Carmen Quintana

Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies.

Cryofixation, cryosubstitution and cryoembedding are a set of low-temperature methods for immunocytochemical and microanalytical ultrastructural studies. This review covers the theoretical and practical aspects of these cryomethods, simple, low-cost, safe devices that provide reproducible results and a summary of recent results. Sections prepared by these three cryomethods can be used to determine elemental composition, molecular composition, functions and 3-D ultrastructure. The information obtained can be treated by multivariate statistical methods. Thus, each cellular compartment can be identified by its morphology, molecular and elemental composition and function and changes in these data during physiological and pathological processes can be monitored.

Could a dysfunction of ferritin be a determinant factor in the aetiology of some neurodegenerative diseases

BACKGROUND The concentration of iron in the brain increases with aging. Furthermore, it has also been observed that patients suffering from neurological diseases (e.g. Parkinson, Alzheimer...) accumulate iron in the brain regions affected by the disease. Nevertheless, it is still not clear whether this accumulation is the initial cause or a secondary consequence of the disease. Free iron excess may be an oxidative stress source causing cell damage if it is not correctly stored in ferritin cores as a ferric iron oxide redox-inert form. SCOPE Both, the composition of ferritin cores and their location at subcellular level have been studied using analytical transmission electron microscopy in brain tissues from progressive supranuclear palsy (PSP) and Alzheimer disease (AD) patients. MAJOR CONCLUSIONS Ferritin has been mainly found in oligodendrocytes and in dystrophic myelinated axons from the neuropili in AD. In relation to the biomineralization of iron inside the ferritin shell, several different crystalline structures have been observed in the study of physiological and pathological ferritin. Two cubic mixed ferric-ferrous iron oxides are the major components of pathological ferritins whereas ferrihydrite, a hexagonal ferric iron oxide, is the major component of physiological ferritin. We hypothesize a dysfunction of ferritin in its ferroxidase activity. GENERAL SIGNIFICANCE The different mineralization of iron inside ferritin may be related to oxidative stress in olygodendrocites, which could affect myelination processes with the consequent perturbation of information transference.

Iron speciation study in Hfe knockout mice tissues: Magnetic and ultrastructural characterisation

Liver, spleen and heart tissues of DBA/2 Hfe knockout mice have been characterised by low temperature AC magnetic susceptibility measurements together with Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction in order to investigate the chemical iron speciation in a murine model of iron overload diseases. With emphasis on ferritin-like species, the temperature dependent in-phase and out-of-phase susceptibility profiles agree with the elemental analysis in that, in this model, iron accumulation takes place in the hepatic tissue while in the spleen and heart tissues no differences have been observed between knockout and wild type animals. The comparison of the magnetic properties between perfused and non-perfused liver tissues has made it possible to estimate the magnetic contribution of usually present blood remains. The TEM observations reveal that, besides the isolated ferritins and ferritin-containing lysosomes-siderosomes present in the hepatocytes, other iron deposits, of heterogeneous size, morphology and crystalline structure (haematite and/or goethite), are present in the cytoplasm, near the membrane, and in extracellular spaces.

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures.

Abstract Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666–9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce “true P-maps”. A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the “useful information” present in the inelastic image series (characteristic signal) from the “non-useful information” (noise and acquisition artifacts). The reconstitution of the original image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation intermediate coronavirus particles found inside infected cells.

About the Presence of Hemosiderin in the Hippocampus of Alzheimer Patients

Ferritin and Hemosiderin (Hm) are the iron-storing “elements” in cells. Whereas Ferritin is a wellcharacterized soluble protein that has been extensively studied [1–5], the term Hemosiderin does not have the same clarity of meaning for the pathologist, biochemist or electron microscopist. For the pathologist [6], Hm represents iron-containing conglomerates, stainable with Perl’s stain. For the biochemist, Hm is a heterogeneous insoluble compound, containing iron, proteins, carbohydrates and lipids. For the electron microscopist, Ferritin and Hm are nanoparticles (5– 7 nm in diameter), recognizable because of the electrondensity of the iron concentrated in their “cores”. Hm has been observed in various tissues and organs, including the liver, spleen, heart, intestines, pancreas and tumors (neuroblastomes), associated with ironoverload pathologies such as primary (PH) and secondary (SH) hemochromatosis and transfusional or local bleeding siderosis [6–8]. Hm is considered a product of degradation of Ferritin localized within siderosomes [4,7]. In Ferritin, iron is mainly stored as ferrihydrite, hydrated Fe iron oxide nanocrystals [1– 3]. In Hm, several different iron oxyhydroxide mineral structures have been identified depending on the disease [9–11]. By employing electron microscopy, Hm can be distinguished from Ferritin when individual particles cannot be resolved or when they come closer than 13 nm (the mean external diameter of one Ferritin protein shell [2]). We have observed, using electron microscopy, Hm in the hippocampus of AD patients [12,13]. Figures 1 and 2 show two examples of Hm rich-regions: (1) the cytoplasm of oligodentrocytes near the nuclei; in these cells Ferritin is often observed in the nucleoplasm [12, 13] and (2) the oligodendrocyte processes associated with myelinated axons devoid of abnormal accumulations of filaments [13]. These oligodendrocyte processes also contain numerous Ferritin molecules and the myelin sheaths of these axons are frayed. What could be the role of Hm in oligodendrocytes ? Let us remind ourselves of some of the known roles of iron in the brain.

Ultramicrotomy for cross-sections of nanostructure

Abstract Ultramicrotomy is a thin sample preparative method for TEM observations. In materials science, it can be used as an alternative method to mechanical polishing and ion-milling for cross-section specimen preparation. This short communication reports results obtained for two different materials: a laser structure based in semiconductor technology (InP/GaInAs) prepared by MBE and a AuSiFe multilayer prepared by co-sputtering. In both cases, Epon embedding medium and a diamond knife were used. The resulting cross-sections revealed good preservation of the different regions of the laser device and the presence of defects in the layers of the AuFeSi structure. It was concluded that ultramicrotomy can be used as a routine preparative method for TEM observations of nanostructures.

Comment to “Mapping and Characterization of Iron Compounds in Alzheimer's Tissue”

The recent article of Collingwood and Dobson [1] is an important article that emphasizes the importance of the characterization and mapping of iron compounds in the iron-rich brain regions with the aim contributing to our understanding of the role of pathological accumulations of iron in the regions of the brain affected by neurodegenerative disease. Nonetheless, below, I discuss ion and electron microprobe techniques for detecting and quantifying iron, not specifically cited by the authors, that allow the mapping of total iron (SIMS and XEDS) at the cellular and sub cellular level and the characterization and mapping of iron compounds (EELS) at ultra structural level. SIMS (Secondary Ion Mass Spectroscopy) imaging technique, allows direct identification of chemical elements with high sensitivity and specificity and, as a consequence, elemental distribution can be visualized (chemical mapping) by SIMS imaging. The physical basis of the method is the following: under the bombardment of the samples by primary ions, the monoatomic or polyatomic species that composed the analyzing object are sputtered. One part of these emitted species is ionized and the SIMS instrument, with the help of a mass spectrometer, sort and maps the ejected ions by their m/e ratio. The latest generation of SIMS instruments, the NanoSIMS-50 instrument, operating in scanning mode, is equipped with a parallel detection system that allows the simultaneous acquisition of five elements which insures a perfect colocalization between simultaneously recorded images. This instrument is particularly useful to identify elements at a sub cellular level, because it is possible to attain resolutions of 50–100 nm with the Cs source and 150–200 nm with the O− source [2]. NanoSIMS microscopy has been already used with success for the visualization of the morphological and chemical alterations taking place in well-characterized regions in pathological brain, in particular in the study of iron distribution in Alzheimer disease tissue [3,4]. Multielemental analysis (nitrogen, phosphorus, sulphur and iron) were performed on semi-thin or ultra-thin sections of Transmission Electron Microscopy (TEM) preparations brain tissue. The possibility of using light microscopy, TEM, and SIMS on the same semi-thin and ultra-thin sections allows correlation between structural and analytical observations at sub-cellular and ultrastructural level. It has been shown that the iron-rich region mapped by nanoSIMS in the hippocampus of AD patients are ferritin and/or hemosiderin rich regions. XEDS (X-Ray Energy dispersive Spectroscopy) and EELS (Electron Energy Loss Spectroscopy) are electron probe nanoanalysis techniques that, associated with transmission electron microscopes (ConventionalTEM, ScanningTEM or ConventionalTEM working in scanning mode, so-called AnalyticalTEM, ATEM), provide compositional maps of ultra-thin sections with nanometric resolution (1–10 nm): XEDS by detecting the element specific X-ray emission under excitation of incident electrons: EELS by detecting element specific energy loss of incident electrons. EELS provides theoretically higher detection sensitivity than EDS due to the larger number of primary events collected in the