant microanatomical features. The SkyScan 2214 system and the Exciscope prototype demonstrated the complementarity of phase information by imaging the eyes of the zebrafish larva. The 3 - µ m thin annual layers in the tooth cementum were identified using Xradia 620 Versa. Conclusions: SkyScan 2214 was the simplest system and was well-suited to visualizing the wealth of anatomical features in the zebrafish larva. Data from the Exciscope prototype with the high photon flux from the liquid metal source showed the spiral nature of the myelin sheaths in the porcine nerve. Xradia 620 Versa, with detector optics as typically installed for synchrotron tomography beamlines, enabled the three-dimensional visualization of the zebrafish larva with comparable quality to the synchrotron data and the annual layers in the tooth cementum.

Application of low-energy scanning transmission electron microscopy for the study of Pt-nanoparticle uptake in human colon carcinoma cells.

High-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) in a scanning electron microscope facilitates the acquisition of images with high chemical sensitivity and high resolution. HAADF STEM at low electron energies is particularly suited to image nanoparticles (NPs) in thin cell sections which are not subjected to poststaining procedures as demonstrated by comparison with bright-field TEM. High membrane contrast is achieved and distinction of NPs with different chemical composition is possible at first sight. Low-energy HAADF STEM was applied to systematically study the uptake of Pt-NPs with a broad size distribution in HT29 colon carcinoma cells as a function of incubation time and incubation temperature. The cellular dose was quantified, that is, the amount and number density of NPs taken up by the cells, as well as the particle-size distribution. The results show a strong dependence of the amount of incubated NPs on the exposure time which can be understood by considering size-dependent diffusion and gravitational settling of the NPs in the cell culture medium.

In vitro toxicity of amorphous silica nanoparticles in human colon carcinoma cells.

The use of nanostructured silica (SiO2) particles is no longer restricted to biomedical and (bio-) technological fields but rather finding applications in products of the food industry. Thus, our studies on the toxicological relevance of SiO2 nanoparticles focused on cytotoxic effects, the modulation of the cellular redox status and the impact on DNA integrity in human colon carcinoma cells (HT29). The results indicate that these SiO2 nanoparticles stimulate the proliferation of HT29 cells, depending on the incubation time and the particle size. The cytotoxicity of the investigated SiO2 nanoparticles was found to depend on the concentration, size and on the FCS content of the culture medium. Furthermore, SiO2 seem to interfere with glutathione biosynthesis. The results indicate further that effects of SiO2 NPs are not mediated by oxidative stress but by interference with the MAPK/ERK1/2 as well as the Nrf2/ARE signalling pathways. Additionally, investigations regarding DNA integrity revealed no substantial (oxidative) DNA damage.

Quantitative high-resolution transmission electron microscopy of single atoms.

Single atoms can be considered as the most basic objects for electron microscopy to test the microscope performance and basic concepts for modeling image contrast. In this work high-resolution transmission electron microscopy was applied to image single platinum, molybdenum, and titanium atoms in an aberration-corrected transmission electron microscope. The atoms are deposited on a self-assembled monolayer substrate that induces only negligible contrast. Single-atom contrast simulations were performed on the basis of Weickenmeier-Kohl and Doyle-Turner form factors. Experimental and simulated image intensities are in quantitative agreement on an absolute intensity scale, which is provided by the vacuum image intensity. This demonstrates that direct testing of basic properties such as form factors becomes feasible.

Platinum nanoparticles and their cellular uptake and DNA platination at non-cytotoxic concentrations.

Three differently sized, highly dispersed platinum nanoparticle (Pt-NP) preparations were generated by supercritical fluid reactive deposition (SFRD) and deposited on a ß-cyclodextrin matrix. The average particle size and size distribution were steered by the precursor reduction conditions, resulting in particle preparations of <20, <100 and >100 nm as characterised by TEM and SEM. As reported previously, these Pt-NPs were found to cause DNA strand breaks in human colon carcinoma cells (HT29) in a concentration- and time-dependent manner and a distinct size dependency. Here, we addressed the question whether Pt-NPs might affect directly DNA integrity in these cells and thus behave analogous to platinum-based chemotherapeutics such as cisplatin. Therefore, DNA-associated Pt as well as the translocation of Pt-NPs through a Caco-2 monolayer was quantified by ICP-MS. STEM imaging demonstrated that Pt-NPs were taken up into HT29 cells in their particulate and aggregated form, but appear not to translocate into the nucleus or interact with mitochondria. The platinum content of the DNA of HT29 cells was found to increase in a time- and concentration-dependent manner with a maximal effect at 1,000 ng/cm(2). ICP-MS analysis of the cell culture medium indicated the formation of soluble Pt species, although to a limited extent. The observations suggest that DNA strand breaks mediated by metallic Pt-NPs are caused by Pt ions forming during the incubation of cells with these nanoparticles.

Cellular uptake of platinum nanoparticles in human colon carcinoma cells and their impact on cellular redox systems and DNA integrity.

Supercritical fluid reactive deposition was used for the deposition of highly dispersed platinum nanoparticles with controllable metal content and particle size distribution on beta-cyclodextrin. The average particle size and size distribution were steered by the precursor reduction conditions, resulting in particle preparations <20, <100, and >100 nm as characterized by transmission electron microscopy and scanning electron microscopy (SEM). These particle preparations of different size distributions were used to address the question as to whether metallic platinum particles are able to invade cells of the gastrointestinal tract as exemplified for the human colon carcinoma cell line HT29 and thus affect the cellular redox status and DNA integrity. Combined focused ion beam and SEM demonstrated that platinum nanoparticles were taken up into HT29 cells in their particulate form. The chemical composition of the particles within the cells was confirmed by energy-dispersive X-ray spectroscopy. The potential influence of platinum nanoparticles on cellular redoxsystems was determined in the DCF assay, on the translocation of Nrf-2 and by monitoring the intracellular glutathione (GSH) levels. The impact on DNA integrity was investigated by single cell gel electrophoresis (comet assay) including the formation of sites sensitive to formamidopyrimidine-DNA-glycosylase. Platinum nanoparticles were found to decrease the cellular GSH level and to impair DNA integrity with a maximal effect at 1 ng/cm(2). These effects were correlated with the particle size in an inverse manner and were enhanced with increasing incubation time but appeared not to be based on the formation of reactive oxygen species.