Ren Minqin

A novel approach to the identification and quantitative elemental analysis of amyloid deposits—Insights into the pathology of Alzheimer's disease

There is considerable interest in the role of metals such as iron, copper, and zinc in amyloid plaque formation in Alzheimers disease. However to convincingly establish their presence in plaques in vivo, a sensitive technique is required that is both quantitatively accurate and avoids isolation of plaques or staining/fixing brain tissue, since these processes introduce contaminants and redistribute elements within the tissue. Combining the three ion beam techniques of scanning transmission ion microscopy, Rutherford back scattering spectrometry and particle induced X-ray emission in conjunction with a high energy (MeV) proton microprobe we have imaged plaques in freeze-dried unstained brain sections from CRND-8 mice, and simultaneously quantified iron, copper, and zinc. Our results show increased metal concentrations within the amyloid plaques compared with the surrounding tissue: iron (85 ppm compared with 42 ppm), copper (16 ppm compared to 6 ppm), and zinc (87 ppm compared to 34 ppm).

Correlation of iron and zinc levels with lesion depth in newly formed atherosclerotic lesions.

Several studies have indicated a relationship between body iron content and cardiovascular disease, although other studies have not. There are also suggestions that zinc has an antioxidant and antiatherosclerotic effect. We have used Nuclear Microscopy, using the combination of Scanning Transmission Ion Microscopy (STIM), Rutherford Backscattering Spectrometry (RBS), and Proton Induced X-ray Emission (PIXE) to map and quantify iron and zinc levels in newly formed atherosclerotic lesions. Sixteen New Zealand White rabbits fed on a high cholesterol diet were divided into four groups of 4 rabbits each. Six weeks into the high cholesterol diet, two groups were treated with the iron chelating agent desferrioxamine, for 2 weeks and 4 weeks, respectively, by surgically implanting with Alzet osmotic pumps (Alza Corporation, Palo Alto, CA, USA) containing desferal (0.5 g/ml). The other two groups served as controls, and were surgically implanted with osmotic pumps containing saline. Tissue sections were taken from the aortic arch, flash frozen, and air-dried. Analysis of atherosclerotic lesions indicated a trend (p =.07) to a reduction in the progression of the lesion after 4 weeks of desferrioxamine treatment. For each of the control and desferrioxamine-treated animals however, the more extensive lesions contained a higher concentration of iron and a lower concentration of zinc. Our results are consistent with the view that early lesion formation may be accelerated by free radical production caused by increased iron levels, that zinc might antagonize such effects, and that more prolonged desferal treatment might have an antiatherosclerotic effect.

Does iron inhibit calcification during atherosclerosis

Oxidative stress has been implicated in the etiology of atherosclerosis and even held responsible for plaque calcification. Transition metals such as iron aggravate oxidative stress. To understand the relation between calcium and iron in atherosclerotic lesions, a sensitive technique is required that is quantitatively accurate and avoids isolation of plaques or staining/fixing tissue, because these processes introduce contaminants and redistribute elements within the tissue. In this study, the three ion-beam techniques of scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle-induced X-ray emission have been combined in conjunction with a high-energy (MeV) proton microprobe to map the spatial distribution of the elements and quantify them simultaneously in atherosclerotic rabbit arteries. The results show that iron and calcium within the atherosclerotic lesions exhibit a highly significant spatial inverse correlation. It may be that iron accelerates the progression of atherosclerotic lesion development, but suppresses calcification. Alternatively, calcification could be a defense mechanism against atherosclerotic progression by excluding iron.

Selenium and mercury are redistributed to the brain during viral infection in mice.

As part of the general host response to coxsackievirus B3 (CB3) infection, the concentration of essential and nonessential trace elements changes in different target organs of the infection. Essential (e.g., Se) and nonessential (e.g., Hg) trace elements are known to interact and affect inflammatory tissue lesions induced by CB3 infection. However, it is unknown whether these changes involve the brain. In the present study, the brain Hg and Se contents were measured through inductively coupled plasma-mass spectrometry and their distribution investigated by means of nuclear microscopy in the early phase (d 3) of CB3 infection in normally fed female Balb/c mice. Because of the infection, the concentration of Hg (4.07±0.46 ng/g wet wt) and Se (340±16 ng/g wet wt) in the brain increased twofold for Hg (8.77±1.65 ng/g wet wt, p<0.05) and by 36% for Se (461±150 ng/g wet wt, ns). Nuclear microscopy of brain sections from mice having elevated Se and Hg concentrations failed to find localized levels of the elements high enough to make detection possible, indicating approximately homogeneous tissue distribution. Although the pathophysiological interpretation of these findings requires further research, the increase of Hg in the brain during infection might have an influence on the pathogenesis of the disease.

Trace elemental distributions in induced atherosclerotic lesions using nuclear microscopy

Nuclear Microscopy, using the combination of scanning transmission ion microscopy, Rutherford backscattering spectrometry and proton induced X-ray emission has the ability to map and accurately quantify localised trace element levels in newly formed atherosclerotic lesions. In this study, New Zealand white rabbits fed on a high cholesterol diet were divided into two groups. One test group was treated with an iron chelating agent desferal, and the second group served as a control model. Tissue sections were taken from the aortic arch, flash frozen and air-dried, and scanned using the nuclear microscope of the Research Centre for Nuclear Microscopy, NUS. Results of this experiment, although not definitive (p ¼ 0:07) indicated that during the treatment with desferal, there was a trend for lesion development to be slowed down. However, analysis of atherosclerotic lesions both from the test and control groups showed that iron concentrations within the lesions exhibit a high degree of correlation with the depth of the lesion in the artery wall, whereas zinc is observed to be anti-correlated to the size of the lesion area. This investigation implies that the observed high iron levels, which can lead to increased free radical damage, may cause premature or accelerated arterial damage. 2003 Elsevier B.V. All rights reserved.

FAST ION BEAM MICROSCOPY OF WHOLE CELLS

The way in which biological cells function is of prime importance, and the determination of such knowledge is highly dependent on probes that can extract information from within the cell. Probing deep inside the cell at high resolutions however is not easy: optical microscopy is limited by fundamental diffraction limits, electron microscopy is not able to maintain spatial resolutions inside a whole cell without slicing the cell into thin sections, and many other new and novel high resolution techniques such as atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM) are essentially surface probes. In this paper we show that microscopy using fast ions has the potential to extract information from inside whole cells in a unique way. This novel fast ion probe utilises the unique characteristic of MeV ion beams, which is the ability to pass through a whole cell while maintaining high spatial resolutions. This paper first addresses the fundamental difference between several types of charged particle probes, more specifically focused beams of electrons and fast ions, as they penetrate organic material. Simulations show that whereas electrons scatter as they penetrate the sample, ions travel in a straight path and therefore maintain spatial resolutions. Also described is a preliminary experiment in which a whole cell is scanned using a low energy (45 keV) helium ion microscope, and the results compared to images obtained using a focused beam of fast (1.2 MeV) helium ions. The results demonstrate the complementarity between imaging using low energy ions, which essentially produce a high resolution image of the cell surface, and high energy ions, which produce an image of the cell interior. The characteristics of the fast ion probe appear to be ideally suited for imaging gold nanoparticles in whole cells. Using scanning transmission ion microscopy (STIM) to image the cell interior, forward scattering transmission ion microscopy (FSTIM) to improve the contrast of the gold nanoparticles, and Rutherford Backscattering Spectrometry (RBS) to determine the depth of the gold nanoparticles in the cell, a 3D visualization of the nanoparticles within the cell can be constructed. Finally a new technique, proton induced fluorescence (PIF), is tested on a cell stained with DAPI, a cell-nucleic acid stain that exhibits a 20-fold increase in fluorescence when binding to DNA. The results indicate that the technique of PIF, although still at an early stage of development, has high potential since there does not seem to be any physical barrier to develop simultaneous structural and fluorescence imaging at sub 10 nm resolutions.

MeV Helium Ion Imaging of Gold Nanoparticles in Whole Cells

Observation of the interior structure of cells and sub-cellular organelles at high spatial resolutions is important for determining the functioning mechanisms of biological cells. Conventional optical microscopies have resolutions limited to around 300 nm due to the diffraction limits of light, and the new super-resolution techniques such as Stimulated Emission Depletion microscopy (STED) have stringent requirements that limit their application areas. Electron microscopy has an important role to play, but is only useful when imaging very thin sections due to electron/electron large angle scattering within the sample. Our results indicate that microscopy using MeV ions has high potential for the imaging of thick samples, e.g. whole cells, at high spatial resolutions. The reason for this is that MeV ions (e.g. protons or alpha particles (helium ions)) maintain a straight trajectory when traversing material, therefore preserving spatial resolution. The interaction of MeV ions with matter is mainly through ion/electron collisions. Due to the high mass mismatch with electrons, ions suffer very low energy transfer for each collision and as a result thousands of collisions can occur before they stop. In addition, there is very little primary ion scattering, and therefore the MeV proton and alpha particle paths are characterised by a straight, deep penetration into the material [1]. As well as ion electron scattering, there is also a much smaller probability of the MeV ion undergoing elastic scattering from an atomic nucleus. In these nuclear collisions the ion can be backscattered (Rutherford backscattering spectrometry RBS), and by measuring the energy of the backscattered ion, the target atom can be identified. RBS is particularly efficient at identifying and measuring heavy metals in low mass matrices such as organic materials. The Centre for Ion Beam Applications, National University of Singapore has recently built up a high resolution single cell imaging facility with the ability of focusing for MeV proton and alpha particle beams down to 30nm spot sizes [2]. The facility incorporates a variety of techniques, including Scanning Transmission Ion Microscopy (STIM) and Rutherford Backscattering Spectroscopy (RBS), with the microanalytical technique of Particle Induced X-ray Emission (PIXE) being added at a later stage. STIM is a technique which relies on the measurement of the energy loss of transmitted ions. MeV helium ions or protons are energetic enough to pass through single whole cells, and by measuring the energy loss of the transmitted ions, a structural image can be assembled which has high density contrast. Gold nanoparticles have great potential uses for medical diagnostics and drug delivery, as tracers and for other biological applications. Although Transmission Electron Microscopy (TEM) has been very successful in identifying nanoparticles in thin cellular sections, it remains difficult to resolve the gold nanoparticles at nanoscale resolution in the whole cell. In this work, we have used STIM and RBS to image normal human lung fibroblast cells (MRC-5) cultured in an environment of 50 nm gold nanoparticles. Cells were grown on 100 nm silicon nitride windows in cell culture medium (RPMI supplemented with fetal bovine serum) containing 50 nm gold nanoparticles (0.2 nM). Following exposure to the nanoparticles for 72 hours, the cells were critically point dried. The STIM measurements were carried out using a silicon surface barrier ion detector positioned behind the cell, and the RBS measurements were carried out using an annular surface barrier detector in front of the cell. The STIM image in Fig. 1 shows the structural view of the whole cell cultured in a gold nanoparticle environment, and Fig. 2 shows a similar control cell grown in normal conditions. The 662 doi:10.1017/S1431927611004181 Microsc. Microanal. 17 (Suppl 2), 2011

Proton microprobe analysis of water trees in underground high voltage cables

The micro-PIXE technique was employed to analyze water trees in the polymeric insulation of some field-aged underground high voltage cables from the Eastern Province of Saudi Arabia. X-ray spectra of water trees, the inner and outer semiconductive compound layers of the cable samples, and the insulation matrix were acquired. Simultaneously, two-dimensional elemental distribution profiles across the water trees were also obtained. The results show how knowledge of the elemental constituents of water trees on a microscopic scale can be useful in attempts to understand premature degradation of underground power cables.