Techniques in Electron Microscopy of Animal Tissue

Abstract

Technical improvements in electron microscopy, both instrumental and preparative, permit increasingly accurate analyses. Digital images for transmission electron microscopy (TEM) can be processed by software programs that automate tasks and create custom tools that allow for image enhancement for brightness, contrast and coloration; for creation of rectangular, ellipsoidal or irregular area selections; and for measurement of mean area and standard deviation. Sample preparation remains a source of error since organelles and spatial arrangements of macromolecules rapidly change after anoxia. Guidelines for maintaining consistency in preparation, examination and interpretation are presented for different electron microscopy (EM) modalities.

Transmission Electron Microscopy

Transmission electron microscopy (TEM) was the major investigation tool in cellular biology from the 1960s to the 1980s. Ultrathin sections of resin-embedded tissues added unique views of the cell and established our modern concepts of organelle morphology. Thereafter, for several decades, the use of TEM declined and with it the number of TEM laboratories and electron microscopists. Pathologists without a working knowledge of TEM failed to include it in their diagnostic and investigative protocols. In publications, it was commonplace to find electron micrographs that had serious artifacts or had been printed at such a low magnification that the reader could not discern in it any component described in the figure legend. Even worse, teachers and textbooks began to use colored cartoons to depict organelle ultrastructure. The end result: an even further detachment from the reality and wonder of cell pathology as revealed by TEM. Despite this, there persisted in veterinary pathology a core of well-trained electron microscopists who continued to produce contributions to our understanding of disease. In the past decade, the wealth of data from new light microscopic technologies has been driving renewed interest in TEM and for new ways to view cell ultrastructure in 3 dimensions at high resolution.

Today, when specimens are properly fixed and embedded and using improvements in existing techniques, both instrumental and preparative, classic TEM continues to reveal striking details of cellular organelles and their alterations in disease. New techniques allow more reproducible data for analysis of cellular structure and function. ¹⁵ Digital images can be processed by an amazing choice of software programs that automate tasks and create custom tools. Image enhancement for brightness, contrast, and coloration; for creation of rectangular, ellipsoidal, or irregular area selections; for measurement of mean area and standard deviation; and for automatic creation of changes using a wand tool all improve analyses of data. ³ Some of these programs (such as ImageJ, an image-processing and analysis program written in Java from the National Institutes of Health, Bethesda, MD) are available online without charge. In the discussion of TEM techniques that follows, bolded terms are candidates for online searches in www.pubmed.gov and other search sites.

Despite remarkable new techniques, the greatest source of error in thin sections for TEM still lies in sample preparation. In conventional fixation and embedment in epoxy resins at ambient temperature, organelle structure and spatial arrangements of macromolecular complexes rapidly change after tissue sampling. For example, the cytoskeleton, Golgi complex, and endomembrane system are remodeled within fractions of a second after tissue anoxia. This requires attention to sample collection, especially times of sampling, trimming, and temperature of fixatives and buffers for fixation. With precise preparation, structures bearing nucleic acids and proteins are preserved.

Although the resolution power of electron microscopes is on the atomic scale, artifacts in cell and tissue specimens introduced by sampling, aldehyde fixatives, dehydration, staining, and section thickness reduces it to nanometers. Recommended guidelines for maintaining consistency in glutaraldehyde immersion fixation are as follows:

• Time of sampling: the interval from tissue anoxia to fixation should be only a few seconds.

• Size of specimen: tissue blocks ≤2 mm² are required (in general, the smaller the better).

• Blade sharpness and stroke: new blade surface for each specimen; rapid, single strokes.

• Fixative and buffer: solutions must be fresh with no denaturation of aldehydes or ion imbalance in buffers.

• Temperature of fixative: cooled (∼4°F), ambient (∼65°F), and body (∼98°F) are options.

Fixation

In the standard preparatory protocol, tissue samples are fixed in 2.5% glutaraldehyde at pH 7.2 (at ambient temperature) for 2 to 3 hours and postfixed in 1% OsO₄ in 0.1 M cacodylate buffer for 60 minutes (Fig. 1). Fixation with glutaraldehyde alone or osmium tetroxide (OsO₄) alone causes artifacts that are substantially avoided when tissue is doubly fixed. Glutaraldehyde fixes proteins well but is harsh on membranes. The reverse is true for OsO₄ which enhances membrane contrast but damages proteins; that is, membrane contrast is enhanced by OsO₄ fixation.

Figure 1.

Standard protocol for tissue in transmission electron microscopy: glutaraldehyde fixation and OsO₄ postfixation. Normal hepatocyte, dog. Nucleus (top left): large nucleolus, many interchromatin granules, sparse chromatin, linear nuclear envelop with many nuclear pores (evidence of an actively transcribing cell). Cytoplasm: plasma membranes of adjacent hepatocytes (bottom) are closely apposed and linear, bear small dense intercellular junctions, and are separated at a biliary canaliculus with normal microvilli. Mitochondria are homogeneous in size and structure with normal membranes, cristae, matrices, and dense calcium-sequestering granules. Golgi complex (center) is associated with transport vesicles. Endoplasmic reticulum (ER) including rough (RER), smooth (SER) and nuclear associated components is normal; dense glycogen particles fill intertubular spaces of smooth ER.

Paraformaldehyde (which produces effects similar to formalin) is used to preserve antigenicity of some proteins in immunolabeling. It is used in combination with glutaraldehyde (referred to as Karnovsky’s solution, which varies from 0.5% to 2% paraformaldehyde with 2% glutaraldehyde).

When fixative takes 1 minute to reach the cell, organelles suffer from autolysis. Even when fixative penetrates in less than 1 second, cross-linking reactions are selective (eg, glutaraldehyde will only react with certain amino acid residues on proteins and will not effectively cross-link nucleic acids and carbohydrate molecules). The result is extraction of unlinked molecules from the cell during rinsing and dehydration. ⁴⁴

Cadodylate and phosphate buffers predominate in fixatives for electron microscopy, but be aware that high concentrations of phosphate damage mitochondria. In cases where phosphate buffers are undesirable, organic buffers such as 1,4 piperazine bis(2-ethanolsulfonic acid) (PIPES) might be considered since it has fewer detrimental effects on cells (Table 1). It is especially important that staining and buffers be appropriate in fixation of calcified soft tissues since redistribution of calcium salts during fixation can lead to misinterpretation. There is some decalcification with OsO₄ fixation, and increasing the time of immersion may give striking results in the differential degrees of calcification in lesions of interstitial tissues (Fig. 2).

Figure 2.

Demineralization of calcium by OsO₄ fixation: extracellular matrix of stomach, uremia, dog. Note: calcification (upper left and right) and phagocytosis by osteoclasts (osteoclast nuclei left and bottom). Calcium has been deposited in foci of proteoglycans in progressively increasing layers that first appear as small dense matrix vesicles, then as small target forms, and finally as massive complex laminar arrangements (asterisk).

Table 1.

Preparatory Techniques in Transmission Electron Microscopy.

Techniques	Uses
Fixation
Glutaraldehyde osmium tetroxide	Standard fixation and postfixation techniques for TEM
Paraformaldehyde	Less destructive than glutaraldehyde on proteins; immunocytochemistry
Microwave acceleration	Short treatment at low-power microwave decreases fixation times 21
Buffers
Cacodylate phosphate	Standard buffers: preserve tissue ion balance; influence fixation penetrance
PIPES	Organic buffer; causes fewer artifacts
Embedding mediums
Epoxy resins	Standard embedding mediums
Methacrylate	Soft resin; applied in section problems
Methyl cellulose	Used for stability in cryofixation, especially in the Tokayasu method
Sectioning
Thick section staining	Toluidine blue is standard for monochrome staining; basic fuchsin, other double stains are options 7,8
Thin section staining	Uranyl acetate and lead citrate are used in standard protocols 7
TEM serial sectioning	Custom TEM camera systems use 4 high-speed cameras to record 3.2 million images that are converted to 3D images 4
SEM serial sectioning	Ribbons of hundreds of images recorded on wafers using SEM backscattered electron with in-lens detector 27
Immunolabeling
Blocking nonspecific staining	Sections floated on glycine followed by commercial blocking solution; sections incubated with primary antibody 62
Antigen retrieval	Heat unmasks protein antigens 4,10
Double labeling	Labels 2 or more antigens
Cryopreparation
Cryosection	Circumvents fixative denaturation 66
Cryobiopsy	Prevents tissue anoxia 50
Metallic labels
Gold	Standard label 23,36,58,62
Ferritin	Little use; difficult to differentiate
Fluoronanogold	Best label for CLEM 12
Microspherules
Polystyrene beads	Clear identification in TEM; beads of varying size useful in differentiation
Composite nanospherules	Complexing of drugs, toxins, and other compounds allows tissue uptake studies
CLEM
Fusion of TEM and light microscopy	Used to detect specific proteins; useful for superresolution microscopy, 28,29 in vivo techniques 55
Fusion of STEM and fluorescence	Detection of specific proteins; greater versatility than TEM alone
Fusion of SEM and fluorescence	Detection of membrane proteins; also used to label cells in liquid media 17

Abbreviations: CLEM, correlative light and electron microscopy; EM, electron microscopy; PIPES, 1,4 piperazine bis(2-ethanolsulfonic acid); STEM, scanning-transmission electron microscopy; TEM, transmission electron microscopy.

Perfusion fixation via cannulae into the vascular system typically is done either by (1) continual gravity-mediated perfusion at 12 cm water pressure or (2) pulsed flow roller pump-mediated perfusion. In either case, perfusion of fixative is preceded by a prefixation buffer perfusion. Both methods give superior fixation of parenchymal cells but lose the relationships with blood cells and lymph vessels. In pulsed flow perfusion fixation of liver, a flow rate of 1 mL/g liver tissue is recommended. ⁷³

Microwave assisted fixation can be used for both light microscopy (LM) and TEM. Microwave energy used in combination with low concentrations of glutaraldehyde (0.05%) or formaldehyde (2.0%) rapidly preserves some lesions; it can be especially useful for fixation of some antigens. ^35,61 For microwave fixation for LM, tissue samples must be no larger than 1 cm³. Criteria for maximum success for electron microscopy include (1) tissue sample of less than 1 mm in 1 dimension, (2) irradiation temperature lower than 50°C, (3) irradiation time less than 50 seconds, (4) immediate replacement of postirradiation solution with cold storage buffer, and (5) tissue fixation within 5 seconds after removal from the blood supply.

Retrieval of formalin-fixed tissue by reprocessing through glutaraldehyde fixative allows for discovery of valuable information for diagnostic TEM. To do this, select 1-mm³ samples of wet tissue fixed in 10% formalin, rinse, and place into glutaraldehyde overnight, and then proceed with the routine electron microscopy protocol.

Formalin-fixed, paraffin-embedded tissue can be retrieved when no other tissue is available or a specific piece of that data is needed for analysis. For this, cut small pieces of tissue from the paraffin block, remove the paraffin in xylene, and rehydrate tissue to buffer, and then place tissues in glutaraldehyde and process according to the routine EM processing schedule. ³⁸ Results are often useful even though artifacts and vacuoles are prominent (Fig. 3).

Figure 3.

Retrieval of formalin-fixed, paraffin-embedded skin lesion; squirrel fibroma virus: tissue processed by removal of paraffin, postfixation in glutaraldehyde, and the routine protocol for paraffin-embedded tissue for transmission electron microscopy. Note condensation of chromatin, protein aggregation, and artifactual vacuolation throughout the section. Inclusions in cytoplasm (of virions and viral material [asterisk]) and nucleus (viral components and lysed nuclear proteins [solid circle]).

Embedding

For standard embedment for TEM, samples are dehydrated through an ethanol series; transferred to several changes of a transitional solvent, propylene oxide; and embedded in epoxy resin. The epoxide resin is intolerant of water and will fail to polymerize properly if the tissue is not totally dehydrated; this results in rubbery blocks that are impossible to cut and tissues that are retrievable to correct the issue. Furthermore, water remaining in the specimen during dehydration will not allow embedding media to completely infiltrate the specimen.

It is critical that embedding mixture ingredients be thoroughly mixed prior to use. Improper mixing of ingredients will result in tissue blocks not hardening sufficiently. Wrong proportions or insufficient time for mixing, as well as defective or insufficient accelerators, may prevent proper infiltration and polymerization. Excessive vigor in mixing for this introduces air bubbles into the resin, which makes cutting difficult and will make samples unstable in the electron beam. ⁶

Sectioning

Ultrathin sections are cut with a diamond knife, placed on grids, and allowed to dry. The number of areas of each tissue processed is recorded and the number of cells evaluated. Correlation of data from the ultrathin section with that from the thick section (by histology) is a critical event in evaluation of electron micrographs. To veterinary pathologists, TEM evaluation of pathologic tissue is inherently compared with histology, typically by examination of alternate thin and thick sections—a technique referred to as parallel light and electron microscopy.

Preparatory cutting of thick sections (1 μ) of resin-embedded tissue allows one to identify lesions and trim the tissue block to an appropriate small size for thin sectioning. Thick sections are cut with a glass knife, stained with 1% toluidine blue (1 minute on a 60°C hotplate), rinsed, dried, and examined by LM. Ultra-thin sections (70 nm) are then cut with a diamond knife from areas selected. Use of a second dye on thick sections provides greater differentiation of tissue components. Basic fuchsin is a useful second stain; for example, after toluidine blue staining, the thick section is stained on the slide for 1 minute with 0.5% basic fuchsin mixed equally with 0.25% borax, and stain is rinsed off with 70% alcohol, followed by a water rinse (Fig. 4).

Figure 4.

Thick section, double stain: toluidine blue and basic fuchsin; skin. This technique produced sections that resemble standard hematoxylin and eosin stains used in histology.

Sectioning of crystals, foreign bodies, and mineralized lesions may require sections that are greater than 70 nm. When these lesions are destroyed by the electron beam after multiple attempts, a resin section overlay procedure may be useful (Fig. 5). New digital technology provides for a less intense beam so destruction of thin section is rarely an issue.

Figure 5.

Double thin section technique for crystals: chromatophore crystal granuloma; skin; butterfly fish. (A) Standard thin section: portions of the cytoplasm of several chromatophores (iridophores) bearing crystalline-reflecting platelets surround a large stellate melanophore. The electron beam has burned away the crystalline-reflecting platelets that surround 1 intact melanophore. (B) Overlay on double thin section cut from the same block: chromatophore-reflecting platelets have been retained.

Staining of Ultrathin Sections

Grids bearing thin sections are processed by positive staining with heavy metals to increase tissue contrast. Staining en bloc (before embedding) can also be done to increase contrast. The most commonly used stains for TEM are uranyl acetate and lead citrate. Uranyl acetate can be used for pre- or postembedding staining. It is typically a 2% aqueous solution, although some resins need an alcoholic (either ethanol or methanol) uranyl acetate to penetrate the resin. The most commonly used lead citrate stain is Reynolds’s lead citrate. Although time-consuming to prepare, Reynolds’s lead is stable as long as it is protected from carbon dioxide and light. Care must be taken during storage and staining since lead citrate will react with atmospheric carbon dioxide to form a fine precipitate of lead carbonate. ⁷

Stain precipitates will be retained on tissue sections when either water or pH is inappropriate. In making the stain, CO₂ free water is required because lead stains are easily precipitated upon contact with CO₂. While staining, it is necessary to protect the staining solution during the staining process as well as during storage. Proper pH is important with this stain and cannot vary by even 0.1 unit from pH 12.0 or poor staining and stain precipitation will occur. ³⁸

Focusing and Photographing the Specimen in the TEM

Careful focusing is one of the most important functions required for successful TEM use. To obtain the best photograph during examination of the stained ultrathin section, one must improve contrast, a task that requires the objectives and condensers of the electron microscope to be in alignment. The TEM uses a high-voltage electron beam emitted by an electron gun to create an image. The beam is accelerated by an anode, focused by electrostatic and electromagnetic lenses, and carried through the specimen, which is transparent to electrons or scatters them out of the beam.

The basic rule of focusing is that the greater the magnification, the more accurate the focusing must be and the closer it should be to the true or near focus setting. True or near focus is where the specimen is exactly in the conjugate plane to the image plane of the objective lens. Optimum results should be obtained from a focused micrograph, but a certain degree of underfocusing is and can be generally favored. Since focusing of the image is affected by variations in the objective lens current, it is best to under- and overfocus to ensure that features in the specimen are genuine.

In producing electron micrographs, the goal is always the generation of valid data, not of electron micrographs of a particular quality. In publishing, electron microscopists face hurdles by reviewers with TEM experience but little understanding of the nature of electron beam focus or deflection and the depth of focus within the ultrathin section. Never “balance the light” to create a uniform background in the micrograph; that manipulation almost always destroys data, especially of tiny vesicles and protein fragments that provide important data for the interpretation of pathologic tissues (Figs. 6, 7).

Figure 6.

Preservation of detail in the dark micrograph: amyloid fibrillogenesis at the surface of an amyloid-secreting reticular cell appears as dense granular areas; renal glomerulus, dog. Surfaces of the amyloid-producing reticular cell contain dense areas of preamyloid polymers. Amyloid fibrils in cross section and longitudinal section. Amyloid P component (arrow) and exosomes release during the process of fibrillogenesis.

Figure 7.

Preservation of background proteins: DNA, ribonucloproteins and viral components, herpesvirus-infected cell. A large DNA/viral nucleoprotein crystal has formed in lattices (bottom) and a large core-generating aggregate of viral proteins has formed a granular inclusion (top right). DNA strands (arrow) at the forming edge of the lattice array are incorporated into immature viral particles. Viral ribonucleoproteins form small vesicular viral cores that incorporate viral DNA. Completed viral capsids are at the periphery of the lesion (top). (Micrograph: Arlis Boothe).

Cryo-electron Microscopy

Ischemic and anoxic artifacts are inevitable due to immersion fixation. The introduction of cryo-electron microscopy (CEM) in the early 1980s allowed improved images and extension of resolution for determining cellular and microbial structures from electron micrographs. Rapid freezing, cryo-preparation, and CEM introduced fewer artifacts during preparation than chemical processing at room temperature and, in addition, allowed images of the hydrated sample. Modern CEM involves cryofixation by high-pressure freezing (HPF), ^32,43,44,67 which, when used in combination with substitution of the vitrified ice by organic solvents at low temperature (freeze substitution), overrides most of the disadvantages of chemical fixation. In vivo cryotechniques, particularly cryobiopsy, ⁵⁰ allow pathologists to obtain tissue specimens from animals without impacts of ischemia or anoxia on cells.

Cryofixation for immunolabeling can be done on Lowicryl sections of high-pressure frozen cells. ^13,26,68 A critical ingredient for fixatives to preserve reactivity is reported to be a cocktail of 0.1% potassium permanganate combined with 0.001% osmium tetroxide. ⁶⁵ To retain fluorescence for LM, samples are embedded in glycol methacrylate. High-pressure freezing is the first step for preserving fluorescence and morphology. HPF and freeze-substitution techniques allow near-instantaneous immobilization of specimens and prevent fixation and dehydration artifacts such as protein aggregation. ⁶⁷

Negative Staining in TEM

Negative staining techniques for TEM have allowed major advances over the past decades by defining fine details on the surface structures of bacteria and viruses (Fig. 8), as well as membranes, lipids, and protein filaments, especially of β-pleated sheets such as amyloid. For staining, a suspension of the particles or fibers to be examined is mixed with a dilute solution of electon-opaque material, such as phophotungstic acid, uranyl acetate, or ammonium molybdate, and dropped onto a suitably coated grid for examination. Negative staining is essential for the identification of viruses in fluids. The study of membrane pores induced by insertion of bacterial toxins into the cellular membrane has revealed the striking complexity of toxin reconfiguration to form pores that cause cytoplasmic leaking and cellular lysis cells in fluids.

Figure 8.

Negative stain: phosphotungstic acid, contagious ecthyma virus. Stain outlines the peripheral tubules that form the outer coat of the virion.

Cryo-negative staining provides useful approaches for high-resolution studies of macromolecules and viral structures. Traditional air-dry negative staining involves specimens absorbed to carbon support films (with or without holes). In cryo-negative staining, the specimens are frozen and hydrated or vitrified using ammonium molybdate. ¹⁴ These methods avoid rouleau formation and other artifacts when lipoproteins are being examined.

Scanning Electron Microscopy

Scanning electron microscopy (SEM) is a surface imaging technique that provides high depth of field with a 3-dimensional appearance (Fig. 9). SEM produces images by probing the specimen with a focused electron beam that is scanned across the surface; the beam in an SEM does not carry an image. In contrast, TEM uses a high-voltage electron beam emitted by an electron gun to create an image.

Figure 9.

Scanning electron micrograph (SEM): front foot of the face fly Musca autumnalis. (A) Various hair types are differentiated in the foot area. (B) Bacterial cells of Brucella abortus are attached to hairs on the foot of the bovine face fly.

Adequate sample preparation used in modern SEMs can provide resolution comparable to that achieved with TEMs—down to ∼3 nm for biological material. Standard SEM equipment uses an electron beam (detected in a raster scan pattern) that interacts with electrons on the sample surface. The beam’s position is combined with the detected signal to produce an image. For nonbiological materials, SEMs can achieve resolution better than 1 nm. The type of signal produced by an SEM can also include back-scatter electrons, characteristic x-rays, and cathodoluminescence. ¹²

High-resolution SEM (HRSEM) is increasingly used to study 3-dimensional organization in biological structures and macromolecular complexes. ^52,54 Environmental scanning electron microscopy (ESEM) enables wet cells to be observed without damage to samples. Newer scanning-transmission electron microscopic (STEM) imaging systems also facilitate observation of wet specimens in ESEM. Correlative fluorescence microscopy TEM enables study of cellular function and structure, and STEM images can achieve spatial resolution of 3 nm. ^17,59

Special Investigative Techniques

EM of Nanoemulsions

Electron microscopy of nanoemulsions is an important tool in the pharmaceutical industry. The size range of colloidal drug delivery systems is at the lower end of the nanometer scale, and the ability to see microparticles and nano-sized structures is indispensible for analysis of nanoemulsions. Use of SEM and TEM techniques to characterize pharmaceutical formulations provides critical information on size, shape, and internal structure of nanoparticulate carrier systems. Current methods are increasingly employed for characterization of pharmaceutical systems such as colloidal lipid emulsions, solid lipid nanoparticulates, polymer-based colloids, nanofibers, and thermotropic and lyotropic liquid crystalline nanoparticles. ^22,30,34

Microspherules

Microspherules of various composition are important investigative tools in both LM and TEM. Uniform-sized polystyrene latex beads, available in dry form and in 10% aqueous solutions in a size range from 0.02 to 10 μm, are commonly used in studies of phagocytosis, thrombosis, inflammation, and intracellular trafficking. For quality control, TEM evaluation of nanocomposite microspherules that are synthesized for many unrelated biomedical uses is essential for determination of particle size, composition, and uniformity. These include a range of diverse polymer microspherules that include chitosan and nanohydroxyapatite used in bone repair and nanocarriers designed to pass the blood-brain barrier. ¹¹ Hollow and porous magnetic iron oxide-based microspherules with hydroxyl group-rich surfaces have been developed as biological sponges that sequester toxic ions such as As, Cr, Cd, and Hg. TEM evaluations are also essential for polymer microspherules used for drug delivery systems and experimentally in rate-controlled, targeted studies for controlled-release protocols. Biodegradable poly-(D, L-lactide-co-glycolide) microspherules are approved for use in humans to stimulate immune responses, typically via dendritic cells. ⁶⁰

Electron-Dense Markers for TEM

Ferritin, colloidal gold, and other heavy metal–based nanoparticles with sufficient contrast for high-resolution TEM have been used for in vivo, in ovo, and in vitro investigations to determine particulate trafficking among organelles. If particles can be counted, their spatial distributions can be subjected to statistical analysis. ^40,42,72 One approach is the detection and section volume localization for particles injected into the animal prior to sampling (ie, particles that are within the cell prior to fixation and embedding). Various sizes of electron-dense particles of colloidal gold are used to study particle transport, especially in blood-tissue, air-tissue, and urine-tissue pathways. For example, when instilled in the trachea of mice, 20-nm diameter colloidal gold particles are found in pulmonary alveolar macrophages within 15 minutes and slightly later are internalized by alveolar epithelium using caveolae.

Immunoelectron Microscopy

Immunoelectron microscopy using heavy metal labels for staining of specific proteins expanded the use of morphology in cellular pathology, and the quantitative analysis of metallic labels in intracellular compartments added precision. Colloidal gold of varying sizes is superior. Ferritin, although useful, suffers from uncontrolled particle size and a confusing similarity to natural ferritin (Fig. 10). Particulate gold labeling on ultrathin sections for antigen localization at the TEM level has had widespread use for 2 decades; most studies have used simple, rapid, and unbiased methods for assessing immunogold labels distributed over different cellular compartments (Fig. 11). ^36,37 Sampling approaches have been developed for stereology in which a regular array of EM fields or linear scans is positioned randomly on labeled sections. Quantitative methods allow estimation of both density and distribution of gold labels in organelles. Spatial mapping, especially important in polarized cells, can be done using a stereologic approach referred to as the rotator. ⁴¹

Figure 10.

Ferritin-labeled Newcastle disease virus: immunolabeling antibody was against the surface hemagglutinin spike on the viral surface. Size of the ferritin particles may result in confusion with natural ferritin in the cell cytoplasm. Metal label is more diffuse than in gold labeling.

Figure 11.

Immunogold-labeled bacteria: Brucella abortus, peritrophic membrane, midgut of the bovine face fly, Musca autumnalis. Gold particles are attached to the bacterial cell surface. The immunolabeling antibody was against lipopolysaccharides on the surface of the bacterial cell. The electron beam is focused on gold particles with deliberate underfocus of the electron lucent peritrophic gel in the background.

Fluoronanogold (FNG) contains 2 different markers, a fluorochrome molecule and a 1.4-nm cluster of gold atoms, and can be seen by both LM and EM. For 2-dimensional TEM, FNG can be seen by both wide-field LM and TEM. For 3-dimensional TEM, FNG is used by confocal LM and EM tomography. ¹² It is especially useful for detection of proteins within cytoplasmic organelles.

Antigen retrieval includes techniques that increase immunoreactivity in tissue. Heat-induced antigen retrieval breaks down protein cross-links formed during aldehyde fixation and unmasks protein epitopes for immunoreactivity (Table 1). ⁵ Techniques are available for epoxy sections. ¹⁰

Morphometry and Stereology in TEM

Precise and accurate quantification of cellular changes in electron micrographs has traditionally used morphometric tools to measure numbers of organelles as well as the surfaces, lengths, and volumes. Development of new models for TEM stereology and computational software for data analysis provide exciting modalities in scientific investigation. ² Electron microscopic tomography, freeze-fracture electron microscopy, and immunolabeling techniques have added new dimensions to TEM. Analytical techniques such as electron energy loss spectroscopy or energy-dispersive x-ray spectroscopy, as well as atomic force microscopy, are additional assets to determine elemental composition of materials. Due to advances in crystallography and magnetic resonance imaging (MRI), almost any protein can be modeled at atomic resolution, yet TEM is still required to place the protein in context of the cell.

Morphometry, the measurement of the form or structure of organelles and cellular components, provided useful data in the past when multiple tissue blocks were taken at necropsy. TEM morphometry was typically done at 2 levels of magnification. At low magnification, micrographs were recorded from each section of random but no overlapping fields of tissue. Classic point-counting morphometry was done using lattice test grids. Relative volume density (V_v ) of nuclei and cytoplasmic organelles was expressed as percentages of the cell, nucleus, or cytoplasm. ² Relative surface densities (S_v ) was expressed as square microns of membrane per cubic micron of cytoplasm. S_v was determined using the equation S_v = I / P_cyt × d, where I is the number of intersections of horizontal test lines with the outer surfaces of the organelle, and P_cyt (total cytoplasmic points) × d (distance between points) is the length of the horizontal test lines overlying the organelle examined. Today, these kinds of data can be acquired and analyzed using software for automatic computerized morphometry.

Stereology is the study of the 3-dimensional structure of objects from 2-dimensional views of them. The 3-dimensional interpretation from planar sections of cells uses random, systematic sampling to provide unbiased and quantitative data. It estimates higher dimensional data from lower dimensional samples. For light microscopy (LM, photon microscopy), stereology has available an astonishing array of equipment, preparatory systems, probes, and software that includes estimating grid size, automated counting, and data sorting. The increasing power of confocal and video LM and the expense and working difficulty of TEM stereology techniques have led to its limited use, particularly in North America. Use of EM stereology is greater in Europe; an annual short course on methods of preserving, visualizing, and quantifying antigens on cellular structures by EM stereology is provided by the European Molecular Biology Organization (see www.cwp.embo.org).

Electron Microscopic Tomography

Electron microscopic tomography (EMT) provides 3-dimensional constructs of organelles. Tomography is the recording of an image at a predetermined plane, and EMT is comparable to medical tomography techniques such as computed axial tomography, positron emission tomography, and MRI. In EMT, the biological sample is imaged with a TEM and a series of images taken from different views. ^1,53 Images are processed and combined to yield the 3-dimensional construct or tomogram. Individual tomograms are calculated from data on images obtained from samples on grids mounted on high-tilt holders; final combined tomograms are viewed with specific software programs.

Computational steps are necessary for interpretation—such as noise reduction, segmentation, and analysis of subvolumes. Computation demands are large in some systems, and high-performance computing (HPC) techniques are useful. ¹⁹ EMT software designed to develop cellular representations provides the ability to see the ultrastructural architecture of organelles and fibers (as well as viruses, bacteria, and other microbes ⁵⁰ ), as well as the clarity of structural relationships. EMT constructs of attachment, entry, and uncoating of herpes viruses have documented the process of viral infection at neuronal synapses. ³⁹

EMT studies using ultra-high-voltage electron microscopes (UHVEM) allow observation of dense and thick specimens. Enamel and other hard tissues can be studied by tomography to create a 3-dimensional ultrastructural image using the focus ion beam (FIB) method and UHVEM. Thin specimens of highly calcified tissue (without demineralization) allow penetration of a UHVEM beam of 2000 kV maximum acceleration voltage into the sample and tomographic projections with a resolution of 2 nm. ⁴⁵

Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) is a fusion of imaging techniques—the combination of methods of different microscopes that use photons or electrons for imaging. ^47,63,65 Modern CLEM techniques focus on the systematized matching of cellular areas between microscope modalities. CLEM means imaging the same structure with 2 or more imaging modalities, typically combining fluorescence and TEM using cells in culture. High-resolution light microscopic images and relocation of the same object at the ultrastructural level using a TEM demand improved ultramicrotomy techniques. ³¹ In most CLEM studies, a 3-dimensional construct of a macromolecular assembly is computed from 2-dimensional TEM images. Single-particle analysis (SPA) requires a large number of images or randomly oriented individual complexes and correction of the control transfer function (CTF) of the microscope. ¹⁶ CLEM is technically challenging and time-consuming, and it is designed for and applied to special approaches to a specific problem.

Cryofixation is required for CLEM on labile or fragile cytoplasmic components such as the cytoskeleton, Golgi complex, and tubules of the endomembrane system. Use of HPF, which is especially important for samples larger than a few micrometers, ⁴³ requires a rapid movement system developed for fast transfer from LM into a cryofixation device (see the Leica EM PACT2 rapid transfer system; Vienna, Austria); this technology is especially useful for CLEM studies. ¹¹

Virtual nanoscopy refers to methods that facilitate CLEM in studies that relate function to structure in large sections. These techniques are used for ultrastructural mapping of cellular regions as large as 1 mm² at nanometer resolution. ⁷⁰ They use standard TEM, rapid automated data collection, and stitching to create large “virtual slices.” For example, complete zebrafish embryo slides scalable in slides up to 281 gigapixels have been used to map endoplasmic reticulum wrapping of mitochondria within the liver. ¹⁸

CLEM expanded with the advent of new fluorescent probes and super-resolution light microscopic techniques, which allowed the study of dynamic processes in living cells. Tracers injected intravenously will label specific cells (such as neurons) and the tracer identified with diaminobenzidine and processing for EM. ⁴⁹ Fluorescent LM provides analytical, quantitative, and 3-dimensional data by using appropriate tags that allow tracking of specific proteins. CLEM strategies have been developed to add ultrastructural analysis. Adding EM tomography to rapid freezing provides new approaches for quantitative 3-dimensional analysis. Glycol methacrylate (GMA), introduced in 1977 for embedment at low magnification, also permits application of immunofluorescence; fluorescence is enhanced by reducing polymerization temperatures, removing 4-methoxyphenol, and adjusting pH of resins solution to alkalinity. ⁷⁴

Green fluorescent proteins (GFPs), which revolutionized light microscopic tracking in living cells, can also be used in TEM. Use of GFP-tagged chimeras allows users to follow organelle movement and to fix at the moment of interest. Correlative microscopy of GFP through photo-oxidation allows for the direct ultrastructural detection of fluorophores upon illumination. Oxygen radicals generated during the GFP bleaching process photo-oxidize diaminobenzidine (DAB) into electron-dense particles that can be seen by routine TEM of thin sections and by electron tomography for 3-dimensional analysis. ²⁴

Super-resolution fluorescence microscopy (SRFM) is used in CLEM to pinpoint the location of proteins at a resolution of 20 nm or even less. Histone proteins and the mitochondrial membrane protein TOM have been localized with SRFM. The ultrathin section is first imaged with SRFM and then with EM. This technique involves (1) high-pressure freezing of tissue, (2) cellular water substituted with acetone, (3) cells fixed with 0.1% potassium permanganate and 0.001% osmium tetroxide, and (4) specimens embedded in GMA plastic, trimmed, and 80-nm slices cut with a diamond knife. The correlative image on fluorescence and EM is aligned based on markers such as 100-nm gold nanoparticles. ⁷¹ Using appropriate software, the fluorescence pattern is correlated with organelles in EM done on the same section.

The SEM is widely used in CLEM studies. One dedicated instrument, the JOEL ClairScope (Tokyo, Japan), is a correlative scanning electron and optical microscope based on an inverted SEM column that allows images of wet samples to be obtained in ambient conditions in a culture dish via a silicon nitride field under the base. A standard inverted optical microscope above the dish holder can take reflected light and epifluorescence images of the same sample. ⁴⁶

Freeze-Fracture Electron Microscopy

Freeze-fracture electron microscopy (FFEM) is a technique for examining the ultrastructure of rapidly frozen biological samples by TEM. FFEM consists of physically breaking apart (fracturing) a frozen biological sample; standard detail exposed by the fracture plane is then seen by vacuum deposition of platinum-carbon to make a replica for examination by TEM. In routine protocols, the steps in FFEM include (1) pretreatment (before freezing) fixation in glutaraldehyde; (2) rapid freezing; (3) fracturing; (4) etching (optional), which involves vacuum sublimation of ice after fracturing; (5) replication; and (6) replica cleaning. ^57,64

The cryofixation replica technique provides a unique view of biological membranes. Plasma membranes are rapidly frozen to the temperature of liquid nitrogen (rapid freezing prevents formation of ice crystals). In this state, the membrane, when given a gentle tap, falls apart along the plane of least resistance, the central plane of the lipid bilayer (ie, the membrane comes apart between the lipid tails). This exposes the 2 half-membranes, each containing half the bilayer. One side of each half-membrane is the true membrane surface; the other is a view of the interior of the membrane, termed the fracture face. The background lipid is smooth, so the large protein molecules stand out as particles or depressions.

Since FFEM provides a planar view of membranes, a major emphasis in its use is to identify integral membrane proteins. In freeze-fracture studies, membrane proteins are seen to be missing or abnormally aggregated in response to several types of injury. In splitting the frozen lipid bilayer membrane into 2 halves, FFEM immobilizes membrane proteins and lipids by the vacuum evaporation of platinum and carbon. After treatment by sodium dodecyl sulfate (SDS) to remove extra-membrane materials, the specimen can be stained using immunogold labeling techniques to provide a 2-dimensional distribution of membrane proteins. ^51,64,69

FFEM cytochemistry embraces a series of techniques that share the chemical identification of structural components in freeze-fracture replicas. These include (1) label-fracture, in which cells in suspension are labeled with immunogold and processed for standard FFEM; (2) fracture-label, where the sample is frozen and fractured, then thawed for labeling; and (3) the freeze-fracture replica immunogold labeling (FRIL). In FRIL, samples are frozen, fractured, and replicated with platinum-carbon as in standard freeze-fracture and then treated with SDS to remove all the biological materials except a fine layer of molecules attached to the replica itself. The Tokuyasu cryo-section immunolabeling of chemically fixed cells is widely used as a sensitive, high-resolution localization technique; the advantages of the Tokuyasu protocol are that antigen accessibility is improved and antigens remain in a hydrated environment. ^13,56

Autoradiography and X-ray Microanalysis

The 2 physical methods to detect and quantify intracellular elements in organelles are autoradiography, which localizes radiolabeled compounds that have been injected systemically and incorporated into the cell, and x-ray microanalysis, which can identify and quantify the total elements in the cell. Once widely used in experimental pathology, autoradiography has largely been abandoned by cell biologists because of its imprecise localization, difficulty in use, and danger of radioactivity. Of all the physical TEM techniques available, electron probe x-ray microanalysis is most used for the study of biological specimens. ^20,48 However, x-ray microanalysis is little used by veterinary pathologists; it should be applied to tissue studies more often, especially to assess intracellular calcium, iron, and other ions that accumulate abnormally in a wide array of lesions. ³³

Atomic Force Microscopy

Atomic force microscopy (AFM) imaging is a high precision type of scanning probe microscopy with resolutions in fractions of a nanometer. Introduced for biological use in the 1990s, AFM provides surface detail of molecular aggregates and domains, membrane-associated structures, receptors, coated pits, and intercellular junctions. AFM also offers nano-manipulation possibilities, strengths and elasticity measurements, force interactions, affinity measurements, cell stiffness, and other physical aspects of membranes and cytoskeleton. Protocols for tissue section AFM techniques are available ²⁵ and have been valuable in studies on acute injury (eg, shape measurements for the quantitative analysis of mitochondrial swelling in myocardial injury). In studies of thin endothelial cells of liver, AFM has been used to characterize the filtration of lipid-carrying particles during transport from blood to hepatocyte. ⁹ In AFM, data are acquired by “feeling” the surface of the section with a mechanical probe—a cantilever with a sharp silicon tip for scanning. Forces between tip and sample deflect the cantilever and are measured using a laser spot reflected from the tip surface of the cantilever into an array of photodiodes. The astonishing precision comes from forces that deflect the cantilever: chemical bonding, van der Waals forces, electrostatic forces, and others.

The Future of Electron Microscopy

The renewed interest for TEM in medical and veterinary pathology arises from several changes over the past decade: (1) requirements that scientific proof of a cellular biochemical change revealed by the new light microscopic techniques ultimately requires evidence of ultrastructural change—the drawing of simple colored cartoons at this level, although instructive, simply doesn’t cut it; (2) improved electron microscopes with instrumentation that includes digitized systems for photography, analytical software programs that solve the problems of sampling, and specimen holders that allow tilting the specimen to a range of angles for unique diffraction angles; and (3) improvements in medical and veterinary education. Bioscientists who shy away from TEM because of the small sampling size, an issue that leads to problems of experimental design, difficulties in interpretation, and defenseless positions in the discussion of results, can now use new technology to prove their point.

Nowadays, a real problem is that electron microscopists are rarely lead scientists in research. Thirty years ago, research project leaders had an intimate knowledge of TEM and actually examined the tissues themselves—the changes that were discovered readily led to new TEM approaches. Now, instead, electron microscopists are asked to contribute after the project design has been completed and to provide data that will support the hypotheses. If the TEM results point in another direction, chances are that the TEM part of the project will be abandoned. Recently, I was asked to consult on an academic TEM study of livers of mice given drugs that disrupt the unfolded protein response. The resulting electron micrographs, despite having striking evidence of a new pathway of dysfunctional lipid metabolism, did not fit with the project’s hypothesis. The TEM part of the study was dropped with no interest in pursuing the astonishing possibilities of what was going on in lipid pathways of treated hepatocytes.

Part of this problem arises from the disturbing move of veterinary education away from critical thinking in basic science over the past decade. Fifty years ago, there was a much-needed push to move clinical material into the basic science curriculum, a push that has continued over the half-century. Today the need is for the reverse—the movement of basic science into the clinical curriculum. Over the long haul, the loss of understanding of abnormal biology is much more important than spending precious educational years teaching future practitioners how to conduct their future marketing and business skills. Changing how students think about cellular and molecular pathology will ultimately lead to thinking about electron microscopy to solve a critical problem.