Effect of specimen preparation and section transfer techniques on the preservation of ultrastructure,lipids and elements in cryosections

Cryofixation, cryoultramicrotomy, and proper transfer of the cryosections into the electron microscope are important for the preservation of good ultrastructure and the measurement of subcellular elemental distributions. These techniques are applicable to tissue systems which can be rapidly frozen so that minimal to no ice damage occurs during the cryofixation step. For the transfer step we have compared the cryotransfer of hydrated sections and subsequent freeze-drying in the electron microscope with the transfer of sections into an external freeze-dryer, followed by exposure to room temperature and humidity before introduction into the electron microscope. The use of a cryotransfer stage for section transfer from the cryoultramicrotome to the electron microscope and low temperature observation of the thin sections avoids the potential problem of rehydration damage to freeze-dried sections as well as provides protection from the possibility of melting of the lipids in the sections. Both of these problems may lead to loss of in situ elemental distribution and morphology. In this report, observations are presented which show the damaging effects of temperatures above 273 K on ultrastructure due to lipid melting in tissues with high lipid content and the redistribution of elements which can be encountered when thin sections become inadvertantly rehydrated.     

Correlated light optical and scanning electron microscopy of Gram smears of bacteria and paraffin sections of cardiac muscle

Light-microscope slides (3 in. × 1 in.) bearing Gram smears of Erysipelothrix rhusiopathiæ, or Staphylococcus aureus, after preliminary examination under the light-optical microscope (LM), were cut down in size, glued onto specimen stubs, coated with gold and examined in the scanning electron microscope (SEM). These preparations served as a control for investigations into bacteria-cell junctions in tissue. Cover-slips from stained sections of staphylococcal or swine erysipelas endocarditis mounted on 3 in. × 1 in. microscope slides (which had been intensively studied previously with conventional light microscopy) were floated off by immersing the slides in xylol. After dehydration of the tissues on the slides, the preparations were treated similarly to the Gram smears, and were examined with the SEM. Lesions of endocarditis were thus examined, and the information gained from these preliminary examinations shed new light on the pathogenesis of the disease. This information had not previously been available by any other technique. Because of this, and in view of the simplicity of preparing sections for scanning electron microscopy, it is suggested that the SEM might be a useful tool to be applied to routine histological sections.     

Potential of CLSM in studying some modern and fossil palynological objects

We have tested possibilities and limitations of confocal laser scanning microscopy to study the morphology of pollen and spores and inner structure of sporoderms. As test objects, we used pollen grains of the modern angiosperm Ribes niveum (Grossulariaceae) and Datura metel (Solanaceae), fossil angiosperm pollen grains of Pseudointegricorpus clarireticulatum and Wodehouseia spinata dated to the Late Cretaceous, fossil gymnosperm pollen grains of Cycadopites‐type dated to the Middle Jurassic, and fossil megaspores Maexisporites rugulaeferus, M. grosstriletus, and Trileites sp. dated to the Early Triassic. For comparative purpose, we studied the same objects with application of conventional light, scanning electron (to entire pollen grains and spores or to semithin sections of their walls), or transmission electron microscopy. The resolution of confocal microscope is much lower than that of electron microscopes, as are its abilities to reconstruct the surface patterns and inner structure. On the other hand, it can provide information that is unreachable by other microscopical methods. Thus, the structure of endoapertures in angiosperm pollen grains can be directly observed. It is also helpful in studies of asymmetrical pollen and pollen grains bearing various appendages and having complicated exine structure, because rotation of 3‐D reconstructions allows one to examine all sides and structures of the pollen grain. The exact location of all visible and concealed structures in the sporoderm can be detected; this information helps to describe the morphology and inner structure of pollen grains and to choose necessary directions of further ultrathin sectioning for a transmission electron microscopical study. In studies of fossil pollen grains that are preserved in clumps and stuck to cuticles, confocal microscope is useful in determining the number of apertures in individual pollen grains. This can be done by means of virtual sections through 3‐D reconstructions of pollen grains. Fossil megaspores are too large and too thick‐walled objects for a confocal study; however, confocal microscope was able to reveal a degree of compression of fossil megaspores, the presence of a cavity between the outer and inner sporoderm layers, and to get some information about sporoderm inner structure.     

Contrast in the transmission mode of a low-voltage scanning electron microscope

The contrast thicknesses (x_k) of thin carbon and platinum films have been measured in the transmission mode of a low-voltage scanning electron microscope for apertures of 40 and 100 mrad and electron energies (E) between 1 and 30 keV. The measured values overlap with those previously measured for E (≥ 17keV) in a transmission electron microscope. Differences in the decrease of x_k with decreasing E between carbon and platinum agree with Wentzel-Kramer-Brillouin calculations of the elastic cross-sections. Knowing the value of x_k allows the exponential decrease ∝ exp(—x/x_k) in transmission with increasing mass-thickness (x = ρt) of the specimen and the increasing gain of contrast for stained biological sections with decreasing electron energy to be calculated for brightfield and darkfield modes.     

Silver impregnation applied to striated muscle: The sarcoplasmic reticulum

The classical black reaction developed by Camillo Golgi is shown to impregnate the tubules and fenestrations of the sarcoplasmic reticulum (SR) in striated muscle. This is a double impregnation of chromate and silver, which usually fills extracellular spaces. The method is difficult insofar as long incubation times are required, and location of the successfully “stained” SR in plastic-embedded tissue blocks is unpredictable. The light microscope is absolutely necessary to find the good regions which can then be cut from the blocks in 1-μm-thick sections and examined in the electron microscope. Stereo pairs give the best results since these resolve overlap problems common to thick sections. A variety of artifacts are illustrated which can help avoid erroneous interpretations. The Golgi-“stained” SR shows this elusive network with unsurpassed contrast and should benefit the morphological studies of muscle-membrane enthusiasts.     

Neuronal imaging with colloidal gold

Colloidal gold is easily prepared, and readily adsorbs to a number of immunoreagents and other proteins for a wide variety of uses for neuronal visualization. Gold probes serve a role as immunolabels for both light and electron microscopy. As an ultrastructural immunocytochemical marker for detection of proteins, peptides or amino acids, gold can be used for immunostaining thick or thin sections prior to embedding, or for immunostaining ultrathin sections after embedding tissue in conventional or unusual embedding matrices. By virtue of its particulate nature, gold as an immunolabel facilitates a semi-quantitative analysis of relative antigen densities on ultrathin sections. Various combinations of different size gold particles or dual immunolabelling with enzymatic immunolabels together with colloidal gold or silver-intensified gold serve well for ultrastructural immunocytochemical localization of two antigens in the same tissue section. Colloidal gold can be detected with light microscopy, transmission and scanning electron microscopy, and with confocal laser microscopy. Silver intensification allows detection of gold at both the light and electron microscope level, and increases the sensitivity of immunogold procedures. Colloidal gold is useful as a tracer for physiological studies of transport and internalization in neurons in vivo and in vitro; computer-assisted video imaging techniques allow detection and tracking of single gold particles in living cells.     

Influence of depth of information and of resolution in stereologic evaluation of surface electron micrographs

Due to the difference in preparation technique, the submicroscopic structure of metals, minerals and ceramics is often easier to evaluate with surface electron micrographs of bulk specimens than with transmission electron micrographs of thin films. As in the latter case, a surface electron micrograph also contains information from a certain depth which for stereologic evaluation necessitates a correction. This correction depends on the relation of grain size, depth of information and resolution. Experimental studies lead to the following results: With 20 keV scanning electron micrographs showing a material contrast resolution between 50 and 200 nm, depending on the material, stereometric analyses are influenced by the depth of information for grain sizes between 1 and 25 μm. Furthermore, the results of analysis are influenced by ‘microscope distortion’ and by ‘distortion of single micrographs’. Depth of information and distortions have the effect that the grain distribution image of polished sections may differ by some percent from an ideal section image. An automatic microstructure analysis seems to be nearly impossible because of variations of photographic density obtained from different particles of one and the same phase. For photo-emission electron micrographs with a material resolution between 15 and 30 nm depending on the material, the influence of the depth of information, microscope distortion and distortions of single micrographs may be neglected. Therefore, such micrographs are well suited for manual or automatic stereometric analysis.     

Contrast in the electron spectroscopic imaging mode of a TEM

The ratio of inelastic-to-elastic total cross-sections has been measured in an energy-filtering electron microscope for different elements. Formulae for the transmission of elastically and inelastically scattered electrons in part I were used to calculate the optimum conditions for a Z-ratio contrast in the electron spectroscopic imaging mode. Structure-sensitive contrast can be observed for all non-carbon atoms in biological sections when filtering with an energy loss at ΔE ～ 250 eV below the carbon K edge. Model experiments with evaporated layers of different elements on a carbon film allow measurement of the contrast increase. Filtering with the carbon plasmon loss shows a lower phase contrast than with zero-loss filtering. This can be explained by calculating contrast transfer functions for inelastically scattered electrons.     

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.     

Optical fringe effects at prism borders in human tooth enamel sections

When ground sections of mammalian teeth are viewed under the light microscope each enamel prism appears to be surrounded by a prism sheath about 0.5 μm] wide. Seen by the electron microscope these sheaths are at most 0.1 μm wide. Because this dimension is beyond the resolving power of the light microscope it is difficult to explain the origin of the optical image. Optical fringes are frequently seen at the borders of transversely cut enamel prisms. The widths of these fringes have been measured under different optical conditions. It is concluded that the fringes are produced due to interference between direct light rays and those reflected from prism borders. A requisite for clear fringe production is that the reflectance of the prism border should be high. This high reflectance could only be achieved if prisms are separated from each other by material having a refractive index which differs significantly from that of the prisms themselves. It is therefore suggested that prisms are separated by a protein layer. It is then possible to explain why prism sheaths appear 0.5 μm wide under the light microscope. Study of mammalian tooth enamel by light microscopy suggests to the observer that the 5 μm wide rods (generally referred to as prisms) which constitute the enamel are each partially surrounded by a ‘sheath’ about 0.5 μm wide. Micro-radiographs indicate that the sheath is considerably less mineralized than the body of the prism (Glas, 1965). In contrast, from direct observation of enamel sections by electron microscopy, it has been concluded that no prism sheath exists and that prisms are separated by an interface which is bordered on its two sides by differently orientated crystals (Helmcke 1960, 1967; Ronnholm, 1962; Meckel, Griebstein & Neal, 1965). Between these two extremes are some electron microscope observations appearing to demonstrate the presence of a 0.1 μm wide membrane-like prism sheath devoid of inorganic crystals (Frank & Nalbandian, 1967) or an irregular region of microporosity where the different orientation of adjacent crystals results in a packing defect (Johnson, 1967). Any enamel section observed with the electron microscope may well have been distorted during its preparation for examination. Shrinkage due to dehydration is one of the most likely artifacts. Because the amount of the distortion cannot be known with accuracy it is difficult to decide which of the above electron-microscope appearances is closest to the actual structure of the prism boundary during life. It has recently been observed that when viewed end-on, the borders of prisms frequently appear as light striations (Osborn, 1968 and in press). In the present investigation the widths of these light striations have been measured under different optical conditions on an enamel section which was maintained in a hydrated condition. It was thought that this data on the optical properties of the hydrated prism boundary could be used to predict the structure of the boundary in vivo. A similar shrinkage is to be expected in sections examined with the light microscope because these have usually been dehydrated prior to mounting. It is therefore reasonable to try to relate the 0.5 μm wide optical image seen with the light microscope to the two different structures observed with the electron microscope. If no prism sheath exists it might be argued that the refractive index variation between the borders of adjacent prisms could in some way account for the production of the optical image. However, because this variation is probably less than 0.02 (Fremlin & Mathieson 1964) it is not clear how it could be used to explain the high contrast of the optical image. Furthermore, even if a 0.1 μm wide prism sheath exists, it is significantly thinner than the wavelength of visible light; again it is not clear how its presence results in the production of an image in the light microscope.     

The use of scanning electron microscope autoradiography to localize the blueberry shoestring virus in its aphid vector

Scanning electron microscopy autoradiography (SEM-AR) in conjunction with light microscope autoradiography (LM-AR) was used to follow the movement of ¹²⁵I-labeled blueberry shoestring virus (BBSSV) through its aphid vector Illinoia pepperi with varying acquisition access periods (AAP). At 6 hr AAP, the virus had reached the stomach; at 12 hr AAP, it had reached the anterior of the intestines, and after 48 hr, AAP was present throughout the aphid. SEM-AR using backscatter electron detection proved very useful because the sample bulk resulted in a shortened exposure time compared to LM sections, correlation of the autoradiograms could be made with fine structure, preparing large numbers of samples was easy, and examining whole longitudinal sections of aphids at low magnification with a clearly visible marker was possible. Limitations were mostly attributed to sample preparation and, in some cases, were easily remedied.     

Histological and ultrastructural features of the rectum in Poecilimon cervus Karabağ, 1950 (Orthoptera: Tettigoniidae)

The morphology and ultrastructure of the rectum in Poecilimon cervus Karaba?, 1950 (Orthoptera, Tettigoniidae) were analyzed by light microscope, scanning (SEM) and transmission electron microscopes (TEM). The rectum is the final part of the digestive tract that plays an important role in water reabsorption in insects and so provides osmoregulation. In the transverse sections, six rectal pads and columnar epithelium can be distinguished. The cuticular intima lines the lumen at the apical side of the epithelium. In the cytoplasm, there are numerous mitochondria, some endocytic vesicles, secreting vesicles whose sizes differ according to the area in the cell, and a nucleus with globular in shape. With this study, we aimed to demonstrate the ultrastructure of the rectum of P. cervus and differences or similarities of with other species.     

The cutting of ultrathin sections with the thickness less than 20 nm from biological specimens embedded in resin blocks

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post‐fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512–517, 2016. © 2016 Wiley Periodicals, Inc.     

The glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3-D reconstruction

Computer-aided 3-D reconstruction of typanosomes from 0·35-μm-thick sections imaged on the Zeiss 902 electron microscope are being used to study the dynamics of cell organization. Segregation of glycolytic enzymes into glycosomes raises questions concerning the distribution and biogenesis of these organelles. Direct counts of glycosomes from Trypanosoma evansi indicate 30–40 per cell and for the closely related T. brucei, 65 per cell. These figures contrast with the estimates of others who have used model-based morphometric methods to obtain a value of 230 per cell.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate-accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). The specimens were embedded in water-soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate-accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

A novel method of Z-contrast imaging in STEM applied to double-labelling

A novel method of Z-contrast imaging in the scanning transmission electron microscope (STEM) is presented. The technique relies on the element dependence of the angular distribution of the scattered electrons, and is realized with a detector consisting of a set of concentric rings. It is possible to discriminate 9-nm colloidal gold and silver specifically distributed on thin sections. In addition to this practical work, numerical evaluations are used to assess the method. With two smaller markers, this approach will be useful in discriminating closely spaced antigenic sites when steric hindrance occurs with double-labelling using probes of different sizes.     

Histology and ultrastructure of the testis and vas deferens in Pseudochorthippus parallelus parallelus (Orthoptera,Acrididae)

Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) is a widespread species in Europe, and also it is localized in some regions in Turkey such as Bursa, Eski?ehir, Ankara, Bolu, Düzce, and Çank?r?. The features of the reproductive organs such as the numbers and shapes of testes and follicles can be used as taxonomical characters. For this purpose, the ultrastructural and histological features of testis and vas deferens in P. parallelus parallelus were examined with using light microscope, scanning electron microscope, and transmission electron microscope. The mature P. parallelus parallelus has two conjugated testes produce spermatozoa. Each testis is composed of numerous testis follicles in which different stages of spermatogenesis and spermiogenesis develop. First, spermatocytes are formed by the mitosis division of the germ cells at the distal end of the follicles. Then, spermatocytes form spermatids by meiosis division in the middle region of the follicles. Finally, spermatids are differentiated to spermatozoa at the proximal region of the follicles. After maturation of the spermatozoa, sperm tails come together as the sperm bundles called as spermatodesm. Each follicle is connected to vas deferens via vas efferens to discharging spermatozoa. In spite of some differences, the testes and the vas deferens in P. parallelus parallelus are highly similar to the those of other species, especially Orthopteran species.