Elemental mapping of Neuromelanin organelles of human Substantia Nigra: correlative ultrastructural and chemical analysis by analytical transmission electron microscopy and nano-secondary ion mass spectrometry.

Neuromelanin (NM) is a compound which highly accumulates mainly in catecholamine neurons of the substantia nigra (SN), and is contained in organelles (NM-containing organelles) with lipid bodies and proteins. These neurons selectively degenerate in Parkinson's disease and NM can play either a protective or toxic role. NM-containing organelles of SN were investigated by Analytical Electron Microscopy (AEM) and Nano-Secondary Ion Mass Spectrometry (NanoSIMS) within human tissue sections with respect to ultrastructure and elemental composition. Within the NM-containing organelle, the single NM granules and lipid bodies had sizes of about 200-600 nm. Energy-Dispersive X-ray microanalysis spectra of the NM granules and lipid bodies were acquired with 100 nm beam diameter in AEM, NanoSIMS yielded elemental maps with a lateral resolution of about 150 nm. AEM yielded the quantitative elemental composition of NM granules and bound metals, e.g., iron with a mole fraction of about 0.15 atomic percent. Chemical analyses by AEM and NanoSIMS were consistent at the subcellular level so that nanoSIMS measurements have been quantitated. In NM granules of SN from healthy subjects, a significant amount of S, Fe, and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved detection limits of nanoSIMS offer new possibilities for chemical mapping, high-sensitivity trace element detection, and reduced acquisition times. Variations between individual NM granules can now be investigated effectively and quantitatively by NanoSIMS mapping Cu and Fe. This should yield new insight into the changes in chemical composition of NM pigments during healthy aging and disease. Neuromelanin-containing organelles of dopamine neurons in normal human substantia nigra were investigated by analytical electron mircoscopy and secondary ion mass spectroscopy (NanoSIMS) yielding the ultrastructure and elemental composition. In neuromelanin granules a significant amount of S, Fe and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved sensitivity of NanoSIMS shows differences in chemical composition between individual neuromelanin granules and allows to study chemical changes of neuromelanin organelles during aging and disease.

Iron accumulation in Bruch's membrane and melanosomes of donor eyes with age-related macular degeneration.

Iron (Fe) accumulation in cytoplasmic storages of the retina and retinal pigment epithelium (RPE) with age has been reported to be a contributing factor to the onset and progression of Age-related Macular Degeneration (AMD). This work investigated whether iron can also be stored in specialized metal-binding melanosomes of the RPE and choroid and in age pigments of the RPE (lipofuscin and melanolipofuscin). As accumulation of debris in Bruch's membrane is an additional hallmark of AMD, the elemental composition of Bruch's membrane was also investigated. Perimacular sections of the retina-choroid complex of six eyes of AMD donors and of seven age-matched healthy controls were investigated using Analytical Electron Microscopy (AEM). The melanosomes of the RPE and choroidal melanocytes of all AMD donors contained about two times higher iron mole fractions (0.06-0.07 at%) compared to the controls, which showed only minor iron mole fractions at or below the detection limit of 0.02 at%. Only melanosomes that contained iron, showed also significant lead peaks (both AMD and control about 0.08 at%). In addition, the electron-dense part of melanolipofuscin granules in the RPE accumulated iron and lead, both for control and AMD donors. Iron in lipofuscin was below the detection limit. The elastic layer of Bruch's membrane of all AMD donors also contained significantly higher iron mole fractions compared to controls (about 0.08 at% Fe), predominantly in areas that were also rich in calcium (Ca) and phosphorus (P), suggesting calcification. Indeed, five of the six AMD donors but only one of the seven controls showed nanocrystalline hydroxyapatite calcifications. Note that such nanocrystalline material can only be detected in EM samples without heavy metal (osmiumtetroxide, uranylacetate) staining. In conclusion, iron accumulation in melanosomal storages and within calcified Bruch's membrane is more pronounced in donors suffering from AMD compared to age-matched controls. This work underlines the common hypothesis that heavy metal homeostasis plays an important role in age-related neuropathy.

Dielectrophoretic investigation of Bi2Te3 nanowires-a microfabricated thermoelectric characterization platform for measuring the thermoelectric and structural properties of single nanowires.

In this article a microfabricated thermoelectric nanowire characterization platform to investigate the thermoelectric and structural properties of single nanowires is presented. By means of dielectrophoresis (DEP), a method to manipulate and orient nanowires in a controlled way to assemble them onto our measurement platform is introduced. The thermoelectric platform fabricated with optimally designed DEP electrodes results in a yield of nanowire assembly of approximately 90% under an applied peak-to-peak ac signal Vpp = 10 V and frequency f = 20 MHz within a series of 200 experiments. Ohmic contacts between the aligned single nanowire and the electrodes on the platform are established by electron beam-induced deposition. The Seebeck coefficient and electrical conductivity of electrochemically synthesized Bi2Te3 nanowires are measured to be -51 µV K(-1) and (943 ± 160)/(Ω(-1) cm(-1)), respectively. Chemical composition and crystallographic structure are obtained using transmission electron microscopy. The selected nanowire is observed to be single crystalline over its entire length and no grain boundaries are detected. At the surface of the nanowire, 66.1 ± 1.1 at.% Te and 34.9 ± 1.1 at.% Bi are observed. In contrast, chemical composition of 64.2 at.% Te and 35.8 at.% Bi is detected in the thick center of the nanowire.

Chemical composition of melanosomes, lipofuscin and melanolipofuscin granules of human RPE tissues.

Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure and determine quantitatively the chemical composition of pigment granules of the choroid and retinal pigment epithelium of two healthy human donors, aged 68 and 85 years. The electron microscopy preparation procedure did not affect the autofluorescence of melanolipofuscin and lipofuscin granules, since staining was omitted during sample preparation. Oval melanosomes, melanolipofuscin and lipofuscin granules were observed, having sizes of about 1.5 µm×0.5 µm, and were analyzed using energy-dispersive X-ray microanalysis and electron energy loss spectroscopy. Up to now, these pigments could only be identified by scattering contrast in bright field images, with melanosomes having dark contrast and lipofuscin being much brighter. High-precision energy-dispersive X-ray microanalysis of pigment granules (>15,000 integrated counts in the oxygen K(α) peak) yielded minimum detectable mole fractions of about 0.02 at% for copper and zinc. For the first time, quantitative analytical electron microscopy yielded the chemical composition of the different pigments without prior isolation from the tissue. This is important to better understand physical and chemical properties of the pigments and their metabolism and turnover. The composition of melanosomes and lipofuscin can clearly be distinguished by the applied methods. Melanosomes were the pigments with largest oxygen (about 5 at%) and nitrogen (about 10 at%) mole fractions. The S/N ratio determination demonstrated a high pheomelanin content of the melanosomes. Lipofuscin had a significantly smaller oxygen mole fraction (about 4 at%) and nitrogen was found to be only slightly above the limit of detection (0.4 at%). For comparison, the cytoplasm contained oxygen and nitrogen mole fractions of 3 at% and 0.8 at%. Bright field images showed melanolipofuscin granules having a core-shell structure with a dark inner and a bright outer fraction. The dark fraction had a chemical composition close to the melanosomes and the composition of the bright fraction could be distinguished from that of lipofuscin due to a significantly increased nitrogen mole fraction in the melanolipofuscin granule. For all pigments observed the oxygen mole fraction yielded a positive correlation with the calcium mole fraction as previously established for melanosomes. Only lipofuscin contained measurable phosphorus mole fractions, which also correlated positively with oxygen. In lipofuscin, mole fractions of nitrogen were significantly smaller than in melanosomes and only indicated a small fraction of proteins. In contrast, the phosphorus mole fraction was significantly larger indicating the presence of significant amounts of phospholipids. Copper and zinc mole fractions were larger than 0.1at% in the melanosomes, but were below the detection limit in the lipofuscin granules. Compared to melanosomes of monkeys and rats analyzed beforehand, human retinal pigment epithelium melanosomes contained the highest amount of zinc, which even exceeded the calcium mole fraction. Trace elements like zinc are of great importance for metabolism and anti-oxidative mechanisms and also play a role in the progression of age related macular degeneration. They can now be investigated by quantitative analytical electron microscopy.

Quantitative high-resolution mapping of phenanthrene sorption to black carbon particles.

Sorption of hydrophobic organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) to black carbon (BC) particles has been the focus of numerous studies. Conclusions on sorption mechanisms of PAH on BC were mostly derived from studies of sorption isotherms and sorption kinetics, which are based on batch experiments. However, mechanistic modeling approaches consider processes at the subparticle scale, some including transport within the pore-space or different spatial pore-domains. Direct evidence based on analytical techniques operating at the submicrometer scale for the location of sorption sites and the adsorbed species is lacking. In this work, we identified, quantified, and mapped the sorption of PAHs on different BC particles (activated carbon, charcoal and diesel soot) on a 25-100 nm scale using scanning transmission X-ray microscopy (STXM). In addition, we visualized the pore structure of the particles by transmission electron microscopy (TEM) on the 1-10 nm-scale. The combination of the chemical information from STXM with the physical information from TEM revealed that phenanthrene accumulates in the interconnected pore-system along primary "cracks" in the particles, confirming an adsorption mechanism.

Coherent light emission in cathodoluminescence when using GaAs in a scanning (transmission) electron microscope.

For most materials science oriented applications incoherent cathodoluminescence (CL) is of main interest, for which the recombination of electron-hole pairs yields the emission of light. However, the incoherent signal is superimposed by coherently excited photons, similar to the situation for X-rays in Energy-Dispersive X-ray spectra (EDX). In EDX two very different processes superimpose in each spectrum: Bremsstrahlung and characteristic X-ray radiation. Both processes yield X-rays, however, their origin is substantially different. Therefore, in the present CL study we focus on the coherent emission of light, in particular Cerenkov radiation. We use a 200µm thick GaAs sample, not electron transparent and therefore not acting as a light guide, and investigate the radiation emitted from the top surface of the sample generated by back-scattered electrons on their way out of the specimen. The CL spectra revealed a pronounced peak corresponding to the expected interband transition. This peak was at 892 nm at room temperature and shifted to 845 nm at 80 K. The coherent light emission significantly modifies the shape of CL spectra at elevated beam energies. For the first time, by the systematic variation of current and energy of primary electrons we could distinguish the coherent and incoherent light superimposed in CL spectra. These findings are essential for the correct interpretation of CL spectra in STEM. The Cerenkov intensity as well as the total intensity in a spectrum scales linearly with the beam current. Additionally, we investigate the influence of asymmetric mirrors on the spectral shapes, collecting roughly only half of the whole solid angle. Different emission behaviour of different physical causes thus lead to changes in the overall spectral shape.

Transition metals and trace elements in the retinal pigment epithelium and choroid: correlative ultrastructural and chemical analysis by analytical electron microscopy and nano-secondary ion mass spectrometry.

Understanding the localisation and abundance of structural elements, trace elements and especially transition metals like Cu and Zn in ocular tissue sections is important for physiology, and also for the characterisation of diseases related to oxidative stress like age-related macular degeneration. Transition metal abundances were investigated in an aged donor eye by nano-secondary ion mass spectrometry (nano-SIMS) elemental mapping using Cs+ and O- primary ions, respectively, and correlated to their respective mole fractions investigated by analytical electron microscopy (AEM). The ultrastructure of the tissue and the elemental composition of melanosomes of the choroid and RPE, and RPE lipofuscin and melanolipofuscin granules can adequately be investigated by nano-SIMS using the secondary ion maps. Melanosomes, 0.5-1 µm in size, yield sulphur maps and maps of stored metals like calcium, sodium and copper. Lipofuscin shows especially high phosphorus signals. Elements with mole fractions of about 0.1 at%, e.g. for P and Cu, as investigated by AEM before, can be validated using simultaneous SIMS maps with an estimated lateral resolution of 66 nm with typical acquisition times of 30 minutes for each area of interest. However, Zn (0.19 at%) was not detected by SIMS. Nano-SIMS imaging of CN-, PO2-, S-, Cu-, Ca+, Fe+ and Na+ ions provides excellent detection limits demonstrating the possibilities for chemical mapping with high-sensitivity trace element detection and reduced acquisition times. Quantification of nano-SIMS data was achieved by correlating mole fractions obtained by AEM to secondary ions per pixel obtained by nano-SIMS. Both methods yield the melanin type in melanosomes and trace metal storage.

Characterization of nano hydroxyapatite/collagen surfaces and cellular behaviors.

Osseointegration at the bone-implant interface is a prerequisite for endosseous implants to succeed in achieving and maintaining their long-term stability in bone tissue. The achievement of osseointegration is significantly affected by surface nature of implants. To optimize osseointegration, this study presents the characterization of synthesized nanocrystalline hydroxyapatite (nano HA) and in vitro studies on nano HA, nano-HA/collagen, and titanium surfaces. Voids were found within the grain of nano HA, which consisted of the shell and the core. The finding assists the clarification of microstructures of nano HA. By low-temperature mixing nano-HA sol with collagen gel (nano-HA/collagen 80:20), nano HA, and nano-HA/collagen coated on pure titanium or porous anodic titanium oxides resulted in higher wettability and lower roughness. The in vitro studies showed that porous structures produced by anodic oxides on titanium served as positive anchorage sites for cell filopodia to connect, and nano HA decreased cell attachment of osteoblasts and induced well-developed long filopodia and broad lamellipodia, thereby enhancing cellular motility. Collagen involvement enhanced cell adhesion to nano HA. Cell reactions to nano HA, nano-HA/collagen, native, and porous titanium surfaces provide some guidance for an optimal osseointegration by their application in surface modifications for implants.

In vivo tracking of Th1 cells by PET reveals quantitative and temporal distribution and specific homing in lymphatic tissue.

UNLABELLED: Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. METHODS: We developed protocols that minimize the inhibitory effects of (64)Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((64)Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ-producing CD4(+) T (Th1) cells with 0.7-2.2 MBq of (64)Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular (64)Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10(7 64)Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of (64)Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10(7 64)Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. RESULTS: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, (64)Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered (64)Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of (64)Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm(3)]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity-diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm(3)). As expected, (64)Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm(3)) when compared with phosphate-buffered saline-challenged animals (4.6 ± 0.5 %ID/cm(3)). CONCLUSION: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for (64)Cu-PTSM-labeled T cells in clinical trials and novel therapy concepts focusing on T-cell-based immunotherapies of autoimmune diseases or cancer.

Phonon spectroscopy in a Bi2Te3 nanowire array.

The lattice dynamics in an array of 56 nm diameter Bi2Te3 nanowires embedded in a self-ordered amorphous alumina membrane were investigated microscopically using (125)Te nuclear inelastic scattering. The element specific density of phonon states is measured on nanowires in two perpendicular orientations and the speed of sound is extracted. Combined high energy synchrotron radiation diffraction and transmission electron microscopy was carried out on the same sample and the crystallinity was investigated. The nanowires grow almost perpendicular to the c-axis, partly with twinning. The average speed of sound in the 56 nm diameter Bi2Te3 nanowires is ~7% smaller with respect to bulk Bi2Te3 and a decrease in the macroscopic lattice thermal conductivity by ~13% due to nanostructuration and to the reduced speed of sound is predicted.

Switching of the natural nanostructure in Bi2Te3 materials by ion irradiation.

In Bi(2)Te(3) materials the natural nanostructure (nns) with a wavelength of 10 nm can be reproducibly switched ON and OFF by Ar(+) ion irradiation at 1.5 and 1 keV. Controlled formation of the nns in Bi(2)Te(3) materials has potential for reducing its thermal conductivity and could increase the thermoelectric figure of merit.

A low zinc diet leads to loss of Zn in melanosomes of the RPE but not in melanosomes of the choroidal melanocytes.

Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composition of melanosomes of the RPE and choroidal melanocytes and RPE lipofuscin granules were investigated using Analytical Electron Microscopy (AEM). In controls the Zn mole fraction of melanosomes was 0.05 at% (RPE) and 0.06 at% (choroid), respectively. For ZD-rats the Zn mole fraction of these granules was almost unchanged in the choroid but decreased and was below the detection limit (0.02 at%) in the RPE. ZD-rats produced also giant melanosomes (diameter 1-3 µm) in the choroid and greatly increased amounts of RPE lipofuscin. The giant melanosomes contained about six times as much Cu as control melanosomes. Lipofuscin granules were identified by AEM and could be clearly distinguished from melanosomes by their chemical composition. Thus, changes of the ultrastructure and transition metal storage of melanosomes due to a ZD can be directly traced using AEM.

Quantitative chemical analysis of ocular melanosomes in stained and non-stained tissues.

Energy-filtered Analytical Electron Microscopy (AEM) was used to image the ultrastructure and determine quantitatively the chemical composition of rat melanosomes of the choroid and the Retinal Pigment Epithelium (RPE). For the first time, the effect of staining in elemental analysis of melanosomes was investigated. Detection limits and accuracies of the applied methods were determined. Compared to previous work applying only quantitative Energy Dispersive X-ray microanalysis (EDX) in the TEM (Eibl, O., et al., 2006. Micron 37, 262), here we present a combined quantitative EDX and Electron Energy Loss Spectroscopy (EELS) analysis, including N. This yields the fraction of eumelanin and pheomelanin in melanosomes by the S/N mole fraction ratio. Melanosomes of the sepia ink sac, used as eumelanin standard, showed an S/N mole fraction ratio of <0.004. Thus, they consist primarily of eumelanin as reported by degradation analysis. In contrast, melanosomes of the rats contained mixed melanin with significant amounts of pheomelanin (S/N 0.02) in the RPE and the choroid. Consistent with the previous publication, it was shown that oxygen mole fractions are especially large in melanosomes (7-10 at.%) compared to other cell compartments, e.g. 2-4 at.% oxygen in the cytoplasm. In the melanosomes of non-stained tissue, the oxygen mole fraction clearly correlated with the Ca mole fraction. EDX spectra used for quantitative analysis had about 15,000 net counts under the oxygen peak, which is necessary to obtain (i) a small statistical error for oxygen and (ii) optimum minimum detectable mole fractions for S, Ca and transition metals. The precise determination of the oxygen mole fraction in melanosomes is important for understanding metabolism. Therefore, a detailed analysis was carried out on the possible errors affecting quantification. While O, S, and N mole fractions yielded similar results in stained and non-stained ocular melanosomes of rats, transition metals can only be determined reliably in non-stained tissues. High-precision EDX analysis of melanosomes yielded minimum detectable mole fractions of less than 0.04 at.% for Cu and Zn, these elements were present in melanosomes with mole fractions of about 0.3 at.% and 0.1at.%, respectively. Zn is of great importance for metabolism and for age related macular degeneration. Its mole fraction in melanosomes of rats is large enough to be detected and to be quantitatively analyzed by EDX spectroscopy. Ultrastructural information can now be correlated to the elemental composition. This is important to better understand the physical and chemical properties of melanosomal metabolism and turnover.

Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats.

BACKGROUND: Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet. METHODOLOGY/PRINCIPAL FINDINGS: Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch's membrane of ZD-LE rats varied between 0.4-3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch's membrane or even inside it. CONCLUSIONS/SIGNIFICANCE: In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch's membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch's membrane.