Heavy Metal Staining Research Articles

Voxel dose-limited resolution for thick beam-sensitive specimens imaged in a TEM or STEM

The resolution limit imposed by radiation damage is quantified in terms of a voxel dose-limited resolution (DLR), applicable to small features within a thick specimen. An analytical formula for this DLR is derived and applied to bright-field mass-thickness contrast from organic (polymer or biological) specimens of thickness between 400 nm and 20 µm. For a permissible dose of 330 MGy (typical of frozen-hydrated tissue), the TEM or STEM image resolution is determined by radiation damage rather than by lens aberrations or beam-broadening effects, which can be restricted by use of a small angle-limiting aperture. DLR is improved by a up to factor of 2 by increasing the primary-electron energy from 300 keV to 3 MeV, or by up to a factor of 3 by heavy-metal staining. For stained samples, a higher electron fluence allows better resolution but the improvement is modest because the voxel DLR is proportional to the 1/4 power of electron dose. The relevance of voxel and columnar DLR is discussed, for both thick and thin samples.

Lipids and Proteins Differentiation in Membrane Fouling Using Heavy Metal Staining and Electron Microscopy at Cryogenic Temperatures.

The detailed characterization of fouling in membranes is essential to understand any observed improvement or reduction on filtration performance. Electron microscopy allows detailed structural characterization, and its combination with labeling techniques, using electron-dense probes, typically allows for the differentiation of biomolecules. Developing specific protocols that allow for differentiation of biomolecules in membrane fouling by electron microscopy is a major challenge due to both as follows: the necessity to preserve the native state of fouled membranes upon real filtration conditions as well as the inability of the electron-dense probes to penetrate the membranes once they have been fouled. In this study, we present the development of a heavy metal staining technique for identification and differentiation of biomolecules in membrane fouling, which is compatible with cryofixation methods. A general contrast enhancement of biomolecules and fouling is achieved. Our observations indicate a strong interaction between biomolecules: A tendency of proteins, both in solution as well as in the fouling, to surround the lipids is observed. Using transmission electron microscopy and scanning electron microscopy at cryogenic conditions, cryo-SEM, in combination with energy-dispersive X-ray spectroscopy, the spatial distribution of proteins and lipids within fouling is shown and the role of proteins in fouling discussed.

Preserving extracellular space for high-quality optical and ultrastructural studies of whole mammalian brains

Analysis of brain structure, connectivity, and molecular diversity relies on effective tissue fixation. Conventional tissue fixation causes extracellular space (ECS) loss, complicating the segmentation of cellular objects from electron microscopy datasets. Previous techniques for preserving ECS in mammalian brains utilizing high-pressure perfusion can give inconsistent results owing to variations in the hydrostatic pressure within the vasculature. A more reliable fixation protocol that uniformly preserves the ECS throughout whole brains would greatly benefit a wide range of neuroscience studies. Here, we report a straightforward transcardial perfusion strategy that preserves ECS throughout the whole rodent brain. No special setup is needed besides sequential solution changes, and the protocol offers excellent reproducibility. In addition to better capturing tissue ultrastructure, preservation of ECS has many downstream advantages such as accelerating heavy-metal staining for electron microscopy, improving detergent-free immunohistochemistry for correlated light and electron microscopy, and facilitating lipid removal for tissue clearing.

Toward Visualizing Genomic DNA Using Electron Microscopy via DNA Metallization

Electron microscopy‐based DNA imaging is a powerful tool that provides a high resolution for observing genomic structures involved in biochemical processes. The first method, heavy metal shadow casting, was developed in 1948. Uranyl acetate has been widely used for DNA electron microscopic imaging since the 1960s. However, for this method, scientists must deal with government regulations for the safety and disposal. Additionally, sample preparation is often complicated and time‐consuming. Recently, nanoparticles and nanowires have emerged as a new way of imaging DNA molecules under both transmission and scanning electron microscopes. However, as this technology is still in its early stages, there is room for further development. In this review, heavy metal staining, nanoparticle staining, and nanowire growth for DNA visualization are introduced. The applications of shadow casting and uranyl acetate staining in the visualization of DNA structures and protein–DNA complexes are discussed. Then, nanomaterial‐based DNA staining methods are covered, including electrostatic interactions, DNA chain modification, reducing‐group‐modified DNA ligands and DNA–peptide/protein interactions. This review provides up‐to‐date information on different DNA staining approaches and their applications in DNA studies. Ultimately, it offers a new direction for genome analysis through DNA visualization.

Immersion Fixation and Staining of Multicubic Millimeter Volumes for Electron Microscopy–Based Connectomics of Human Brain Biopsies

Connectomics allows mapping of cells and their circuits at the nanometer scale in volumes of approximately 1 mm3. Given that the human cerebral cortex can be 3 mm in thickness, larger volumes are required. Larger-volume circuit reconstructions of human brain are limited by 1) the availability of fresh biopsies; 2) the need for excellent preservation of ultrastructure, including extracellular space; and 3) the requirement of uniform staining throughout the sample, among other technical challenges. Cerebral cortical samples from neurosurgical patients are available owing to lead placement for deep brain stimulation. Described here is an immersion fixation, heavy metal staining, and tissue processing method that consistently provides excellent ultrastructure throughout human and rodent surgical brain samples of volumes 2 × 2 × 2 mm3 and up to 37 mm3 with one dimension ≤2 mm. This method should allow synapse-level circuit analysis in samples from patients with psychiatric and neurologic disorders.

Utilizing micro-CT imaging to define collagen modeling in murine aorta.

We aim to demonstrate local aortic tissue biodynamics via collagen and elastin fiber remodeling in murine aorta at varying distension using microcomputed tomography (microCT). Ten week-old C57BL/6 mice were injected via tail vein with in-vivo collagen hybridizing peptide, which reforms the triple-helix structure of denatured collagen, and is visualized via fluorescein tag. Careful dissection and removal of the thoracoabdominal aorta was performed, which was pressurized with formalin at varying distensions. Samples were then prepared via heavy-metal staining, and fixed in epoxy resin. These were subsequently imaged at the microCT scanner at University of Chicago, as well as at the 2-BM-B beamline using the Advanced Photon Source at Argonne National Laboratory to obtain micron and sub-micron resolution images and thus provide multi-level views of the tissue. Clear visualization of distinct collagen and elastin layers was observed with microCT. The images also clearly illustrate the effect on individual collagen and elastin fiber organization created by overdistension and shear-stress on the aorta. Clear demonstration of the geometric structure of aortic elastin fibers was seen upon initial segmentation. Preliminary experiments demonstrate that microCT provides an excellent visualization of murine aorta at micron and submicron levels. Next steps will be to segment and mesh this data in order to simulate local tissue responses to stress via finite element analysis. This method will produce a multi-scale imaging model of aortic failure.

Automated large volume sample preparation for vEM.

New developments in electron microscopy technology, improved efficiency of detectors, and artificial intelligence applications for data analysis over the past decade have increased the use of volume electron microscopy (vEM) in the life sciences field. Moreover, sample preparation methods are continuously being modified by investigators to improve final sample quality, increase electron density, combine imaging technologies, and minimize the introduction of artifacts into specimens under study. There are a variety of conventional bench protocols that a researcher can utilize, though most of these protocols require several days. In this work, we describe the utilization of an automated specimen processor, the mPrep™ ASP-2000™, to prepare samples for vEM that are compatible with focused ion beam scanning electron microscopy (FIB-SEM), serial block face scanning electron microscopy (SBF-SEM), and array tomography (AT). The protocols described here aimed for methods that are completed in a much shorter period of time while minimizing the exposure of the operator to hazardous and toxic chemicals and improving the reproducibility of the specimens' heavy metal staining, all without compromising the quality of the data acquired using backscattered electrons during SEM imaging. As a control, we have included a widely used sample bench protocol and have utilized it as a comparator for image quality analysis, both qualitatively and using image quality analysis metrics.

Sorption studies of Cr (VI) ions from synthetic waste water using chitosan embedded in calcium alginate beads

In current scenario, heavy metal stains of aquatic media have become a solemn ecological issue for which much concern has been expressed. These heavy metals include chromium, lead, mercury, arsenic, and cadmium. Industrialization, agricultural activities, the rapid growth of the global population, excessive use of chemicals, and uncontrolled urbanization are the main causes of heavy metal pollution. The excellent adsorption behavior of Chitosan and its modifications to various toxic heavy metals from aqueous solutions has garnered a lot of attention over the past few decades. In the present work Chitosan and immobilized Chitosan were used to compare the amount of chromium (VI) removal by altering limiting variables like Cr VI concentration, adsorbent loading, Hydrogen ion concentration and time of contact. Kinetic studies were also studied along with the isotherm models like Langmuir, Freundlich and Temkin models to analyze the experimental data and understand the adsorption performance. The efficiency of the adsorbents was checked by carrying out reusability studies by subjecting the adsorbents for repeated cycles of sorption studies. Results revealed that the immobilized biopolymers exhibited higher sorption capacity of 12.7 mg/g whereas the unmodified Chitosan exhibited 7.3 mg/g and also the efficiency of the immobilized biopolymers did not decrease with 2 – 3 cycles.

A versatile enhanced freeze-substitution protocol for volume electron microscopy.

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (Hordeum vulgare), nematode (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.

One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM.

Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an “all in one” sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.

Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM) for the identification of synaptic contacts in neural tissue. Peroxidases, in particular, can efficiently metabolize 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in a multiplexed manner in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) that are favored for the large-scale ultrastructural assessment of synaptic architecture However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) can efficienty deposit EM-visible DAB products in thick tissue without detergent treatment opening the possibility for the multiplex labeling of genetically defined cell-types in combination with volume EM methods. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

Whole-cell observation of ZIO-stained Golgi apparatus in rat hepatocytes with serial block-face scanning electron microscope, SBF-SEM.

The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with serial block-face scanning electron microscope (SBF-SEM), a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae, and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of zinc, iodine and osmium (ZIO) staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: (i) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; (ii) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.

New Workflow for Cryo-Electron Tomography Delivers Accurate Three-Dimensional Data Without Need of Averaging Multiple Experiments, Therefore Generating New Evidence of Mechanisms of Action

It has been recently demonstrated experimentally that, after a temperature conditioning prior exposure to the electron beam, flashfrozen biological samples (whether or not also exposed to a heavy-metal stain) resist high electron doses inside a Transmission Electron Microscope. This first proof of concept has been limited only by the resolution of the instrument used, not equipped with aberration corrector. In such conditions, published data on individual samples cannot match the Å-resolution commonly shown by cryo-electron tomograms, which data are averages over thousands of low-resolution images. Indeed, corresponding calculated structures in publications show the most probable position of each atom in the imaged proteins, obtained with a statistical Angstrom-level uncertainty. Within such instrumental limitations, the data shown here on cryo-immobilized samples are instead the first three-dimensional images documenting precisely the organization of proteins in a solution, flash-frozen in action and imaged directly, without the need to average many experiments. Therefore, these figures show only instantaneous molecular configurations. Actually, it is rather well-known that during action (i.e. during a protein-protein interaction) atoms are not necessarily in their average positions. Therefore, this new sample pre-conditioning, done at low-temperature and before exposure to the electron beam, delivers for the first-time accurate 3D measurements of protein organization in solution. This direct experimental evidence altogether, if confirmed, may constitute a basis to revisit some theoretical models describing some protein functions, e.g. for the two cases presented here, the influenza virus and tubulin, i.e. the most abundant protein in the human body.

New Workflow for Cryo-Electron Tomography Delivers Accurate Three-Dimensional Data Without Need of Averaging Multiple Experiments, Therefore Generating New Evidence of Mechanisms of Action

It has been recently demonstrated experimentally that, after a temperature conditioning prior exposure to the electron beam, flashfrozen biological samples (whether or not also exposed to a heavy-metal stain) resist high electron doses inside a Transmission Electron Microscope. This first proof of concept has been limited only by the resolution of the instrument used, not equipped with aberration corrector. In such conditions, published data on individual samples cannot match the Å-resolution commonly shown by cryo-electron tomograms, which data are averages over thousands of low-resolution images. Indeed, corresponding calculated structures in publications show the most probable position of each atom in the imaged proteins, obtained with a statistical Angstrom-level uncertainty. Within such instrumental limitations, the data shown here on cryo-immobilized samples are instead the first three-dimensional images documenting precisely the organization of proteins in a solution, flash-frozen in action and imaged directly, without the need to average many experiments. Therefore, these figures show only instantaneous molecular configurations. Actually, it is rather well-known that during action (i.e. during a protein-protein interaction) atoms are not necessarily in their average positions. Therefore, this new sample pre-conditioning, done at low-temperature and before exposure to the electron beam, delivers for the first-time accurate 3D measurements of protein organization in solution. This direct experimental evidence altogether, if confirmed, may constitute a basis to revisit some theoretical models describing some protein functions, e.g. for the two cases presented here, the influenza virus and tubulin, i.e. the most abundant protein in the human body

Energy dispersive X-ray spectrometry reveals an unexpected variability in electron microscopy stain distribution within cells and tissues

Heavy metal staining of biological material remains an important topic for electron microscopists, particularly with the rise of three-dimensional (3D) scanning electron microscopy (SEM) techniques and the ultrastructural study of large tissue volumes, which are often not compatible with cryo-imaging methods. There are a huge variety of stains that have been used to generate contrast in biological samples and many publications have gone into detail about how to apply these stains and what structures they bind or have affinity to in different sample types.

UA-Zero as a Uranyl Acetate Replacement When Diagnosing Primary Ciliary Dyskinesia by Transmission Electron Microscopy.

Primary ciliary dyskinesia (PCD) is a disorder affecting motile cilia. An early accurate diagnosis helps prevent lung damage and preserve lung function. To make a diagnostic assessment, one of the commonly used methods that allows for the examination of ciliary ultrastructure is transmission electron microscopy (TEM). This allows for a quantitative assessment of ciliary components to identify defects associated with PCD. Heavy metal staining is required to provide a contrast when imaging cilia in the TEM. One of the most commonly used stains is uranyl acetate (UA). UA can be applied to cellular material before embedding (en bloc), or to ultrathin sections of embedded samples (grid staining). UA is radioactive and, due to growing safety concerns and restrictions by government bodies, universities and hospitals, it is essential to find a suitable alternative. We show UA-zero (UAZ), when used en bloc, provides a high contrast and is a suitable replacement for UA. PCD diagnostic experts, having reviewed ciliary cross-sections stained with UAZ en bloc, are confident that the staining and PCD defects are readily detectable similar to samples that have been stained with UA.

Laboratory phase-contrast nanotomography of unstained Bombus terrestris compound eyes.

Imaging the visual systems of bumblebees and other pollinating insects may increase understanding of their dependence on specific habitats and how they will be affected by climate change. Current high-resolution imaging methods are either limited to two dimensions (light- and electron microscopy) or have limited access (synchrotron radiation x-ray tomography). For x-ray imaging, heavy metal stains are often used to increase contrast. Here, we present micron-resolution imaging of compound eyes of buff-tailed bumblebees (Bombus terrestris) using a table-top x-ray nanotomography (nano-CT) system. By propagation-based phase-contrast imaging, the use of stains was avoided and the microanatomy could more accurately be reconstructed than in samples stained with phosphotungstic acid or osmium tetroxide. The findings in the nano-CT images of the compound eye were confirmed by comparisons with light- and transmission electron microscopy of the same sample and finally, comparisons to synchrotron radiation tomography as well as to a commercial micro-CT system weredone.

Super-resolution correlative light-electron microscopy using a click-chemistry approach for studying intracellular trafficking.

Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM

Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

Strategies for Optimizing Heavy Metal Staining in Freeze-substituted Resin Embedded Plants Samples for Electron and X-ray Microscopy

Journal Article Strategies for Optimizing Heavy Metal Staining in Freeze-substituted Resin Embedded Plants Samples for Electron and X-ray Microscopy Get access Kirk Czymmek, Kirk Czymmek Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Keith Duncan, Keith Duncan Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Ningning Zhang, Ningning Zhang Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Ru Zhang Ru Zhang Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 26, Issue S2, 1 August 2020, Pages 142–143, https://doi.org/10.1017/S1431927620013550 Published: 01 August 2020