The structure of the fruit peel in two varieties of Malus domestica Borkh. (Rosaceae) before and after storage

The structure of fruit peel of two apple varieties ‘Szampion’ and ‘Jonagold’ was investigated using light microscopy as well as scanning and transmission electron microscopy. The samples were taken immediately after harvest and after 6-month controlled atmosphere storage. The Szampion and Jonagold fruit differed in terms of the surface type, number of lenticels, thickness of the cuticular epithelium, height of epidermal cells and thickness of the hypodermis as well as the amount of crystalline wax and the number of microcracks formed on the fruit surface. The 6-month storage resulted in fruit weight loss, increased numbers and depth of microcracks, thickening of the amorphous wax layer and enhanced production of platelet forms of crystalline wax, which filled the microcracks abundantly. Compared with Jonagold, the Szampion fruit exhibited a fewer lenticels, a bigger number of microcracks, smaller amounts of crystalline wax and more substantial weight loss. The apple varieties studied had a reticulate–lamellate cuticle, and at harvest, the epidermal and hypodermal cells contained numerous amyloplasts filled with starch grains, which were not found after the storage period. Additionally, after storage, the cell protoplasts in the apple peel displayed a disorganised structure, and their vacuoles contained fragments of cell membranes, intravacuolar precipitates and deposits, and spherical bodies. The results may facilitate better understanding of changes occurring in fruits of Szampion and Jonagold during storage and help choose the best storage conditions to reduce loss of weight and prevent impairment of fruit quality.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.     

Insect morphology in the age of phylogenomics: innovative techniques and its future role in systematics

A brief account of the history of insect morphology is given. Different techniques and analytical methods used in current projects on insect morphology and phylogeny and their optimized combined application are described. These include fixation, dissection, maceration, histology (microtome sectioning), scanning electron microscopy (SEM), transmission electron microscopy (TEM), serial block‐face scanning electron microscopy (SBFSEM), focused ion beam scanning electron microscopy (FIB/SEM), confocal laser scanning microscopy (CLSM), bleaching, micro‐computed tomography (μCT), computer‐based three‐dimensional reconstruction, focus stacking of digital images, geometric morphometrics and the storage of morphological metadata. The role of insect morphology in the “age of phylogenomics” is discussed.     

Rose leaf structure in relation to different stages of micropropagation

Summary The Mme Isaac Pereire rose was investigated in an attempt to establish how micropropagated roses might best be weaned into normal growth conditions. Leaves of in vitro grown plants, weaned plants and the stock plant were studied, using light microscopy and different scanning and transmission electron microscopical techniques. Features that varied in the different growing conditions were leaf size and thickness, amount of wax, thickness of cuticle and external epidermal cell wall, number and aperture of the stomata, size of the epidermal cells, number of layers of the palisade cells, and size of the chloroplasts in the mesophyll. The rose in the present study had wax on the in vitro cultured plants; this wax was of similar ultrastructural appearance to that of the stock plant, even though in smaller quantities. Weaned plants had an intermediate amount of wax. The cuticle was thin, ranging from 0.04 m on plants growing in vitro to 0.3-0.6 m on weaned plants and stock plants. Stomata were always wide-open on leaves taken from cultures with a relative humidity of 100%. After four weeks in a humidity lowered to 85% stomata had closed.Abbreviations BAP 6-benzyl-aminopurine - CPD critical point drier - CTEM conventional transmission electron microscopy - NAA a-naphthaleneacetic acid - psi pounds per square inch - SEM scanning electron microscopy - TEM transmission electron microscopy     

Novel methodology utilizing confocal laser scanning microscopy for systematic analysis in arthropods (Insecta)

The use of confocal laser scanning microscopy (CLSM) for imagingarthropod structures has the potential to profoundly impactthe systematics of this group. Three-dimensional visualizationof CLSM data provides high-fidelity, detailed images of minusculestructures unobtainable by traditional methods (for example,hand illustration, bright-field light microscopy, scanning electronmicroscopy). A CLSM data set consists of a stack of 2-D images("optical slices") collected from a transparent, fluorescentspecimen of suitable thickness. Small arthropod structures areparticularly well suited for CLSM imaging owing to the autofluorescentnature of their tissues. Here, we document the practical aspectsof a methodology developed for obtaining image stacks via CLSMfrom autofluorescent insect cuticular structures.     

Measuring the thickness of an outer layer of viable bacteria in an oral biofilm by viability mapping

Bacterial biofilms have been reported to contain distinct regions of viable and nonviable bacteria. The purpose of this study was to identify such regions in biofilms of oral bacteria and to determine their dimensions. Oral biofilms were grown aerobically in a constant-depth film fermenter (CDFF) and studied using confocal laser scanning microscopy (CLSM) incorporating viability staining with water immersion lenses. A variety of viability distributions were observed, including biofilm "stacks" possessing an outer layer of viable bacteria surrounding an internal core of nonviable bacteria. Using image analysis tools, we measured the thickness of this outer viable region, in the x-y plane, from single confocal optical sections, and determined the mean angle (theta) of these portions of the biofilm stack (10.93 degrees ). x-y plane thickness data in conjunction with the data on the angle of the stack returned the thickness of the outer viable layer perpendicular to the bulk medium flow as 36.62 microm (31.61-42.21 microm accounting for 95% confidence for variation in both the x-y plane thickness and theta). We have shown that CLSM, in conjunction with vital stains and image analysis techniques, can reveal viability patterns in biofilms and where appropriate can be used to measure the dimensions of these structures.     

The egg-shell of Drosophila melanogaster. VI, Structural analysis of the wax layer in laid eggs

Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 A thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.5-1 microns in diameter) which are partially overlapping and highly compressed between the vitelline membrane and the innermost chorionic layer. The evidence for this layer being a wax are the facts that a) it is not preserved in conventional fat-extracting electron microscopy methods, b) it directs laterally the fracture planes during freeze-fracturing and reveals smooth fracture faces. Analysis of the structural features of wax in mature egg-shell in various species of Drosophilidae have shown that the wax layer exhibits indistinguishable (among the species) hydrophobic plaques, which have the same size and thickness with Drosophila melanogaster. These data provide structural evidence explaining the physiological resistance of the insect eggs studied, against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. In addition, the data demonstrate how an extracellular substance can be organized to perform that function.     

Improved method for visualizing cells revealed dynamic morphological changes of ventral neuroblasts during ventral cleft closure of Caenorhabditis elegans

The formation of intricate and functional biological structures depends on the dynamic changes of cellular morphology. Confocal laser scanning microscopy (CLSM) is a widely used method to reveal the three-dimensional (3-D) structure of cells during the development of Caenorhabditis elegans (C. elegans) and other model organisms. Improving the efficiency and image quality of CLSM would benefit studies using this method. We found that CED-10::GFP::CED-10, a green fluorescent protein (GFP) marker, is intensely expressed beneath the cell surface, facilitating visualization of cellular morphology in C. elegans embryos. By combining the unique properties of this marker, and with the help of direct 3-D rendering of images obtained by CLSM, we developed a simple but powerful method for investigating cellular morphology in developing embryos. Using this method we, for the first time, document the dynamic changes in the morphology of ventral neuroblasts in vivo during ventral cleft closure.     

Study of lichens with different state of hydration by the combination of low temperature scanning electron and confocal laser scanning microscopies.

The use of techniques such as low temperature scanning electron microscopy (LTSEM) and confocal laser scanning microscopy (CLSM) allows the study of lichen thalli in different states of hydration and also near the natural state. The spatial organization of desiccated thalli, with reduced, very compact algal layers, is different from that of hydrated ones. Sometimes, the observation with transmission electron microscopy (TEM) of photobiont pyrenoids from desiccated thalli reports pyrenoids with a central part of a weak stained matrix lacking pyrenoglobuli, named "empty zones". "Empty zones" are not distinguishable with LTSEM and do not present immunolabelling with rubisco antibody in TEM. These zones could be originated by an expansion process during rehydration produced in chemical fixation.     

Ultrastructural characterization of the epithelium that constitutes the cardiac gland epithelial 'honeycomb' in the stomach of the babirusa (Babyrousa babyrussa)

The cardiac gland region in the stomachs of eleven babirusa (Babyrousa babyrussa) from zoological collections was studied by scanning and transmission electron microscopy. An array of tightly-packed, thin-walled tubes, closely resembling a 'honeycomb' covered this region. The tubes were between twelve and sixteen cells (200 and 260 microm) in height and usually two cells (8-20 microm) in thickness. Mucus granules present in the cytoplasm of cells in the lower half of the tube were largely absent from the ad-luminal half of the tube. Instead, open goblet-like structures lined the tube, apparently giving additional strength. Bacteria were present in the lumen of each thin-walled tube and in the underlying gland pit. The evidence suggests that this large area of the babirusa stomach may be structured to form a stable, self-refreshing environment to house and multiply autochthonous commensal bacteria.     

Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta

Scanning electron microscopy of the epicuticle of the tobacco hornworm, Manduca sexta, showed that pupae in diapause possess thicker wax layers than non-diapausing pupae. Extractions of the epicuticle with CHCl₃ established that diapausing pupae secreted over three times as much wax as non-diapausing pupae, and that the total period of wax secretion in diapausing pupae was two to three times longer than that of non-diapausing pupae. Apparently, the extra thickness of the wax layer of diapausing pupae protects the insect from desiccation during diapause. The deposition of additional wax may result from hormonal changes accompanying entry into diapause.     

Ultrastructure of the nonhuman primate vaginal mucosa: epithelial changes during the menstrual cycle and pregnancy

Ultrastructural changes in the vaginal epithelium of the rhesus monkey during the menstrual cycle and pregnancy were studied by scanning and transmission electron microscopy. During the menstrual cycle, the epithelium was keratinized but varied in thickness. Cells of the basal and parabasal layers were polyhedral in shape but as they differentiated they accumulated glycogen and filaments. Cells in the intermediate layers had keratohyaline and membrane-coating granules. Cells in the superficial layers had a thickened cell envelope, abundant keratin filaments, electron-dense intercellular material, and focal tight junctions. The epithelial surface had numerous microridges and numerous adherent bacteria; bacteria were rare on desquamating cells. The epithelium remained keratinized for about the first month of gestation, then underwent "mucification." The cells contained abundant granules and Golgi apparatus. Concomitant with this transformation, bacteria were no longer adherent to the epithelial surface and the surface cells had microvilli instead of microridges. The epithelial changes during pregnancy were roughly associated with the changing pattern of steroid hormone secretion during gestation.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Scanning transmission X-ray,laser scanning,and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Ultrastructure of <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> × <Emphasis Type="Italic">E. urophylla</Emphasis> plants cultivated ex vitro in greenhouse and field conditions

The young and expanded leaf micromorphology and ultrastructure of Eucalyptus grandis 2 E. urophylla juvenile plants, cultivated in greenhouse and field conditions, were analyzed by scanning and transmission electron microscopy. In greenhouse leaves epicuticular wax needles covered the abaxial and adaxial surfaces. On the adaxial surface, the needles form an atypical arrangement in lines, mainly over the anticlinal wall of epidermis cells. After plant transfer to field conditions, the organization of epicuticular wax was altered forming amorphous layers on the adaxial leaf surface, in contrast to the abaxial surface, which maintained the wax needle cover. In both culture conditions the lamellar cuticle formed on the young leaves surface disappeared during leaf enlargement. The ex vitro environment induced the development of hypostomatic leaves. The dorsiventral organization of greenhouse leaves was replaced by an isobilateral arrangement in field conditions with concomitant aerial space reduction. Results suggest that those structural changes may be some of the strategies to avoid excessive plant transpiration during Eucalyptus hybrid plants' acclimatization.     

Analysis by Confocal Microscopy of the Structure of Cambium in the Hardwood Kalopanax pictus

An understanding of the morphology and the developmental changesin the shapes and dimensions of cambial cells requires three-dimensional(3-D) analysis of thick slices of tissue. We devised a simpleprotocol using confocal laser scanning microscopy (CLSM), withsafranin and acridine orange as fluorescent dyes and glycerolas the clearing and mounting medium, to examine the 3-D structureof the dormant cambium in Kalopanax pictus, a ring-porous hardwood.Optical sections and high contrast images provided clear informationabout the shapes and nuclear status of cambial cells, whichhave previously been difficult to determine using conventionalmicroscopy. The axially-oriented cambial cells were found tovary in shape, in particular around the rays, and were not alwaystypically fusiform. We evaluated the reliability of our methodby comparing results with those of a parallel study of the samematerial by standard analysis of serial sections of epoxy-embeddedspecimens. The images of optical sections obtained by CLSM wereof high quality and similar to images obtained by conventionallight microscopy of semi-thin mechanical sections. Use of theconfocal microscope provided a quick and easy method for visualizationof the structure of the cambium in thick hand-cut sections andfor studies of the developmental changes in cells from the cambiumto the xylem. Copyright 2000 Annals of Botany Company Confocal laser scanning microscopy, Kalopanax pictus, three-dimensional reconstruction, vascular cambium     

The structure of integument and wax glands of Phenacoccus fraxinus (Hemiptera: Coccoidea: Pseudococcidae)

Using scanning electron microscopy and optical microscopy,we studied the structure of the integument and wax glands of the mealybug,Phenacoccus fraxinus Tang(Hemiptera:Coccoidea:Pseudococcidae).We observed the ultrastructure of four wax pores including trilocular,quinquelocular,and multilocular pores as well as tubular ducts,recording characteristics of their structure,size and distribution.We found that that the integument of the mealybug consists of three main layers-the procuticle,epidermis and basement membrane-and four sub-layers of the procuticle-the epicuticle,exocuticle,endocuticle and formation zone.The waxsecreting gland cells were closely arranged in epidermis.All of them were complex and composed of one central cell and two or more lateral cells.These complex cells possess a large common reservoir for collection and storage.Synthesized by the glandular cells,the wax is excreted outside integument through canals.     

The fine structure of the microconidium of blastomyces dermatitidis

Aspects of the fine structure of the microconidium of the mycelial phase of the dimorphic fungal pathogenBlastomyces dermatitidis as seen by techniques of scanning and transmission electron microscopy are described and illustrated by electron micrographs. The conidia ofB. dermatitidis undergo changes in the ultrastructural appearance of the cell wall as the spore matures. The cell wall becomes irregular in its thickness and possesses two distinct layers. No discrete or unique surface spines or projections were evident when the conidium ofB. dermatitidis was viewed by scanning electron microscopy. Upon maturity there is a marked deposition of lipid material in large, multiple storage bodies which occupy much of the cytoplasmic area. However, the cytoplasmic organelles appear to retain their structural integrity.