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Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been established as a powerful technique for the determination of metal and nonmetal distributions within biological systems with high sensitivity. An imaging LA-ICP-MS technique for Fe, Cu, Zn, and Mn was developed to produce large series of quantitative element maps in native brain sections of mice subchronically intoxicated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) as a model of Parkinson’s disease. Images were calibrated using matrix-matched laboratory standards. A software solution allowing a precise delineation of anatomical structures was implemented. Coronal brain sections were analyzed crossing the striatum and the substantia nigra, respectively. Animals sacrificed 2 h, 7 d, or 28 d after the last MPTP injection and controls were investigated.
We observed significant decreases of Cu concentrations in the periventricular zone and the fascia dentata at 2 h and 7d and a recovery or overcompensation at 28 d, most pronounced in the rostral periventricular zone (+40%). In the cortex Cu decreased slightly to −10%. Fe increased in the interpeduncular nucleus (+40%) but not in the substantia nigra. This pattern is in line with a differential regulation of periventricular and parenchymal Cu, and with the histochemical localization of Fe, and congruent to regions of preferential MPTP binding described in the rodent brain.
The LA-ICP-MS technique yielded valid and statistically robust results in the present study on 39 slices from 19 animals. Our findings underline the value of routine micro-local analytical techniques in the life sciences and affirm a role of Cu availability in Parkinson’s disease.
Polarised light imaging (PLI) utilises the birefringence of the myelin sheaths in order to visualise the orientation of nerve fibres in microtome sections of adult human post-mortem brains at ultra-high spatial resolution. The preparation of post-mortem brains for PLI involves fixation, freezing and cutting into 100-μm-thick sections. Hence, geometrical distortions of histological sections are inevitable and have to be removed for 3D reconstruction and subsequent fibre tracking. We here present a processing pipeline for 3D reconstruction of these sections using PLI derived multimodal images of post-mortem brains. Blockface images of the brains were obtained during cutting; they serve as reference data for alignment and elimination of distortion artefacts. In addition to the spatial image transformation, fibre orientation vectors were reoriented using the transformation fields, which consider both affine and subsequent non-linear registration. The application of this registration and reorientation approach results in a smooth fibre vector field, which reflects brain morphology. PLI combined with 3D reconstruction and fibre tracking is a powerful tool for human brain mapping. It can also serve as an independent method for evaluating in vivo fibre tractography.
Bioimaging of Metals by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS)
(2010)
The distribution analysis of (essential, beneficial, or toxic) metals (e.g., Cu, Fe, Zn, Pb, and others), metalloids, and non‐metals in biological tissues is of key interest in life science. Over the past few years, the development and application of several imaging mass spectrometric techniques has been rapidly growing in biology and medicine. Especially, in brain research metalloproteins are in the focus of targeted therapy approaches of neurodegenerative diseases such as Alzheimer's and Parkinson's disease, or stroke, or tumor growth. Laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS) using double‐focusing sector field (LA‐ICP‐SFMS) or quadrupole‐based mass spectrometers (LA‐ICP‐QMS) has been successfully applied as a powerful imaging (mapping) technique to produce quantitative images of detailed regionally specific element distributions in thin tissue sections of human or rodent brain. Imaging LA‐ICP‐QMS was also applied to investigate metal distributions in plant and animal sections to study, for example, the uptake and transport of nutrient and toxic elements or environmental contamination. The combination of imaging LA‐ICP‐MS of metals with proteomic studies using biomolecular mass spectrometry identifies metal‐containing proteins and also phosphoproteins. Metal‐containing proteins were imaged in a two‐dimensional gel after electrophoretic separation of proteins (SDS or Blue Native PAGE). Recent progress in LA‐ICP‐MS imaging as a stand‐alone technique and in combination with MALDI/ESI‐MS for selected life science applications is summarized.
For accurate registration of histological sections blockface images are frequently used as three dimensional reference. However, due to the use of endocentric lenses the images suffer from perspective errors such as scaling and seemingly relative movement of planes which are located in different distances parallel to the imaging sensor. The suggested correction of those errors is based on the estimation of scaling factors derived from image registration of regions characterized by differing distances to the point of view in neighboring sections. The correction allows the generation of a consistent three dimensional blockface volume.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinson’s disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.
Polarized light imaging (PLI) enables the evaluation of fiber orientations in histological sections of human postmortem brains, with ultra-high spatial resolution. PLI is based on the birefringent properties of the myelin sheath of nerve fibers. As a result, the polarization state of light propagating through a rotating polarimeter is changed in such a way that the detected signal at each measurement unit of a charged-coupled device (CCD) camera describes a sinusoidal signal. Vectors of the fiber orientation defined by inclination and direction angles can then directly be derived from the optical signals employing PLI analysis. However, noise, light scatter and filter inhomogeneities interfere with the original sinusoidal PLI signals. We here introduce a novel method using independent component analysis (ICA) to decompose the PLI images into statistically independent component maps. After decomposition, gray and white matter structures can clearly be distinguished from noise and other artifacts. The signal enhancement after artifact rejection is quantitatively evaluated in 134 histological whole brain sections. Thus, the primary sinusoidal signals from polarized light imaging can be effectively restored after noise and artifact rejection utilizing ICA. Our method therefore contributes to the analysis of nerve fiber orientation in the human brain within a micrometer scale.