Ultramicrotome Sectioning Research Articles

Automated stain-free histomorphometry of peripheral nerve by contrast-enhancing techniques and artificial intelligence

BackgroundTraditional histopathologic evaluation of peripheral nerve using brightfield microscopy is resource-intensive, necessitating complex sample preparation. Label-free imaging techniques paired with artificial intelligence-based image reconstruction and segmentation may facilitate peripheral nerve histomorphometry. New methodHerein, the utility of label-free phase contrast techniques paired with artificial intelligence-based image processing for imaging of mammalian peripheral nerve is demonstrated. ResultsFresh frozen murine sciatic nerve sections were imaged in transmission modalities using differential interference and phase contrast microscopy and in epifluorescent modality following staining with myelin-specific dye. Deep learning was employed to predict epifluorescent images from transmitted phase contrast images, and machine learning employed for automated segmentation of myelinated axons for reporting of axons counts and g-ratios. Comparison with existing methodsConventional peripheral nerve histomorphometry employs resource intensive resin embedding, ultra-microtome sectioning, and staining steps. Herein we demonstrate feasibility of high-throughput nerve histomorphometry via label-free phase contrast imaging of frozen sections. ConclusionsClinical applications of label-free phase contrast microscopy paired with deep learning algorithms are discussed.

Imaging the inner structure of chromosomes: contribution of focused ion beam/scanning electron microscopy to chromosome research.

Visualization of the chromosome ultrastructure has revealed new insights into its structural and functional properties. The use of new methods for revealing not only the surface but also the inner structure of the chromosome has been emerged. Some methods have long been used, such as conventional transmission electron microscopy (TEM). Although it has indispensably contributed to the revelation of the ultrastructure of the various biological samples, including chromosomes, some challenges have also been encountered, such as laborious sample preparation, limited view areas, and loss of information on some parts due to ultramicrotome sectioning. Therefore, a more advanced method is needed. Scanning electron microscopy (SEM) is also advantageous in the surface visualization of chromosome samples. However, it is limited by accessibility to gain the inner structure information. Focused ion beam/scanning electron microscopy (FIB/SEM) provides a way to investigate the inner structure of the samples in a direct slice-and-view manner to observe the ultrastructure of the inner part of the sample continuously and further construct a three-dimensional image. This method has long been used in the material science field, and recently, it has also been applied to biological research, such as in showing the inner structure of chromosomes. This review article presents the contributions of this new method to chromosome research and its recent developments in the inner structure of chromosome and discusses its current and potential applications to the high-resolution imaging of chromosomes.

Ultra-Microtome for the Preparation of TEM Specimens from Battery Cathodes.

With the wide application of ultra-microtome sectioning in the preparation of transmission electron microscopy (TEM) specimens with bio- and organic materials, here, we report an ultra-microtome-based method for the preparation of TEM specimens from cathodes of Li-ion batteries. The ultra-microtome sectioning reduces the sample thickness to tens of nanometers and yields atomic resolution from the core region of particles of hundreds of nanometers. Analysis indicates that the mechanical cross-sectioning introduces no observable microstructural artifacts or structural damage, such as microcracking and nanoporosity. These results demonstrate the high efficiency of the ultra-microtome approach in preparing well-thinned specimens of particulate materials that allow for atomic-scale TEM imaging of a large number of sectioned particles in one single TEM specimen, thereby providing statistically significant results of the TEM analysis.

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology.

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2μm thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy.

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.

A novel design of a scanning probe microscope integrated with an ultramicrotome for serial block-face nanotomography.

We present a new concept of a combined scanning probe microscope (SPM)/ultramicrotome apparatus. It enables "slice-and-view" scanning probe nanotomography measurements and 3D reconstruction of the bulk sample nanostructure from series of SPM images after consecutive ultrathin sections. The sample is fixed on a flat XYZ scanning piezostage mounted on the ultramicrotome arm. The SPM measuring head with a cantilever tip and a laser-photodiode tip detection system approaches the sample for SPM measurements of the block-face surface immediately after the ultramicrotome sectioning is performed. The SPM head is moved along guides that are also fixed on the ultramicrotome arm. Thereby, relative dysfunctional displacements of the tip, the sample, and the ultramicrotome knife are minimized. The design of the SPM head enables open frontal optical access to the sample block-face adapted for high-resolution optical lenses for correlative SPM/optical microscopy applications. The new system can be used in a wide range of applications for the study of 3D nanostructures of biological objects, biomaterials, polymer nanocomposites, and nanohybrid materials in various SPM and optical microscopy measuring modes.

Silver Enhancement for Transmission Electron Microscopy Imaging of Antibody Fragment-Gold Nanoparticles Conjugates Immobilized on Ordered Mesoporous Silica.

Ordered mesoporous silica (OMS) materials are receiving great attention as possible carriers for valuable but unstable drugs as, for example, therapeutic proteins. A key issue is to prove that the therapeutic protein is effectively able to penetrate the pores of OMS during the adsorption step. Here, we immobilized an antibody fragment [F(ab')GAMIgG] conjugated with ultrasmall gold nanoparticles (GNPs) onto amino-functionalized SBA-15 (SBA-NH2) mesoporous silica. The aim of this work is the visualization of the location of the conjugates adsorbed onto SBA-NH2 with transmission electron microscopy (TEM). Because of the ultrasmall size of GNPs (<1 nm), we use the silver enhancement procedure to amplify their size. In this procedure, ultrathin sections of conjugate-loaded SBA-NH2 particles are prepared by a ultramicrotome sectioning technique. The ultrasmall GNPs located on the top side of the 70-90 nm thick slices act as microcrystallization nucleation sites for the deposition of reduced metallic silver. Consequently, the ultrasmall GNPs increase their size. This allows for the direct imaging of the conjugates adsorbed. We clearly localize the F(ab')GAMIgG-GNPs conjugates either on the external surface of the particles or inside the mesopores of SBA-NH2 through TEM.

STEM mode in the SEM for the analysis of cellular sections prepared by ultramicrotome sectioning

The use of the dual imaging capabilities of a scanning electron microscope fitted with a transmitted electron detector is highlighted in the analysis of samples with importance in the field of nanotoxicology. Cellular uptake of nanomaterials is often examined by transmission electron microscopy of thin sections prepared by ultramicrotome sectioning. Examination by SEM allows for the detection of artefacts caused by sample preparation (eg. nanomaterial pull-out) and the complementary STEM mode permits study of the interaction between nanomaterials and cells. Thin sections of two nanomaterials of importance in nanotoxicology (cadmium selenide quantum dots and single walled carbon nanotubes) are examined using STEM mode in the SEM.

STEM mode in the SEM: A practical tool for nanotoxicology

The addition of a transmitted electron detector to a scanning electron microscope (SEM) allows the recording of bright and dark field scanning transmission electron microscope (STEM) images and the corresponding in-lens secondary electron images from the same region of a thin sample. These combined imaging techniques have been applied here to the analysis of ultrathin sections of cells exposed in vitro to nanomaterials for toxicology investigation. Electron microscopy in general permits the exact nature of the interaction of nanomaterials and cells to be elucidated, and in addition the use of STEM mode in the SEM enables the easy identification and exclusion of artefacts produced by ultramicrotome sectioning. The imaging and analysis obtained by using the STEM mode in the SEM configuration from three different nanomaterial systems of importance (iron oxide nanoparticles, single-walled carbon nanotubes and cadmium selenide quantum dots) indicate that it is a simple, practical and cost-effective tool for nanotoxicological research.

Array Tomography: Production of Arrays

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time consuming and require some practice to perfect. This protocol describes the sectioning of embedded tissues and the mounting of the serial arrays. The procedures require some familiarity with the techniques used for ultramicrotome sectioning for electron microscopy.

Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy

We describe the use of transmission electron microscopy (TEM) for cellular ultrastructural examination of nanoparticle (NP)-exposed biomaterials. Preparation and imaging of electron-transparent thin cell sections with TEM provides excellent spatial resolution (approximately 1 nm), which is required to track these elusive materials. This protocol provides a step-by-step method for the mass-basis dosing of cultured cells with NPs, and the process of fixing, dehydrating, staining, resin embedding, ultramicrotome sectioning and subsequently visualizing NP uptake and translocation to specific intracellular locations with TEM. In order to avoid potential artifacts, some technical challenges are addressed. Based on our results, this procedure can be used to elucidate the intracellular fate of NPs, facilitating the development of biosensors and therapeutics, and provide a critical component for understanding NP toxicity. This protocol takes approximately 1 week.

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT-MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3-D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.

A TEM study of samples from acid mine drainage systems: metal-mineral association with implications for transport

Transmission electron microscopy (TEM), with energy dispersive X-ray (EDX) analysis and energy filtered transmission electron microscopy/electron energy loss spectroscopy (EFTEM/EELS), as well as powder X-ray diffraction (XRD) and scanning electron microscopy (SEM), have been used to study bed sediments from two acid mine drainage (AMD) sites in western Montana, USA. TEM and associated techniques, including sample preparation via epoxy impregnation and ultramicrotome sectioning, afford the opportunity to better interpret and understand complex water-rock interactions in these types of samples. For the sample taken from the first site (Mike Horse mine), ferrihydrite is the dominant phase, Si and Zn are the most abundant elements sorbed to ferrihydrite surfaces, and Pb is notably absent from ferrihydrite association. Three additional important metal-containing phases (gahnite, hydrohetaerolite, and plumbojarosite), that were not apparent in the powder XRD pattern because of their relatively low concentration, were identified in the TEM. The presence of these phases is important, because, for example, gahnite and plumbojarosite act as sinks for Zn and Pb, respectively. Therefore, the mobility of Pb from this part of the drainage system depends on the stability of plumbojarosite and the ability of ferrihydrite to sorb the released Pb. From thermodynamic data in the literature, we predict that Pb will be released by the dissolution of plumbojarosite above a pH of 4 to 5, but it will then be recaptured by ferrihydrite if the pH continues to rise to 5.5 and higher, irrespective of competition effects from other metals. Therefore, only a relatively narrow pH window exists in which Pb can escape this portion of the system as an aqueous species. For the sample taken from the other site included in this study (the Carbonate mine), jarosite and quartz are the dominant phases. Interestingly, however, the jarosites are both Pb-poor and Pb-enriched. In addition, TEM reveals the presence of microcrystalline hematite with Si, S, and P sorbed to its surfaces, a nearly pure amorphous Si, Al oxyhydroxide, and an amorphous silica phase containing minor amounts of Al, Ca, and Fe. Pb will probably be released from these mixed K-Pb jarosites above pH 4 to 5, but the Pb may be retarded by the strongly adsorbing microcrystalline hematite in this pH range. The sink for Al in this system is the amorphous Si, Al oxyhydroxide, not Al(OH)3 which is typically used in AMD modeling schemes.

Pretreatment of aluminium: topography, surface chemistry and adhesive bond durability

This paper reports on work in which bonded joints between a clad aluminium alloy (L165, comprising Cu 4.4%, Mg 0.5%, Si 0.8%, Mn 0.8%), with different pretreatments, and an epoxy resin (Ciba-Geigy's Redux 312/5) have been examined. The datum for comparison of the pretreatments is the Boeing phosphoric acid anodization (BAC 5555). Other treatments include sulfuric acid anodizing in combination with a phosphoric acid dip and a sulfuric acid/ferric sulfate etch. The topographical structure of the surface layers formed has been examined using scanning and transmission electron microscopy in conjunction with ultramicrotome sectioning. The chemical composition of pretreated surfaces has been characterized by X-ray photoelectron spectroscopy and depth profiling using argon-ion etch performed. Adhesive bonds have been prepared and their durability assessed using wedge test specimens. The data collected have been used to calculate crack growth and strain energy release rate as functions of time for the bonds produced. Surfaces of failed specimens have been examined to establish the locus of failure.

A combined scanning and transmission electronmicroscopic investigation of human (secondary) cataract material

Cataract lenses from patients of advanced age were processed for SEM by standard pre-fixation followed by treatment by the Tannin-Arginine-Osmium-tetroxide (TAO) method and critical point drying, and for TEM by standard pre-fixation followed by vibratomation, standard post-fixation, ultramicrotome sectioning and staining with uranyl acetate/lead citrate. Secondary cataract material was brought onto a Millipore filter, fixed by standard methods, dried in air and sputter-coated with Au. Both SEM and TEM images revealed degeneration processes in lensfibre material, such as swelling of the lensfibre, protrusion of the cytoplasm, fibrillation of the cell membrane, loss of the nucleus, spherical bodies of various sizes between 0.5-1.5 microns, sometimes surrounded by a (double) membrane with different contrast but without cellular evidence, and small and large vacuoles partly filled with granular material both in and at the periphery of the lensfibre-body. The secondary cataract material on the Millipore filter revealed erythrocytes and more or less spherical bodies with high contrast, measuring between 0.5-1.5 microns, often referred to as Elschnig's pearls, besides non-definable organic material. The SEM and TEM micrographs of the cataract lens material strongly suggest that the spherical bodies with sizes of approximately 0.5-1.5 micrometer and high contrast without cellular evidence, are similar to the more or less spherical bodies found in the secondary cataract material on the filter, referred to as Elschnig's pearls.

High-Resolution Imaging of Ordered Mixed-Layer Clays

AbstractHigh-resolution transmission electron microscopy (HRTEM) has been used to examine illite/smectite from the Mancos Shale; rectorite from Garland County, Arkansas; illite from Silver Hill, Montana; Na-smectite from Crook County, Wyoming; corrensite from Packwood, Washington; and diagenetic chlorite from the Tuscaloosa Formation. Thin specimens were prepared by ion milling, ultramicrotome sectioning, and/or grain dispersal on a holey carbon substrate. Some smectite-bearing clays were also examined after intercalation with dodecylamine hydrochloride (DH). Intercalation of smectite with DH proved to be a reliable method for HRTEM imaging of expanded smectite (d (001) = 16 Å) which could then be distinguished from unexpanded illite (d (001) = 10 Å). Lattice fringes of basal spacings of DH-intercalated rectorite and illite/smectite showed a 26-Å periodicity. These data support X-ray powder diffraction (XRD) studies which suggest that these samples are ordered, interstratified varieties of illite and smectite. The ion-thinned, unexpanded corrensite sample showed discrete crystallites containing 10-Å and 14-Å basal spacings corresponding to collapsed smectite and chlorite, respectively. Regions containing disordered layers of chlorite and smectite were also noted. Crystallites containing regular alternations of smectite and chlorite layers were not common. These HRTEM observations of corrensite did not corroborate XRD data. Particle sizes parallel to the c axis ranged widely for each sample studied, and many particles showed basal dimensions equivalent to more than five layers. For all illite, smectite, and illite/ smectite particles examined, crystallite sizes of about 20 Å in the basal dimension were not observed.

Techniques for preparing fossil palynomorphs for study with the scanning and transmission electron microscopes

An improved mounting technique involving a simplified micromanipulator is described which makes it easier to examine fossil palynomorphs with scanning and transmission electron microscopes. For scanning electron microscopy, individual fossil palynomorphs are picked from dried residues with the micromanipulator and arranged on adhesive-coated coverslips. A polyvinyl chloride adhesive and a polyurethane adhesive proved to be superior among the twenty-three adhesives tested. After mounting, the coverslips are cemented on SEM specimen stages, and the fossils are coated with a thin film of gold-palladium alloy in a vacuum evaporator. For transmission electron microscopy, the fossil palynomorphs are placed directly on an agar cube, covered with an additional agar drop, and then impregnated with an epoxy resin for ultramicrotome sectioning.