Synaptic morphology of inner and outer hair cells of the human organ of Corti

Innervations of inner and outer hair cells of the organ of Corti of the human cochlea were studied by serial section electron microscopy. At the base of inner hair cells, presumed afferent fibers were of varying size and demonstrated synaptic specialization consisting of a presynaptic body, vesicles, and asymmetrical synaptic membrane specialization. Two types of neurons, vesiculated presumably efferent and nonvesiculated presumably afferent, synapsed at the base of outer hair cells. The synaptic specialization of afferent fibers included presynaptic body, vesicles, and asymmetrical membrane thickening, whereas efferent synapses demonstrated presynaptic vesicles and a subsynaptic cisterna. Some presumably afferent nerve terminals formed a reciprocal synapse with outer hair cells in both the human and the chimpanzee. Such a synaptic relationship demonstrated morphologic specialization consistent with both hair cell-to-neuron and neuron-to-hair cell transmission between the same outer hair cell and nerve terminal. The innervation density of inner and outer hair cells and the comparative anatomy of the afferent and efferent innervation are discussed.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Surface and internal structure correlation: high-voltage and scanning electron microscopies of wholemount alveolar walls of human lung

We found that the high-voltage electron microscope (HVEM) operating at 1–5 MeV was able to transilluminate and form a focused transmission image of whole-mounts of alveolar walls from human lung, a tissue sufficiently thin to require no embedment and sectioning. Resultant micrographs resembled a composite of scanning and transmission electron microscope images: surface and internal structure of the alveolar wall were visualized in a single micrograph. Although the scanning electron microscope extracts some subsurface information in the secondary electron mode, the HVEM produced better images of both surface and subsurface features. Lungs were fixed, dehydrated, critical point dried, and metal coated as for conventional scanning electron microscopy, then individual alveolar walls were excised by hand and mounted on transmission electron microscope grids. Regions of the alveolar wall up to 10 μm thick were delineated with the high-voltage electron microscope. Cell surface characteristics were correlated with cell type as identified by underlying cell internal structure. Whole white blood cells within capillaries of the alveolar wall were identified by the configuration of their nuclei. Features of the nucleus and surface of alveolar type II cells were recorded simultaneously. Whole red blood cells were imaged within intact capillaries that branched and wove from one alveolar surface to the other. HVEM analysis of excised alveolar septa allows definitive correlation of surface and underlying structures in single micrographs of broad portions of the alveolar wall and is an alternative to embedment, microtomy and serial section reconstruction for this uniquely thin tissue.     

The ultrastructure of neuronal-pinealocytic interconnections in the monkey pineal.

Recent ultrastructural studies of neuronal-pinealocytic interconnections in the monkey pineal are reviewed. The pinealocytes in the adult monkey show almost all of the cytological specializations known in subprimate mammals. Adjacent pinealocytes are functionally coupled through ribbon synapses on cell bodies and gap junctions on cell bodies and cell processes. The pinealocytes receive direct synpatic contacts of nerve fibers with cholinergic terminal morphology. Nerve cells restricted to the central portion of the pineal receive synaptic contacts with more than three different morphologically defined types of nerve terminals. In addition to nerve terminals containing small clear vesicles or vesicles of pleomorphic morphology, a pinealocyte's terminal process containing the synaptic ribbon forms a true synaptic contact on the nerve cell body. The diversity of synapses on these nerve cells strongly suggests multiple origins of these neurons rather than a single peripheral parasympathetic origin. The possible involvement of pineal neurons in an intrinsic circuit that regulates the function of pinealocytes and integrates the neural input from the central as well as the peripheral nervous systems is discussed.     

Neuromodulation of arthropod mechanosensory neurons

Arthropod mechanosensory afferents have long been known to receive efferent synaptic connections onto their centrally located axon terminals. These connections cause presynaptic inhibition by attenuating the action potentials arriving at the axon terminals, thus reducing the synaptic potentials in the postsynaptic neurons. This type of inhibition can specifically reduce the excitation of selected postsynaptic neurons while leaving others unaffected. However, recent research has demonstrated that sensory signals detected by arthropod mechanosensory neurons can also be synaptically modulated before they ever arrive at the axon terminals. In arachnids and crustaceans, wide and complex networks of synapses on all parts of the afferent neurons, including the somata and dendrites, provide mechanisms to inhibit or enhance the responses to mechanical stimuli as they are being detected. This modulation will affect the signal transmission to all axonal branches and postsynaptic cells of the affected receptor neuron. In addition to the increased complexity of mechanosensory information transmission produced by these synapses, a variety of circulating neuroactive substances also modulate these neurons by acting on their postsynaptic receptors.     

The use of high-voltage electron microscopy and semi-thick sections for examination of cyanobacterial thylakoid membrane arrangements

High-voltage electron microscopy (HVEM) of semi-thick sections was evaluated as a technique for studying thylakoid membrane arrangements in cyanobacterial cells. Semi-thick sections (0·25 μm) provided important information that was relatively difficult or impractical to obtain by viewing either randomly or serially cut thin sections. Specifically, the semi-thick sections were better suited for visualizing (i) overall thylakoid arrangements and (ii) interconnections between the thylakoids and the cytoplasmic membrane. By comparison, randomly cut thin sections frequently yielded deceptively incomplete or inconsistent data in regard to these specific features. Tilting of thick sections about two perpendicular axes served to improve the clarity of complex membranous intersections and other cell features.     

Electron diffraction in porous silicon

Electron diffraction patterns, obtained in a high-voltage electron microscope (HVEM), of silicon containing a high density of small pores (diameter ca. 5 nm) produced by anodic etching show an enhancement of high-order Bragg reflections at the expense of low-order Bragg reflections. A theory of this effect is developed based on multiple scattering of Bloch waves at the pores. The theory allows the determination of the volume fraction of the pores from quantitative diffraction data. The results of this analysis are in good agreement with those obtained from HVEM images.     

CTEM versus STEM versus HVEM: Evaluation of the three-dimensional morphology of arsenic inclusions

Electron-dense arsenic inclusions appeared in the nucleus of parenchymal hepatocytes from fish exposed to arsenate, but were absent in fish exposed under identical conditions to solutions lacking arsenic. Images of these inclusions were compared using conventional transmission electron microscopy (CTEM), scanning transmission electron microscopy (STEM), and high-voltage electron microscopy (HVEM). Stereo pairs from the same individual inclusions were examined using each of these methods to provide a more complete understanding of their three-dimensional organization and to evaluate the relative merits of each technique in the study of similar electron-dense structures. Comparable results were obtained with the three types of instrumentation. Although HVEM is the technique of choice for the analysis of three-dimensional images of such high electron-dense structures, STEM proved to be a good alternate technique for the selection and general evaluation of samples in preparation for HVEM.     

Immunoelectron microscopic studies on protein gene product 9.5 and calcitonin gene-related peptide in vallate taste cells and related nerves in the guinea pig

On the basis of our previous report that protein gene product 9.5 (PGP 9.5)-immunoreactive nerve fibers and taste cells and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers are found in guinea pig vallate papillae [Huang and Lu (1996b) Arch. Histol. Cytol. 59:433-441]. We speculated that PGP 9.5 might be a marker for taste receptor cells and that CGRP might play an important role in taste transmission. We, therefore, performed an immunohistochemical and ultrastructural analysis of taste cells and related nerves in guinea pig vallate papillae. In the connective tissue of the vallate papilla, the ultrastructural data revealed that the PGP 9.5-immunoreactive nerve fibers were both myelinated and unmyelinated. The CGRP-immunoreactive nerve fibers were unmyelinated and surrounded by the cytoplasm of Schwann cells as were the non-immunoreactive fibers. In the vallate taste buds, only type III cells, which make synaptic contacts with intragemmal nerves, were PGP 9.5-immunoreactive, while the nerve terminals making synaptic contact with the underlying type III cells were CGRP-immunoreactive. From these observations, we conclude that: (1) PGP 9.5 might be a useful specific marker for type III cells in guinea pig vallate taste buds; and (2) CGRP-containing nerve fibers might be primarily involved in the neural transmission of taste stimuli.     

Application of the Golgi/electron microscopy technique for cell identification in immunocytochemical, retrograde labeling, and developmental studies of hippocampal neurons.

In this study the Golgi/electron microscopy (EM) technique has been used for an analysis of the fine structure, specific synaptic connections, and differentiation of neurons in the hippocampus and fascia dentata of rodents. In a first series of experiments the specific synaptic contacts formed between cholinergic terminals and identified hippocampal neurons were studied. By means of a variant of the section Golgi impregnation procedure, Vibratome sections immunostained for choline acetyltransferase, the acetylcholine-synthesizing enzyme, were Golgi-impregnated in order to identify the target neurons of cholinergic terminals in the hippocampus. It could be shown with this combined approach that cholinergic septohippocampal fibers form a variety of synapses with different target structures of the Golgi-impregnated and gold-toned hippocampal neurons. In this report cholinergic synapses on the heads of small spines, the necks of large complex spines, dendritic shafts, and cell bodies of identified dentate granule cells are described. The variety of cholinergic synapses suggests that cholinergic transmission in the fascia dentata is a complex event. Next, the Golgi/EM technique was applied to Vibratome sections that contained retrogradely labeled neurons in the hilar region of the fascia dentata following horseradish peroxidase (HRP) injection into the contralateral hippocampus. With this combined approach some of the hilar cells projecting to the contralateral side were identified as mossy cells by the presence of retrogradely transported HRP in thin sections through these Golgi-impregnated and gold-toned neurons. Our findings suggest that the mossy cells are part of the commissural/associational system terminating in the inner molecular layer of the fascia dentata. They are mainly driven by hilar collaterals of granule cell axons that form giant synapses on their dendrites. Finally, the Golgi/EM procedure was used to study the differentiation and developmental plasticity of hippocampal and dentate neurons in transplants and slice cultures of hippocampus. Under both experimental conditions, the differentiating neurons are deprived of their normal laminated afferent innervation but develop their major cell-specific characteristics including a large number of postsynaptic structures (spines). As revealed in thin sections of gold-toned identified cells, all these spines formed synapses with presynaptic boutons suggesting sprouting of the transplanted and cultured neurons, respectively. Altogether, the present report demonstrates the usefulness of the Golgi/EM technique, particularly of the section impregnation procedure, for a variety of studies requiring the identification of individual neurons at the ultrastructural level.     

Organization of microtubules in cochlear hair cells

The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.     

The inner ear ultrastructure from the paddlefish (Polyodon spathula) using transmission electron microscopy

The structure of the hair cells on each sensory macula from the inner ear of the paddlefish (Polyodon spathula) was studied using scanning and transmission electron microscopy, revealing the nucleated cell bodies and peripheral nerve fibres of the saccule utricle and lagena. Examination of the structures within the cell body revealed comparable features with those found in the inner ear hair cells from bony fish species, although in P. spathula the afferent cell body is almost twice the size. This is the first time that the inner ear hair cells from an Acipenseriform fish have been studied using transmission microscopy, thus providing benchmark anatomical information in relation to the cellular morphology of the afferent receptors from a ‘healthy’P. spathula ear. Structural information is of assistance in the study of aquatic animal hearing for environmental monitoring purposes, as morphological data can be used to confirm if evidence of raised hearing thresholds from animals exposed to intense anthropogenic noise or other destructive agents (determined using electrophysiological or behavioural techniques) are a direct result of damage to the ultrastructure of the inner ear.     

Technique to study three-dimensional spatial arrangement of synaptic vesicles using data from single sections

Synaptic vesicles are membrane-bound organelles storing neurotransmitters in presynaptic terminals and releasing them into the synaptic cleft. Coordinated movements of synaptic vesicles relate to synaptic function and their spatial arrangement can provide useful information about the activity of a synapse. This article presents a technique to extract quantitative information about three-dimensional (3D) spatial arrangement of synaptic vesicles from measurements performed on single ultrathin random sections of a presynaptic terminal. The technique presumes quantification of a 2D density as well as 2D spatial pattern formed by vesicle profiles using a minimum spanning tree (MST) algorithm, in digitized micrographs of a presynaptic terminal. Further, original software was used to simulate a 3D spatial arrangement of synaptic vesicles and their random sectioning. A 3D density and pattern of synaptic vesicles were used as basic input parameters of the model, while a 2D density and MST quantities for vesicle profiles served as output, model-derived parameters allowing one to compare and fit simulated distributions to experimental ones. Pilot simulations performed to check the validity of the technique have shown that a 2D density and MST quantities of vesicle profiles closely relate to a 3D density and spatial pattern of vesicles. The technique was demonstrated in the analysis of spatial distribution of synaptic vesicles in axonal terminals forming asymmetric synaptic densities in the stratum radiatum of the CA1 subfield of the murine hippocampus.     

Freeze-fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/μm². In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/μm². A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.     

Immunoelectron microscopic localization of neurotransmitters in the cochlea

This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to γ-aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA-like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA-like, and sometimes met-enkephalin-like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.     

Effects of atopic dermatitis on the morphology and water content of scalp hair

The effects of atopic dermatitis (AD) on scalp hair properties, such as morphology and water content, were investigated using atomic force microscopy (AFM) and thermogravimetric analyzer. Hairs from lesional and nonlesional scalp regions of eight patients with AD were investigated. The severity of the disease, which was evaluated using the SCORing Atopic Dermatitis index, was 48.75 (range, 40-80). Hairs from 15 normal adults were also examined as controls. The surface images were taken in an area of 20 × 20 μm(2) with 512 × 512 pixels and a scan speed of 0.8 line/s. AD affected the cuticle structures and scales of scalp hair. The edges of cuticles were torn and collapsed, and the scales were very thick. The water contents of both types of AD hair were less than the control: 12% ± 0.7%, 11.7% ± 0.4%, and 13% ± 0.8% for lesional AD hair, nonlesional AD hair, and control hair, respectively. The scalp hair of patients with AD can be characterized by thick and globular scale patterns. The hair of patients with AD has less water content than normal hair showing a good agreement with the property of skin having AD.     

A new method to correlate acoustic spectroscopic microscopy (30 MHz) and light microscopy

A powerful new method is used to investigate the correlation between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into successive slices, 250 μm and 10 μm thick. The thick sections were investigated acoustically, the thin sections by means of light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2·5 × 2·5 mm). Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30 MHz, the slope of the attenuation spectrum (between 10 and 50 MHz), backscattering at 30 MHz, the slope of the backscattering spectrum (between 10 and 50 MHz) and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 μm. The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen-rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 × 250 μm. These histological features were correlated with the acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.     

Scar formation in mice deafened with kanamycin and furosemide

In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium–thiocarbohydrazide–osmium (OTO), tannic acid–arginine–osmium, and the conventional method with gold‐coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech. 79:766–772, 2016. © 2016 Wiley Periodicals, Inc.     

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.