Embedding Protocol Research Articles

Alterations of mouse lung tissue dimensions during processing for morphometry: A comparison of methods

Preservation of original tissue dimensions is an essential prerequisite for morphometric studies. Shrinkage occurring during tissue processing for histology may severely influence the appearance of structures seen under the microscope and stereological calculations. Therefore, shrinkage has to be avoided so that estimates obtained by application of unbiased stereology are indeed unbiased. The present study investigates the alterations of tissue dimensions of mouse lung samples during processing for histology. Different fixatives as well as embedding protocols are considered. Mouse lungs were fixed by instillation of either 4% formalin or a mixture of 1.5% glutaraldehyde/1.5% formaldehyde. Tissue blocks were sampled according to principles of stereology for embedding in paraffin, glycol methacrylate without treatment with osmium tetroxide and uranyl acetate, and glycol methacrylate including treatment with osmium tetroxide and uranyl acetate before dehydration. Shrinkage was investigated by stereological measurements of dimensional changes of tissue cut faces. Results show a shrinkage of the cut face areas of roughly 40% per lung during paraffin embedding, 30% during "simple" glycol methacrylate embedding, and <3% during osmium tetroxide/uranyl acetate/glycol methacrylate embedding. Furthermore, the superiority of the glutaraldehyde-containing fixative regarding shrinkage is demonstrated. In conclusion, the use of a glutaraldehyde-containing fixative and embedding in glycol methacrylate with previous treatment of the samples with osmium tetroxide and uranyl acetate before dehydration is recommended for stereological studies of the mouse lung.

Solvent‐free tissue processing using supercritical carbon dioxide

Xylene is most often employed in tissue processing protocols for paraffin embedding, but poses a health hazard. The aim of this study was to evaluate a solvent-free processing protocol that uses supercritical carbon dioxide (scCO(2) ) as an intermediate. A series of tests (with bovine tissues) was run, evaluating dehydration and tissue shrinkage in our new scCO(2) -based protocol as compared with routine processing using a graded ethanol and xylene series. A series of tests was then run to evaluate the significance of processing parameters for the outcome. Finally, a validation series was performed with optimal conditions, testing various human tissues with several staining methods. The tissue water content after paraffination was the same with our new scCO(2) -based protocol and the routine xylene-based protocol. Tissue shrinkage was similar with the two methods, at ∼15%, which is also similar to values in the literature. In the validation series, the human tissues showed good morphology with strong staining, probably because of stronger antigenicity. This scCO(2) -based protocol has been shown to be a good solvent-free, alternative form of tissue processing. Although not the focus of this article, the time needed for tissue processing with this new protocol is within 4 h, and there is no need to change macroscopy/sectioning protocols.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Imaging of articular cartilage – Data matching using X-ray tomography, SEM, FIB slicing and conventional histology

The study was aimed at demonstrating a true cellular resolution for articular cartilage using synchrotron radiation-based X-ray microcomputed tomography (SR-μCT) with a sample-specific optimization of the phase contrast. The generated tomographic data were later used to prepare a matching histological sample from the full volume specimen.We used highly coherent and monochromatic X-rays from a synchrotron source to image a tissue sample of bovine articular cartilage after deparaffinization. Phase contrast enhancement was achieved by using five different sample to detector distances for the same X-ray energy. After tomography, the sample was re-embedded into resin while retaining a dedicated sample orientation for subsequent sectioning and polishing, which was conducted until a previously defined spatial position was achieved. The protocol for resin embedding was developed to inhibit morphological changes during embedding. Giemsa staining was applied for better structural and morphological discrimination. Data from tomography and lightmicroscopy were exactly matched and finally compared to results from FIB/SEM imaging. Image detail was achieved at a single cell resolution. Image detail was achieved at a single cell resolution, which has been estimated to be 0.833μm/voxel in the tomographic data.SR-μCT with optimized phase contrast properties represents a method to investigate biological tissues in certain areas of interest, where true cellular resolution or enhanced volumetric imaging is needed. In this study, we demonstrate that this method can compete with conventional histology using light microscopy but even surpasses it due to the possibility of retrieving volumetric data.

Improved biocytin labeling and neuronal 3D reconstruction

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

Characterization of membranous (M) cells in normal feline conjunctiva‐associated lymphoid tissue (CALT)

To characterize conjunctival lymphoid nodules obtained from the nictitans of healthy cats to determine if the follicle-associated epithelium (FAE) of conjunctiva-associated lymphoid tissue (CALT) in this species contains membranous (M)-cells analogous to those described in other regions of mucosa-associated lymphoid tissue (MALT). Lymphoid follicles from nictitan bulbar surfaces of 10 healthy cats (20 eyes total) were examined. Nictitans from five cats were harvested immediately post-mortem and a minimum of 12 lymphoid nodules from each third eyelid were isolated using a Zeiss operating microscope. At least three lymphoid follicles from each eye were examined using light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) using standard fixation and embedding protocols. Nictitan-lymphoid follicles from another five healthy cats were processed for immunohistochemistry to characterize the distribution of T- and B-lymphocytes present beneath the FAE. The FAE overlying CALT from 10 healthy cats demonstrated morphology characteristic of M-cells including attenuated apical cell surface with blunted microvilli and microfolds, invaginated basolateral membrane forming a cytoplasmic pocket, and diminished distance between the apical and pocket membrane. Immunohistochemistry of lymphoid tissue subtending the FAE demonstrated B-cell dependent regions in the germinal centers surrounded by T-cell dependent interfollicular zones. Healthy feline CALT contains morphologic features analogous to those described in other regions of MALT. Documentation of feline conjunctival M-cells is of clinical relevance in the study of primary infectious, allergic, and autoimmune ocular diseases, as well as a potential means of vaccination or drug delivery.

Improvement of Ultrastructural Preservation of Eimeria Oocysts by Microwave-assisted Chemical Fixation or by High Pressure Freezing and Freeze Substitution

Fixation and preparation for electron microscopy of coccidian oocysts is a general problem. Especially in sporulated oocysts, proper fixation and resin infiltration are hindered by the robust oocyst wall. Conventional chemical fixation therefore leads to artefacts that obscure cellular details in the oocysts. In this study, sporulated oocysts of Eimeria nieschulzi were subjected to different fixation and embedding protocols: conventional chemical fixation and embedding in Spurr's resin, microwave-assisted fixation and processing followed by embedding in epon, and high pressure freezing followed by freeze substitution and epon embedding. The samples were finally studied by transmission electron microscopy. Many ultrastructural features of the oocyst wall and the sporozoites were already substantially improved after microwaved-assisted fixation and processing. However, the fine structural preservation still suffered from shrinkage and artificial extraction, which occured during dehydration and infiltration. High pressure freezing (HPF) and freeze substitution (FS) revealed much better preservation. Oocyst walls retained their ovoid shape, and the ultrastructure of sporozoites was well preserved with no signs of shrinkage or extraction. HPF and FS are therefore a suitable method for the ultrastructural analysis of coccidian oocysts.

Projection Neuron Circuits Resolved Using Correlative Array Tomography

Assessment of three-dimensional morphological structure and synaptic connectivity is essential for a comprehensive understanding of neural processes controlling behavior. Different microscopy approaches have been proposed based on light microcopy (LM), electron microscopy (EM), or a combination of both. Correlative array tomography (CAT) is a technique in which arrays of ultrathin serial sections are repeatedly stained with fluorescent antibodies against synaptic molecules and neurotransmitters and imaged with LM and EM (Micheva and Smith, 2007). The utility of this correlative approach is limited by the ability to preserve fluorescence and antigenicity on the one hand, and EM tissue ultrastructure on the other. We demonstrate tissue staining and fixation protocols and a workflow that yield an excellent compromise between these multimodal imaging constraints. We adapt CAT for the study of projection neurons between different vocal brain regions in the songbird. We inject fluorescent tracers of different colors into afferent and efferent areas of HVC in zebra finches. Fluorescence of some tracers is lost during tissue preparation but recovered using anti-dye antibodies. Synapses are identified in EM imagery based on their morphology and ultrastructure and classified into projection neuron type based on fluorescence signal. Our adaptation of array tomography, involving the use of fluorescent tracers and heavy-metal rich staining and embedding protocols for high membrane contrast in EM will be useful for research aimed at statistically describing connectivity between different projection neuron types and for elucidating how sensory signals are routed in the brain and transformed into a meaningful motor output.

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

Hybrid porous resist with sensing functionality

Natively porous hybrid organic–inorganic sol–gel systems have been engineered to be used as functional positive photoresists, aimed to the realization of microsensors in a single-step process.Interesting results have been obtained combing three main sol–gel system features: the direct patternability through X-ray lithography, being processable without the addition of a photocatalyst; the functionalizability, properly designing the organic component of the hybrid network or incorporating active species; the open micro or mesoporosity, tailored by the synthesis process and precursor choice.The photoprocessable porous films have been synthesized starting from a Bridged Polysilsesquioxane (BPS) precursor, 1,4-bis(triethoxysilyl)benzene. The correlation between chemical properties of the sol–gel material and its patternability is described in detail. X-ray exposure leads to a progressive alkyl and aromatic compound elimination and promotes inorganic condensation in the system, allowing the selective dissolution of irradiated cross-linked films in suitable etchants. Patterns of final resolution lower than 100nm have been realized on BPS-based films synthesized in acid conditions, a procedure that allows to take advantage of a straightforward embedding protocol for active species in the sol–gel matrix. The BPS-based system has been doped with a covalently linked quinolinium dye, obtaining thin sensing films patternable by X-ray lithography. A feasibility test for the fabrication of optical microdevices, where fluorescence properties are obtained directly on the patterned coatings, has been provided.

Exact nonadditive kinetic potentials for embedded density functional theory

We describe an embedded density functional theory (DFT) protocol in which the nonadditive kinetic energy component of the embedding potential is treated exactly. At each iteration of the Kohn-Sham equations for constrained electron density, the Zhao-Morrison-Parr constrained search method for constructing Kohn-Sham orbitals is combined with the King-Handy expression for the exact kinetic potential. We use this formally exact embedding protocol to calculate ionization energies for a series of three- and four-electron atomic systems, and the results are compared to embedded DFT calculations that utilize the Thomas-Fermi (TF) and the Thomas-Fermi-von Weisacker approximations to the kinetic energy functional. These calculations illustrate the expected breakdown due to the TF approximation for the nonadditive kinetic potential, with errors of 30%-80% in the calculated ionization energies; by contrast, the exact protocol is found to be accurate and stable. To significantly improve the convergence of the new protocol, we introduce a density-based switching function to map between the exact nonadditive kinetic potential and the TF approximation in the region of the nuclear cusp, and we demonstrate that this approximation has little effect on the accuracy of the calculated ionization energies. Finally, we describe possible extensions of the exact protocol to perform accurate embedded DFT calculations in large systems with strongly overlapping subsystem densities.

Characterization of isolated polyamide thin films of RO and NF membranes using novel TEM techniques

Achieving a better understanding of transport and rejection mechanisms in RO and NF membranes requires more detailed information of the nanostructure of polyamide thin films. This study reports on two novel transmission electron microscopy (TEM) techniques for characterizing polyamide nanostructure. The first technique produces cross-sectional images of isolated polyamide thin films by removing the polysulfone support from regular TEM cross-sections. In the second technique called “projected area” TEM (PA-TEM), isolated polyamide thin films are placed with their surface perpendicular to the electron beam. The resulting images capture the thickness, morphology and mass density of the entire thin film. In combination, these new techniques provide information on polyamide nanostructure that is not evident using conventional methods. For the commercial RO membrane ESPA3, the cross-sectional view of the isolated polyamide thin film shows a 30–60 nm thick base of nodular polyamide (presumably the separation barrier) that forms a relatively smooth interface with the polysulfone support. Above this, a more open structure of loose polyamide extends outward giving rise to the ridge-and-valley surface structure. In PA-TEM images, the ridges and valleys correspond to the dark and bright regions, respectively; the polyamide nodular base appears as round features forming an irregular honeycomb pattern throughout the images. Membrane cross-sections were prepared with a simple resin embedding protocol using the acrylic resin LR White. The protocol did not require dehydration steps, and was applicable to both dry and wet membrane samples. Artifacts that may be produced during sample preparation were also documented.

Automated and Optimized Embedding of Proteins into Membranes for Molecular Dynamics Simulations using Griffin

As new atomic structures of membrane proteins are resolved, they reveal increasingly complex transmembrane topologies, and often highly irregular surfaces with crevices and pores. In many cases, specific interactions with the lipid membrane are formed and are functionally crucial, as is the overall lipid composition. Compounded with increasing protein size, these characteristics pose a challenge for the construction of high-quality simulation models of membrane proteins in lipid bilayers; that these models are sufficiently realistic is of obvious importance for the reliability of simulation-based studies of these systems. To automate and optimize this process, we have developed GRIFFIN (GRId-based Force-Field INput). In the initial steps of this embedding protocol, the program carves lipid and water molecules out of the protein volume as necessary to conserve the system density. In the main optimization phase GRIFFIN adds an implicit, grid-based protein force field to the molecular simulation of the carved membrane-water system. In this force field, molecules inside the implicit protein volume experience an outward force that will expel them from that volume, whereas molecules outside are subject to electrostatic and van-der-Waals attractive interactions with the implicit protein. At each step of the simulation, these are updated by GRIFFIN and combined with the intermolecular forces of the explicit membrane-water system, to derive a trajectory of the atomic positions. This procedure enables the construction of realistic and reproducible starting configurations of the protein-membrane interface within a reasonable timeframe and with minimal intervention. GRIFFIN is a standalone tool it is designed to work with any existing molecular dynamics package, such as NAMD or GROMACS. Examples of challenging applications are presented.

Microwave‐assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h - with only a few steps that have to be performed manually - until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material (Arabidopsis thaliana, Nicotiana tabacum and Picea abies) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.

Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0 degrees C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.

Easy flat embedding of oriented samples in hydrophilic resin (LR White) under controlled atmosphere: Application allowing both nucleic acid hybridizations (CARD-FISH) and ultrastructural observations

Hydrophilic resins present the advantage of making possible both hybridization experiments involving either antibodies or oligonucleotide probes and ultrastructural observations. Whereas various embedding protocols are available, only very few concern flat-embedded preparations. In this study we describe an easy protocol for flat embedding of small-oriented biological samples in hydrophilic resins (LR White). The most important constraints are (i) to polymerize the samples under argon-saturated atmosphere (avoiding oxygen which is an inhibitor of LR White polymerization) and (ii) to use transparent flat embedding molds. Two kinds of samples were analyzed: small pieces of large tissue that need to be accurately oriented for a valuable analysis and very small organisms such as free-living nematodes, which are very hard to investigate with conventional paraffin wax embedding techniques. Semi-thin sections strongly reinforce the quality of the observations from oligonucleotidic in situ hybridization experiments by reducing the background usually encountered in oligonucleotide probe hybridization experiments from sections. Such protocols could also permit a cheap alternative to the use of laser scanning confocal microscopes for oligonucleotidic in situ hybridization as in FISH and CARD-FISH experiments from histological sections. The interest of this embedding protocol is reinforced by the fact that molecular in situ hybridization experiments and ultrastructural observations from thin sections can be carried out from a single-small individual (<1mm in length) sample.

Radical prostatectomy specimen processing: A critical appraisal of sampling methods

Radical prostatectomy gives the most accurate and detailed information available for determining prognosis and deciding about adjuvant therapy for prostate cancer. The consistency and reproducibility of observations from these specimens requires a standardised protocol for tissue fixation, sampling, embedding and processing. Although the publication of processing protocols in recent years has led to a convergence of opinions, differences still exist in methods of handling, sampling and processing specimens. In this review article, we incorporate the processing methods for radical prostatectomy addressed in the previous reports and propose a comprehensively standardised approach to evaluate radical prostatectomy specimens.

Histological Processing of pH-Sensitive Hydrogels Used in Corneal Implant Applications

Hydrogels are hydrophilic, crosslinked polymers that are being used with increasing frequency in a variety of biomedical applications. The ability to obtain high-quality histological sections, without artifacts, after in vivo implantation is critical to their evaluation. Unfortunately, because of their high water content and environmental sensitivities, many hydrogels are unable to be successfully processed for histology using typical tissue processing methods. In this work, we present a method for successfully processing pHsensitive hydrogels for histological analysis. A modified glycol methacrylate (GMA) processing and embedding protocol is described. One cornea each from 10 New Zealand Red rabbits was implanted with a poly(ethylene glycol) and poly(acrylic acid) interpenetrating double network hydrogel disc for 2 weeks and processed for histology. Maintaining a neutral pH during fixation with glutaraldehyde is a critical initial step for processing. In addition, typical tissue processing methods require the use of alcohol and/or xylene for specimen dehydration, causing severe distortion of hydrogel morphology. The substitution of these chemicals with deionized water during the processing steps also proved to be vital for maintaining hydrogel structure and morphology. Slides stained with cresyl violet showed excellent tissue and hydrogel morphology with minimal histological artifacts or distortion (The J Histotechnol 30:157, 2007)Submitted August 27, 2006; accepted with revisions March 12, 2007.

ONIOM Studies of Esterification at Oxidized Carbon Nanotube Tips

Esterification of oxidized carbon nanotubes (CNTs) can open a new route for the separation of zigzag and armchair nanotubes. We studied theoretically (by using hybrid DFT within the ONIOM embedding protocol) the reactions of monocarboxy-substituted oxidized tips of zigzag and armchair single-walled CNTs (SWCNTs) with methanol. According to the calculated values of activation energy, Gibbs free-activation barriers, and enthalpies of formation for the SWCNT-(COOH)H5 models, the zigzag nanotube isomer is more reactive as compared to its armchair counterpart. For other models we obtained variable results.

Bottom-up optimization approach and nonlinear model for oilfield output programming

In the process of oilfield development, it is unable to separate the marginal effect of each developing measure from the aggregate effect of all developing measures, so both marginal analysis method and Markowitz portfolio theory are helpless for the oilfield output programming. Function Simulation is proved to be a feasible tool to model the input-output relationship of oilfield development. By training with historical development data, it possesses extrapolation capability in some range, which forms the basis for the oilfield output programming. The purpose of this paper is to report on the use of bottom-up optimization mechanism which is embedded by Function Simulation to optimize the distribution of output in oilfield. Following presentation of the features of oilfield development, the top-down approach and the Function Simulation as a tool for oilfield output programming are reviewed. The process of designing the bottom-up optimization framework embedded with Function simulations, determining the embedding protocol and coordinating the corresponding relationship between the subentry outputs and the subentry costs is described. Subsequently, models and an example are given. Finally, the paper concludes that above optimization method is suitable to solve a class of programming problems.