Application of Ion Beam Methods in Biomedical Research: Quantitative Microscopy with Trace Element Sensitivity

The methods of analysis with a focused ion beam, commonly termed as nuclear microscopy, include quantitative physical processes like PIXE and RBS. The element concentrations in a sample can be quantitatively mapped with a sub-micron spatial resolution and a sub-ppm sensitivity. Its fully quantitative and non-destructive nature makes it particularly suitable for analysing biological samples. The applications in biomedical research are manifold. The iron overload hypothesis in Parkinson\\\''s disease is investigated by a differential analysis of human substantia nigra. The trace element content is quantified in neuromelanin, in microglia cells, and in extraneuronal environment. A comparison of six Parkinsonian cases with six control cases revealed no significant elevation in iron level bound to neuromelanin. In fact, a decrease in the Fe/S ratio of Parkinsonian neuromelanin was measured, suggesting a modification in its iron binding properties. Drosophila melanogaster, or the fruit fly, is a widely used model organism in neurobiological experiments. The electrolyte elements are quantified in various organs associated with the olfactory signalling, namely the brain, the antenna and its sensilla hairs, the mouth parts, and the compound eye. The determination of spatially resolved element concentrations is useful in preparing the organ specific Ringer\\\''s solution, an artificial lymph that is used in disruptive neurobiological experiments. The role of trace elements in the progression of atherosclerosis is examined in a pilot study. A differential quantification of the element content in an induced murine atherosclerotic lesion reveals elevated S and Ca levels in the artery wall adjacent to the lesion and an increase in iron in the lesion. The 3D quantitative distribution of elements is reconstructed by means of stacking the 2D quantitative maps of consecutive sections of an artery. The feasibility of generating a quantitative elemental rodent brain atlas by Large Area Mapping is investigated by measuring at high beam currents. A whole coronal section of the rat brain was measured in segments in 14 h. Individual quantitative maps of the segments are pieced together to reconstruct a high-definition element distribution map of the whole section with a subcellular spatial resolution. The use of immunohistochemical staining enhanced with single elements helps in determining the cell specific element content. Its concurrent use with Large Area Mapping can give cellular element distribution maps

Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging

AbstractAdministration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore–drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0±0.6cm−1 at 716nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice

Application of Ion Beam Methods in Biomedical Research: Quantitative Microscopy with Trace Element Sensitivity

The methods of analysis with a focused ion beam, commonly termed as nuclear microscopy, include quantitative physical processes like PIXE and RBS. The element concentrations in a sample can be quantitatively mapped with a sub-micron spatial resolution and a sub-ppm sensitivity. Its fully quantitative and non-destructive nature makes it particularly suitable for analysing biological samples. The applications in biomedical research are manifold. The iron overload hypothesis in Parkinson\\\''s disease is investigated by a differential analysis of human substantia nigra. The trace element content is quantified in neuromelanin, in microglia cells, and in extraneuronal environment. A comparison of six Parkinsonian cases with six control cases revealed no significant elevation in iron level bound to neuromelanin. In fact, a decrease in the Fe/S ratio of Parkinsonian neuromelanin was measured, suggesting a modification in its iron binding properties. Drosophila melanogaster, or the fruit fly, is a widely used model organism in neurobiological experiments. The electrolyte elements are quantified in various organs associated with the olfactory signalling, namely the brain, the antenna and its sensilla hairs, the mouth parts, and the compound eye. The determination of spatially resolved element concentrations is useful in preparing the organ specific Ringer\\\''s solution, an artificial lymph that is used in disruptive neurobiological experiments. The role of trace elements in the progression of atherosclerosis is examined in a pilot study. A differential quantification of the element content in an induced murine atherosclerotic lesion reveals elevated S and Ca levels in the artery wall adjacent to the lesion and an increase in iron in the lesion. The 3D quantitative distribution of elements is reconstructed by means of stacking the 2D quantitative maps of consecutive sections of an artery. The feasibility of generating a quantitative elemental rodent brain atlas by Large Area Mapping is investigated by measuring at high beam currents. A whole coronal section of the rat brain was measured in segments in 14 h. Individual quantitative maps of the segments are pieced together to reconstruct a high-definition element distribution map of the whole section with a subcellular spatial resolution. The use of immunohistochemical staining enhanced with single elements helps in determining the cell specific element content. Its concurrent use with Large Area Mapping can give cellular element distribution maps

Application of Ion Beam Methods in Biomedical Research: Quantitative Microscopy with Trace Element Sensitivity

The methods of analysis with a focused ion beam, commonly termed as nuclear microscopy, include quantitative physical processes like PIXE and RBS. The element concentrations in a sample can be quantitatively mapped with a sub-micron spatial resolution and a sub-ppm sensitivity. Its fully quantitative and non-destructive nature makes it particularly suitable for analysing biological samples. The applications in biomedical research are manifold. The iron overload hypothesis in Parkinson\\\''s disease is investigated by a differential analysis of human substantia nigra. The trace element content is quantified in neuromelanin, in microglia cells, and in extraneuronal environment. A comparison of six Parkinsonian cases with six control cases revealed no significant elevation in iron level bound to neuromelanin. In fact, a decrease in the Fe/S ratio of Parkinsonian neuromelanin was measured, suggesting a modification in its iron binding properties. Drosophila melanogaster, or the fruit fly, is a widely used model organism in neurobiological experiments. The electrolyte elements are quantified in various organs associated with the olfactory signalling, namely the brain, the antenna and its sensilla hairs, the mouth parts, and the compound eye. The determination of spatially resolved element concentrations is useful in preparing the organ specific Ringer\\\''s solution, an artificial lymph that is used in disruptive neurobiological experiments. The role of trace elements in the progression of atherosclerosis is examined in a pilot study. A differential quantification of the element content in an induced murine atherosclerotic lesion reveals elevated S and Ca levels in the artery wall adjacent to the lesion and an increase in iron in the lesion. The 3D quantitative distribution of elements is reconstructed by means of stacking the 2D quantitative maps of consecutive sections of an artery. The feasibility of generating a quantitative elemental rodent brain atlas by Large Area Mapping is investigated by measuring at high beam currents. A whole coronal section of the rat brain was measured in segments in 14 h. Individual quantitative maps of the segments are pieced together to reconstruct a high-definition element distribution map of the whole section with a subcellular spatial resolution. The use of immunohistochemical staining enhanced with single elements helps in determining the cell specific element content. Its concurrent use with Large Area Mapping can give cellular element distribution maps

The Density of Cell Nuclei at the Materno-Fetal Exchange Barrier is Sexually Dimorphic in Normal Placentas, but not in IUGR

Abstract Placental sexual dimorphism is of special interest in prenatal programming. Various postnatal diseases with gender dependent incidence, especially neuropsychiatric disorders like schizophrenia and autism spectrum disorders, have prenatal risk factors established. However, the functional relevance of placental microarchitecture in prenatal programming is poorly investigated, mainly due to a lack of statistically efficient methods. We hypothesized that the recently established 3D microscopic analysis of villous trees would be able to identify microscopic structural correlates of human placental sexual dimorphism. We analyzed the density of cell nuclei of villous trophoblast, i.e. the materno-fetal exchange barrier, in placentas from term pregnancies. The cell nuclei were grouped into proliferative and non-proliferative nuclei by detection of a proliferation marker (PCNA). Normal female placentas showed a higher density of non-proliferating nuclei (PCNA-negative) in villous trophoblast than normal male placentas. The density of PCNA-negative cell nuclei was higher in placentas of pregnancies with intrauterine growth retardation (IUGR) than in control placentas. The data of the present study shows that the density of non-proliferative cell nuclei in the syncytial layer of villous trophoblast is influenced by fetal sex and by IUGR, while proliferation remains unchanged. A novel concept of post-fusion regulation of syncytial structure and function is proposed