Quantifying nanodiamonds biodistribution in whole cells with correlative iono-nanoscopy.

Correlative imaging and quantification of intracellular nanoparticles with the underlying ultrastructure is crucial for understanding cell-nanoparticle interactions in biological research. However, correlative nanoscale imaging of whole cells still remains a daunting challenge. Here, we report a straightforward nanoscopic approach for whole-cell correlative imaging, by simultaneous ionoluminescence and ultrastructure mapping implemented with a highly focused beam of alpha particles. We demonstrate that fluorescent nanodiamonds exhibit fast, ultrabright and stable emission upon excitation by alpha particles. Thus, by using fluorescent nanodiamonds as imaging probes, our approach enables quantification and correlative localization of single nanodiamonds within a whole cell at sub-30 nm resolution. As an application example, we show that our approach, together with Monte Carlo simulations and radiobiological experiments, can be employed to provide unique insights into the mechanisms of nanodiamond radiosensitization at the single whole-cell level. These findings may benefit clinical studies of radio-enhancement effects by nanoparticles in charged-particle cancer therapy.

Dosimetric uncertainties impact on cell survival curve with low energy proton.

There is an increasing number of radiobiological experiments being conducted with low energy protons (less than 5 MeV) for radiobiological studies due to availability of sub-millimetre focused beam. However, low energy proton has broad microdosimetric spectra which can introduce dosimetric uncertainty. In this work, we quantify the impact of this dosimetric uncertainties on the cell survival curve and how it affects the estimation of the alpha and beta parameters in the LQ formalism. Monte Carlo simulation is used to generate the microdosimetric spectra in a micrometer-sized water sphere under proton irradiation. This is modelled using radiobiological experiment set-up at the Centre of Ion Beam Application (CIBA) in National University of Singapore. Our results show that the microdosimetric spectra can introduce both systematic and random shifts in dose and cell survival; this effect is most pronounced with low energy protons. The alpha and beta uncertainties can be up to 10% and above 30%, respectively for low energy protons passing through thin cell target (about 10 microns). These uncertainties are non-negligible and show that care must be taken in using the cell survival curve and its derived parameters for radiobiological models.

Myeloperoxidase is a potential molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque.

Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque. Methods and results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene. Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

Element-based prognostics of occupational pneumoconiosis using micro-proton-induced X-ray emission analysis.

Pneumoconiosis is an occupational disease accompanied by long-term lung impairment, for which prediction of prognosis is poorly understood because of the complexity of the inhaled particles. Micro-proton-induced X-ray emission (micro-PIXE) analysis, which is advantageous for high-sensitivity, two-dimensional element mapping of lung tissues, was used to investigate element-based predictive factors of prognosis in Chinese patients with welder's and coal miner's pneumoconiosis. Chest radiographs and lung function tests showed that most of the coal miners deteriorated, whereas symptoms in some welders were alleviated after 5 yr, as determined by comparing percent vital capacity (%VC) and forced expiratory volume in the 1st second over forced vital capacity (FEV1.0/FVC) to values taken at the initial diagnosis. Micro-PIXE analysis suggested that the most abundant particulates in welder's pneumoconiosis were Fe, Mn, and Ti (metallic oxide),which were accompanied by particulates containing Si, Al, and Ca (aluminum silicate) or only Si (SiO2); the most abundant particulates in coal miner's pneumoconiosis were composed of C, Si, Al, K, and Ti, which were accompanied by particulates containing Ca or Fe. Particulates containing Al, Si, S, K, Ca, and Ti (orthoclase and anorthite) were correlated with severity of fibrosis. Multivariable linear regression suggested that long-term FEV1.0/FVC decrease was independently associated with Si and smoking index, whereas %VC decrease was associated with Si and Ti. A risk index comprised of these factors was developed to predict the prognosis of pneumoconiosis. Micro-PIXE analysis is feasible for the evaluation of elemental composition and dust exposure, especially for patients whose exposure is mixed or uncertain.

High-resolution 3D imaging and quantification of gold nanoparticles in a whole cell using scanning transmission ion microscopy.

Increasing interest in the use of nanoparticles (NPs) to elucidate the function of nanometer-sized assemblies of macromolecules and organelles within cells, and to develop biomedical applications such as drug delivery, labeling, diagnostic sensing, and heat treatment of cancer cells has prompted investigations into novel techniques that can image NPs within whole cells and tissue at high resolution. Using fast ions focused to nanodimensions, we show that gold NPs (AuNPs) inside whole cells can be imaged at high resolution, and the precise location of the particles and the number of particles can be quantified. High-resolution density information of the cell can be generated using scanning transmission ion microscopy, enhanced contrast for AuNPs can be achieved using forward scattering transmission ion microscopy, and depth information can be generated from elastically backscattered ions (Rutherford backscattering spectrometry). These techniques and associated instrumentation are at an early stage of technical development, but we believe there are no physical constraints that will prevent whole-cell three-dimensional imaging at <10 nm resolution.

Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy.

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (µg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240±9µM in wet weight level (135±5µg/g dry weight) in the stratum lucidum (SL) compared to 144±6µM in wet weight level (81±3µg/g dry weight) in the stratum pyramidale (SP) and 78±10µM in wet weight level (44±5µg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice.

Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.

WITHDRAWN: Measurement of beam focus quality in biomedical nuclear microscopy.

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Zinc supplementation inhibits lipid peroxidation and the development of atherosclerosis in rabbits fed a high cholesterol diet.

Developing atherosclerotic lesions in hypercholesterolemic rabbits are depleted in zinc, while iron accumulates. This study examined the influence of zinc supplementation on the development of atherosclerosis and used isotope dilution gas chromatography-mass spectrometry techniques to measure biomarkers of oxidative lipid damage in atherosclerotic rabbit aorta. Our previous method for F(2)-isoprostane measurement was adapted to include the quantitation of cholesterol oxidation products in the same sample. Two groups of New Zealand white rabbits were fed a high cholesterol (1% w/w) diet and one group was also supplemented with zinc (1 g/kg) for 8 weeks. Controls were fed a normal diet. Zinc supplementation did not significantly alter the increase in total plasma cholesterol levels observed in animals fed high cholesterol. However, in cholesterol-fed animals zinc supplementation significantly reduced the accumulation of total cholesterol levels in aorta which was accompanied by a significant reduction in average aortic lesion cross-sectional areas of the animals. Elevated levels of cholesterol oxidation products (5,6-alpha and beta cholesterol epoxides, 7beta-hydroxycholesterol, 7-ketocholesterol) in aorta and total F(2)-isoprostanes in plasma and aorta of rabbits fed a cholesterol diet were significantly decreased by zinc supplementation. Our data indicate that zinc has an antiatherogenic effect, possibly due to a reduction in iron-catalyzed free radical reactions.

Promotion of atherogenesis by copper or iron--which is more likely?

Iron levels increase in atherosclerotic lesions in cholesterol fed-rabbits and play a role in atherosclerosis. We investigated whether copper also rises. Male New Zealand White rabbits were fed high-cholesterol diets for 8 weeks. After sacrifice, lesion sizes were determined, and elemental analyses of the lesion and unaffected artery wall performed using nuclear microscopy. Unlike iron, lesion copper is decreased by about half compared with the unaffected artery wall, and much less copper than iron is present. Our data suggest that iron may be more likely to play a role in the promotion of atherosclerosis than copper.

Zinc supplementation decreases the development of atherosclerosis in rabbits.

Developing atherosclerotic plaques in cholesterol-fed rabbits are enriched in iron but depleted in zinc. In order to examine further the role of zinc, New Zealand White rabbits were fed a high-cholesterol 1% (w/w) diet with zinc (1 g/kg) supplementation for 8 weeks. After the 8-week period, the average atherosclerotic lesion cross-sectional areas in the aortas of the animals fed with the zinc supplement were significantly decreased (1.0 mm2) compared with lesion areas of the animals fed only on the high-cholesterol diet (3.1 mm2). Using nuclear microscopy, a technique for mapping and measuring trace elements in tissue sections, lesion zinc levels (24 ppm) were observed to be unchanged in the zinc-fed rabbits compared to controls. However, average lesion Fe levels in the zinc-fed group were measured at 32 ppm, whereas in the control group the average Fe levels were significantly higher at 43 ppm (P = 0.03). Our data support the concept that zinc may have an antiatherogenic effect by decreasing iron levels in the lesion, possibly leading to inhibition of iron-catalyzed free radical reactions.

A nuclear microscopic and histochemical study of iron concentrations and distribution in the midbrain of two age groups of monkeys unilaterally injected with MPTP.

The present study was carried out to elucidate the concentration and distribution of iron in the substantia nigra of two age groups of monkeys after experimental hemi-Parkinsonism induced by unilateral internal carotid injections of MPTP. Iron levels and distribution were detected using the nuclear microscope, which is able to provide structural and quantitative elemental analysis of biological tissue down to the parts per million (ppm) level of analytical sensitivity. Five weeks after unilateral lesioning with MPTP, we observed a 30-65% loss of neurons in the injected substantia nigra of each monkey, compared with the contralateral control 'non-lesioned' side. In monkeys less than 7 years of age, the iron was distributed fairly uniformly and showed little evidence of focal deposits. In monkeys greater than 7 years of age, we observed many dense focal deposits of iron in the substantia nigra. A comparison between iron distributions in nuclear microscopic scans and cell distributions in the same sections stained by the Nissl technique showed that areas containing high iron concentrations were present not where large-diameter neurons with abundant Nissl substance (presumed dopaminergic neurons) were located but in a region ventral to these cell bodies, i.e., in the substantia nigra pars reticulata. These distributions were present on the control side as well as the MPTP-injected side. Since a previous study has shown that unilateral MPTP injection results in lesions of the substantia nigra of the same side but negligible injury to the opposite side, this implies that the iron deposits existed in the older monkeys before MPTP injections (i.e. they occurred normally). The accumulation of iron in the substantia nigra with age suggests the possibility of localised damage to neurons through the catalysis of free radicals.