On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

Immunogold staining procedure for the localisation of regulatory peptides

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.     

Microbial cell disruption for improving lipid recovery using pressurized CO2: Role of CO2 solubility in cell suspension,sugar broth,and spent media

The study of in situ gas explosion to lyse the triglyceride‐rich cells involves the solubilization of gas (e.g., carbon dioxide, CO₂) in lipid‐rich cells under pressure followed by a rapid decompression, which allows the gas inside the cell to rapidly expand and rupture the cell from inside out. The aim of this study was to perform the cell disruption using pressurized CO₂ as well as to determine the solubility of CO₂ in Rhodotorula glutinis cell suspension, sugar broth media, and spent media. Cell disruption of R. glutinis was performed at two pressures of 2,000 and 3,500 kPa, respectively, at 295.2 K, and it was found from both scanning electron microscopy (SEM) and plate count that a substantial amount of R. glutinis was disrupted due to the pressurized CO₂. We also found a considerable portion of lipid present in the aqueous phase after the disruption at P = 3,500 kPa compared to control (no pressure) and P = 2,000 kPa, which implied that more intracellular lipid was released due to the pressurized CO₂. Solubility of CO₂ in R. glutinis cell suspension was found to be higher than the solubility of CO₂ in both sugar broth media and spent media. Experimental solubility was correlated using the extended Henry's law, which showed a good agreement with the experimental data. Enthalpy and entropy of dissolution of CO₂ were found to be ?14.22 kJ mol^?1 and 48.10 kJ mol^?1 K^?1, 9.64 kJ mol^?1 and 32.52 kJ mol^?1 K^?1, and 7.50 kJ mol^?1 and 25.22 kJ mol^?1 K^?1 in R. glutinis, spent media, and sugar broth media, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:737–748, 2017     

The adenohypophysis of the flounder,Pleuronectes flesus,and the minnow,Phoxinus phoxinus

Summary The pituitary gland of the flounder, Pleuronectes flesus, showed several unusual cytological features. Between the RPD and the PPD was a zone of cells that stained purple with Alcian blue—PAS—orange G. Many of these cells were apparently degenerating. In the PPD the strands and coils of presumptive STH cells showed a tremendous variation in both size and staining properties. In the PI there were two cell types, the PAS-positive one bordering the neurohypophysis. Around the periphery of the PI was a zone of chromophobic cells, and throughout the PI were numerous intracellular and extracellular acidophil spheres.No well defined ACTH cells were found in the RPD of the minnow (Phoxinus phoxinus). The Alcian blue—PAS—orange G technique distinguishes between blue TSH cells and purple GTH cells in the RPD and PPD. GTH cells from animals collected in the winter were vacuolated. The PI contained two cell types whose staining reactions and ultrastructure were extremely variable. Intra- and extra-cellular acidophil spheres were present.I should like to thank Dr. T. Kerr of the Zoology Department, Leeds University, for his help and encouragement, and Mr. J. Dingley of the Department of Zoology, Aberystwyth, for collecting the flounders.     

Electron microscopy of dry peanut (Arachis hypogaea L.) seeds crushed for oil removal. Fixation and embedding of anhydrously prepared tissues

Summary Aqueous fixatives caused dry seed tissues to swell; mashed peanuts, crushed to remove oil, swelled even more. Use of anhydrous, organic solvents as vehicles for fixatives enabled maintenance of dimensional stability during fixation of dry seed tissues; even crushed seed tissue did not swell significantly when processed anhydrously. However, anhydrously processed specimens proved difficult to section. The difficulty was due to imperfect permeation of plastic into the seed tissues during embedding. An explanation of why anhydrously processed dry seed tissues are so difficult to embed in plastic is offered.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

The basement membrane of the insect and crustacean compound eye: Definition,fine structure,and comparative morphology

Summary The basement membrane of the compound eye of four insect species and three crustacean species was investigated employing electron microscopy. The basement membrane consists of an extracellular (basal lamina) and a cellular portion, the latter being composed of the flattened terminal extensions of cone cells and accessory pigment cells in insects and distal pigment cells in crustaceans. Other cells can also contribute to the basement membrane. It is thus a complex structure in all well-developed compound eyes. The cellular contributions vary in different species and were found to correlate to specific taxonomic units.     

Plasmodium iophurae: cationized ferritin staining, an electron microscope cytochemical method for differentiating malarial parasite and host cell membranes

The surface membranes of erythrocyte-free Plasmodium lophurae and its host cell, the duckling erythrocyte, stain differentially when exposed to cationized ferritin (CF). At low CF concentrations (0.18 mg/ml) only the outer surface of the red cell stains, whereas at the standard concentration (0.7 mg/ml) both the red cell and the parasitophorous vacuolar membranes (PVM) were stained on their outer faces. By using a high CF concentration (3.7 mg/ml), the parasite's plasma membrane (PM) could be distinguished from that of the PVM: The former did not bind CF, whereas the latter was stained on its outer surface. At this level of CF the red cell membrane stained on both faces if these surfaces were exposed to stain. Although the PVM is formed by red cell endocytosis of the parasite, it can be distinguished from the membrane of the erythrocyte as well as that of the PM.     

The use of electron microscopy and immunocytochemistry to characterise spontaneously-arising,transplantable rat tumors

When examined by light microscopy, transplanted animal tumors frequently bear little resemblance to the original neoplasm. If such tumors are to be used as models of human cancer they should be characterised as regards extant rather than historical features. Consequently, we have examined, by electron microscopy and immunocytochemistry, five spontaneously arising tumors transplantable in the WAB/Not rat that are currently diagnosed on the basis of historical features only. A typical sarcoma was used for comparison. Of four spontaneously arising tumors previously classified as carcinoma, Sp4 possessed epithelial features on both ultrastructural and immunocytochemical analysis, Sp107 on ultrastructural analysis only and Sp15 and Sp22 by neither technique. Expression of vimentin was most marked with Sp15 and Sp107. The putative sarcoma, Sp24, showed clear evidence of epithelial differentiation but no evidence of vimentin expression. This study (a) records the phenotypic drift of experimental tumors on transplantation (most clearly with Sp107) and the co-expression of cytokeratins and vimentin in putative carcinomas, (b) confirms the inadequacy of routine histology for accurate characterisation of such tumors and (c) details techniques for a more thorough assessment of state of differentiation that should guide the choice of experimental model.     

The influence of embedding media and fixation on the postembedment ultrastructural demonstration of complex carbohydrates. III. High iron diamine staining for sulfated glycoconjugates

The high iron diamine (HID) method for detection of sulfated complex carbohydrate has been applied directly on thin sections of variably fixed tissues embedded in epoxy and nonepoxy resins. Results with postembedment HID staining in mouse intestinal epithelium are compared to those previously obtained using preembedment methods. Sections from epoxy-embedded tissues have been found to exhibit the weakest staining intensity. Intense, specific staining was obtained in tissues not postfixed with osmium tetroxide and embedded in polystyrene, polyester resins, styrene-methacrylate, and especially the styrene-Vestopal W embedding mixture. Postosmication of tissues abolished HID staining in epoxy resins and the styrene-Spurr's resin embedding mixture, but only reduced the staining intensity in tissues embedded in nonepoxy resins.     

Spermatogenesis and sperm structure of Acerella muscorum, (Ionescu, 1930) (Hexapoda, Protura)

The general organization of the male genital system, the spermatogenesis and the sperm structure of the proturan Acerella muscorum have been described. At the apex of testis apical huge cells are present; their cytoplasm contains a conventional centriole, a large amount of dense material and several less electron-dense masses surrounded by mitochondria. Spermatocytes have normal centrioles and are interconnected by cytoplasmic bridges. Such bridges seem to be absent between spermatid cells and justify the lack of synchronization of cell maturation. Spermatids are almost globular cells with a spheroidal nucleus and a large mass of dense material corresponding to the centriole adjunct. Within this mass a centriole is preserved. Mitochondria of normal structure are located between the nucleus and the plasma membrane. The spermatids are surrounded by a thick membrane. No flagellar structure is formed. Sperm have a compact spheroidal nucleus, a large cap of centriole adjunct material within which a centriole is still visible. A layer of mitochondria is located over the nucleus. The cytoplasm is reduced in comparison to spermatids; many dense bodies are interspersed with sperm in the testicular lumen. The sperm are small, immotile cells of about 2.5-3 μm in diameter.     

The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta)

Spermatogenesis and the sperm structure of the terebrantian Aeolothrips intermedius Bagnall are described. Spermatogenesis consists of two mitotic divisions; the second is characterized by the loss of half of the spermatids, which have pyknotic nuclei. Early spermatids have two centrioles, but when spermiogenesis starts, a third centriole is produced. The three basal bodies give rise to three flagella; later these fuse into a single flagellum which contains three 9 + 0 axonemes. The basal bodies are surrounded by a large amount of centriole adjunct material. During spermiogenesis this material contributes to the shifting of the three axonemes towards the anterior sperm region parallel to the elongating nucleus, and it is transformed into a dense cylinder. In the mature spermatids the three axonemes amalgamate to create a bundle of 27 doublet microtubules. Near the end of spermiogenesis the dense cylinder of the centriole adjunct lies parallel to the nucleus and the axonemes. It ends where the mitochondrion appears at half-sperm length. We confirm that Terebrantia testes have a single sperm cyst; their sperm are characterized by a cylindrical nucleus, three axonemes fused into one, a small mitochondrion and a short cylindrical centriole adjunct which corresponds to the dense body described in a previous work. The acrosome is lacking. At the midpoint of the anterior half of the sperm the outline of the cross-section is bilobed, with the nucleus contained in a pocket evagination of the plasma membrane. These characters are discussed in light of a comparison between Tubulifera and Terebrantia.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

The innervation of the salivary gland of the moth,Manduca sexta

Summary The salivary glands of the moth, Manduca sexta (Insecta: Sphingidae), are unlike most other salivary glands in that they are innervated from one source only. Vital staining of nerves with methylene-blue reveals numerous fine nerves extending to the glands from the oesophageal nerve, a part of the stomatogastric or visceral nervous system. Light and electron microscopy confirm that only the fluid-secreting cells, confined to a discrete region in these glands, are innervated. Axons with or without glial wrappings are found in intercellular spaces between fluid-secreting cells. Axons lacking a glial sheath contain, after glutaraldehyde-osmium tetroxide fixation, large granular and small agranular vesicles. In nerve endings in glands fixed with permanganate these smaller vesicles are granular, having the electron-dense cores characteristic of monoamine-containing neurons. These nerve endings with synaptoid areas are in close (direct) contact with the fluid-secreting cells.I am grateful to Professor T. Weis-Fogh for accommodation in the department of Zoology and to Dr. Nancy Lane for use of A.R.C. facilities and advice. Thanks are also due to Drs. M. J. Berridge, S.H.P. Maddrell, and W. T. Prince and Mr. R. A. Leslie for helpful discussion. Financial assistance from Clare College, Cambridge is gratefully acknowledged.     

The ultrastructure of dentinogenesis and amelogenesis in rat molar tooth germs grown as organ cultures in vitro

Summary Tooth germs from foetal rats of 17 days post-insemination were maintained in vitro for 12 days. Odontoblasts and ameloblasts differentiated and secreted their respective matrices in which mineralization occurred. The ultrastructure of the cells was qualitatively similar to that observed in normal development. Odontoblasts contained more lysosome-like bodies and were found to degenerate in some sites. Mantle dentine was formed but few von Korff fibres were observed. Calcospherites were rarely seen and the mineralizing front of dentine was predominantly linear, associated with numerous small early foci of mineral formation. Enamel showed prism formation associated with the Tomes' process of the ameloblast but some local disturbances in the pattern of enamel formation were observed.Work at the Strangeways Research Laboratory was carried out under the supervision of Dame Honor Fell, F.R.S. with the support of the Medical Research Council.     

Fine structure of the vomeronasal organ in the grass lizard, Takydromus tachydromoides

The squamates are composed of many taxa, among which there is morphological variation in the vomeronasal organ (VNO). To elucidate the evolution of chemoreception in squamate reptiles, morphological data from the VNO from a variety of squamate species is required. In this study, the morphology of the VNO of the grass lizard Takydromus tachydromoides was examined using light and electron microscopy. The VNO consists of a pair of dome-shaped structures, which communicate with the oral cavity. There are no associated glandular structures. Microvilli are present on the apical surfaces of receptor cells in its sensory epithelium, as well as on supporting cells, and there are centrioles and ciliary precursor bodies on the dendrites. In addition to ciliated cells and basal cells in the non-sensory epithelium, there is a novel type of non-ciliated cell in T. tachydromoides. They have constricted apical cytoplasm and microvilli instead of cilia, and are sparsely distributed in the epithelium. Based on these results, the variation in the morphology of the VNO in scincomorpha, a representative squamate taxon, is discussed.     

Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.