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The ability to visualize cell and tissue morphology at a high magnification using scanning electron microscopy (SEM) has revolutionized plant sciences research. In plant–insect interactions studies, SEM-based imaging has been of immense assistance to understand plant surface morphology including trichomes [plant hairs; physical defense structures against herbivores], spines, waxes, and insect morphological characteristics such as mouth parts, antennae, and legs, that they interact with. While SEM provides finer details of samples, and the imaging process is simpler now with advanced image acquisition and processing, sample preparation methodology has lagged. The need to undergo elaborate sample preparation with cryogenic freezing, multiple alcohol washes, and sputter coating makes SEM imaging expensive, time consuming, and warrants skilled professionals, making it inaccessible to majority of scientists. Here, using a desktop version of SEM (SNE- 4500 Plus Tabletop), we show that the “plug and play” method can efficiently produce SEM images with sufficient details for most morphological studies in plant–insect interactions. We used leaf trichomes of Solanum genus as our primary model, and oviposition by tobacco hornworm (Manduca sexta; Lepidoptera: Sphingidae) and fall armyworm (Spodoptera frugiperda; Lepidoptera: Noctuidae), and leaf surface wax imaging as additional examples to show the effectiveness of this instrument and present a detailed methodology to produce the best results with this instrument. While traditional sample preparation can still produce better resolved images with less distortion, we show that even at a higher magnification, the desktop SEM can deliver quality images. Overall, this study provides detailed methodology with a simpler “no sample preparation” technique for scanning fresh biological samples without the use of any additional chemicals and machinery.

The primary responsibility of the clinical electron microscopy laboratory is to produce, and deliver to the pathologist or clinician, high quality ultrastructural images of pathological specimens in a timely manner. In order to facilitate this process, medical EM labs are converting from traditional chemical processing of photographic film and paper to the acquisition and transmission of electron images by computer. Ideally the electron microscope would incorporate a digital camera in the column to collect the images, thus bypassing chemical processing of film and paper altogether. However, due mainly to the high cost of the in-column cameras, many labs continue to use film, but replace the time consuming enlarging/printing in the darkroom with digital techniques at the computer.

We have determined the magnitude of flexural rigidity of flagellar hooks of peritrichous and single polar flagellated bacteria. Several physical parameters, such as contour length and straight length, were measured from electron micrographs of curved flagellar hooks. We correlated the thermal bending motion of the flagellar hooks with their elastic properties and by employing the measured parameters we determined the elastic parameters of the flagellar hooks, such as bend persistence length and flexural rigidity. The extremely low magnitude of the flexural rigidity of the flagellar hooks clearly indicates that the flagellar hooks can flex easily in any desired direction. Interestingly, the flagellar hooks of single polar flagellated bacteria also possess an extremely low flexural rigidity. These results will help elucidate the role of flagellar hooks in the swimming process of single polar flagellated bacteria.

Analysis of Experimental Error in High Resolution Electron Micrographs

Surface area and volume measurements were made for ram and human spermatozoa. Measurements were made using different techniques in an attempt to confirm estimates arrived at by independent methodologies. Sperm head projected surface areas were calculated from published formulae using linear micrometry measurements or were obtained directly by image analysis. Total surface areas were calculated as twice the projected head area plus the flagella area, estimated from published dimensions. Spermatozoon surface area was also measured from electron micrographs using a stereological method. Micrometric methods gave values of 135 microns2 for ram spermatozoa and 106 microns2 for human spermatozoa. Stereology methods gave values of 142 microns2 for ram spermatozoa and 106.5 microns2 for human spermatozoa, offering good agreement between the two methods. Sperm volumes were estimated by stereology and by radiolabel volume exclusion methods. From stereology, ram sperm volume was 13.3 microns3 and human sperm volume was 22.2 microns3. From the volume exclusion method, ram sperm volume was estimated as 25 microns3. Attempts to measure total volume and water volume simultaneously using and adaptation of this method were unsuccessful. Separate estimation of water volume for ram spermatozoa gave a value or 12.8 microns3, suggesting a 50% water space. Results from this study are compared with existing published values.

The myelin sheath of peripheral nerves was observed by transmission electron microscopy (TEM) using plastic-embedded sections and ultrathin frozen sections. Repeat distances of myelin sheaths were measured in high-powered electron micrographs. The ultrathin frozen sections showed a longer repeat distance than the plastic-embedded sections. The ultrathin frozen sections were thought to contain fewer artefacts, as they had not been subject to dehydration and embedding. It is known that broken myelin sheaths are often observed under conventional TEM. It is thought that these procedures cause contraction and partial destruction of the myelin sheath.

A taxonomic account of the Elaphoglossum species occurring on the Tristan de Cunha, Gough Island and Marion Island groups in the South Atlantic and Southern Ocean are provided. Two new species, Elaphoglossum lasiolepium J. P. Roux from Gough Island and E. gracilifolium J.P. Rotix from Inaccessible Island, are described. A key and descriptions are given where they previously were not available or were incomplete. Notes on habitats, distribution and relationships are provided. The spores are illustrated by means electron micrographs.

Heterozygotes for three long inversions on chromosome 1 were analyzed by serial reconstruction from electron micrographs. Measurements of loop lengths at different meiotic prophase substages revealed that the homologous synapsis of the inverted region was gradually replaced by nonhomologous synapsis as loops were eliminated during pachytene. This synaptic adjustment was apparently not affected by crossovers which occurred within the 150- and 160-cM long loops.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Two-dimensional (2-D) Fourier analysis was used to quantify the structural organization of collagen fibers in the dermis of the human skin. The 2-D Fourier spectra of electron micrographs of collagen fibers contained large amplitude Fourier components (1 fundamental and 3 harmonic peaks), representing the predominant structural parameter of the fibers, the center-to-center spacing of collagen fibrils. The average center-to-center spacing of the collagen fibrils was found from the position of the peaks. For a normal fiber of reticular dermis, this spacing was 116 nm. The fibril diameter appeared to modulate the peak heights.

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.

The publisher regrets that in the above-mentioned article Fig. 3 was printed wrong. The correct version is reprinted here. Figure 3 Electron micrographs of …

For several years, we have been involved in developing a completely automated system for cryoelectron microscopy, which will allow a 3D electron density map of a macromolecular structure to be generated from a specimen inserted into the microscope with no manual intervention required by an operator. Towards this goal, we have developed a system, called Leginon, to automatically acquire images from the electron microscope. In order to integrate the 3D reconstruction process with the data acquisition step we need then to automatically identify and segment objects from the electron micrographs. This presents a challenge, particularly in the case when micrographs are acquired very close to focus, which results in an image with extremely low contrast. We present here the results of a two-stage automated process for detecting and selecting filamentous structures from images of this kind. Images are acquired in defocus pairs; a near-to-focus (NTF) image is acquired first, followed by a far-from-focus (FFF) image. In the first stage of the process, a threelevel perceptual organization algorithm is used to identify and segment filaments in the FFF image. In the second stage the NTF image is aligned to the FFF image using phase correlation techniques and the filaments are selected at the same coordinates as were identified in the FFF image.In humans, perceptual organization is the ability to immediately detect structural relationships such as co-linearity, parallelism, and connectivity among image elements. Researchers in human vision perception have long recognized the importance of the common rules by which our visual system attempts to group information. Some of these rules include proximity, similarity, common fate, continuation and closure.

Several structures of single particles have been resolved to 7-9 Å using electron cryomicroscopy. This field is now poised to make the next step, to near atomic (3-4 Å) resolution. Signal to noise ratio is the critical factor determining the amount of data required for a structural determination at higher resolution. When properly weighted, signal to noise ratio is additive as image data is averaged coherently, however, this relies on perfect alignment of the noisy images. Typical refinement algorithms require as high a signal to noise ratio as possible for accurate alignment. We report a method, which allows accurate, routine determination of signal to noise ratio and other image parameters, both for data quality assessment and microscope quality tracking.Figure la shows a typical 400 kV electron image of ice-embedded herpesvirus capsids. The incoherent sum of the Fourier transforms of particle images (Fig. lb) shows that the data contains information out to a resolution of 7 Å. When rotationally averaged, this power spectrum can be expressed as

Molecular microscopy is one of the most important structural approaches in cell biological investigations and can provide insight into complex biological questions that no other technique can provide. Currently, the technique typically requires the acquisition of very large numbers of transmission electron micrographs from frozen hydrated specimens using low dose techniques. The field is constrained by manual data acquisition methods that are slow, labor-intensive and result in a very low percentage of suitable images. We have developed a system, called Leginon, for automatically acquiring images from a transmission electron microscope. Our first prototype of this system demonstrated that we could acquire 1000 high magnification images per day from negatively stained catalase crystals. We have now extended this system to acquire low dose images of specimens embedded in vitreous ice.Methods: Specimens were prepared on Quantifoil grids using techniques which have been described previously. The Leginon system uses a Philips CM200 TEM and a Gatan MSC CCD camera and is controlled by the emScope software library. The overall acquisition protocol requires (i) obtaining a low magnification image [660x] of a grid square from a Quantifoil grid (fig. 1a); (ii) automatically identifying holes containing ice of suitable thickness; (iii) acquiring an intermediate magnification image [6600x] of the identified hole (fig. 1b); (iv) identifying features of interest within the hole; (v) focusing at high magnification [38,000x] and finally (vi) acquiring a high magnification image (fig 1c,d).

We examine the possibility that loss of photosynthesis is related to the decline in the chloroplast content of leaves during senescence. For this purpose, a procedure which provides quantitative information on the number as well as the size of chloroplasts was introduced. (Computer-assisted analysis of electron micrographs of leaf sections)

The present-day interactive manual selection of biological molecules from digitized micrographs for single particle averaging and reconstruction requires substantial effort and time. Thus a computer algorithm capable of recognition of structural content and selection of particles would be desirable.A few approaches have been proposed in the past. The method by Frank and Wagenknecht is based on the principle of correlation search. Van Heel's method is based on the computation of the local variance over a small area around each point of the image field. The method by Harauz and Fong-Lochovsky is based on edge-detection. The present work was focussed on the detection and classification of particlesby exploiting the standard statistical methods of discriminant analysis.The proposed technique is described in the block diagram (Fig.1). As illustrated in the figure, the program consists of three distinct segments devoted to, respectively, preparation of the data, training session and automatic selection based on a discriminant function set up in the training, hi the data preparation segment, the micrograph is (i) reduced four-fold in size, (ii) low-pass filtered and (iii) run through a peak search algorithm.

The traditional method of preparing high quality projection slides of one's best electron micrographs was to “print” negatives onto Kodak 2″ x 2″ emulsified glass slides using a standard enlarger fitted with a reduction lens and then to tray develop the slide to the desired contrast. As A. Kent Christensen pointed out in the June/July 1994 issue of this publication, the cost of 2″ x 2″ slides has become prohibitive. In any event, I always found adjusting the reduction lens on the enlarger to be a major hassle and searching for the correct exposure and developing times to be a ffustratingly inexact experience with much plate wastage.

w Aedev cilrcifiaSpus group is made up of 2 species rather than 4 subspecies. From biological crones in the labo- ratory, il was shown that A. a. epaclins. A. d. pen'cfiares ami A. a. nieheni crosses have a high genetic aETmity, while A. a. niropnlfiu^ has a low genetic .ininily when crowd with the other 3. In addition to this. A. a. atropalfms is autogenous while the other 3 subspecies are anautogenous. Scanning .Electron Micrographs of (he chorionic sculpturing of the eggs support the concept o*' 2 species. The egg of -i1. n. atropalpus is distinct I'rom that of the 01 her 3 subspecies. The evidence yirongly supports ihe eleva- tion of/I. a. cili-ofialpus and A. a. efmctius ro spccic.s stains. A. a. nielsem and A. a. penchares do not differ biologic;tlly from A. a. epactiw. and should be treated ;>s 1 specie's.

Complete isolation of Fraction 1 protein from alfalfa leaves was achieved by a combination of ammonium sulfate fractionation and gel filtration. Analytical ultracentrifugation gave a S20°,w value of 18.0. Judging from the CD spectrum the protein contains a large amount of β-form as well as tobacco F-l protein. Electron micrographs showed closely similar appearances for the two F-l proteins. The F-l protein (from alfalfa leaves) was separated to large subunits (53,000 daltons) and small subunits (14,000 daltons) on SDS gel electrophoresis. Further the amino acid composition of the large subunit was found similar to those of tobacco and spinach, but considerably different from them in small subunits.

A single dictyosome from a suspension cultured sycamore cell has been reconstructed on an enlarged scale using electron micrographs obtained from a series of 12 thin sections as the basis for the moulding of profile slices. Slices were hardened and stuck together to form single cisternae, tubules and vesicles. These structural elements were joined, true-to-scale, to give a three-dimensional figure. The model thus obtained is more realistic than other well-known dictyosomal models previously presented in the literature. In contrast to these our model exhibits occasional membrane continuities between adjacent cisternae of the dictyosomal stack and entertains the possibility of similar connections between cisternae of the endoplasmic reticulum and the Golgi apparatus.

Electron micrographs of 45 sural nerves from patients with acquired (22) or heredodegenerative neuropathy (23) were analysed with respect to the number of unmyelinated nerve fibres, 37 nerves with respect to the number of Schwann cell sub-units and of structures connected with Schwann cells. Findings were compared with those in 6 nerves from control subjects and referred to the total number rather than to the number per mm2 to eliminate error due to increase in the transverse endoneurial area, present in more than half the diseased nerves. Ninety-one per cent of the diseased nerves showed one or several abnormalities in unmyelinated fibres of their Schwann cells. The best indicator of fibre loss was an increase in the number of Schwann cell sub-units devoid of axons, found in more than half the nerves. This was the only abnormality related with decrease in number of myelinated fibres. The increase in number of empty Schwann cell sub-units was due both to loss of unmyelinated nerve fibres and to proliferation of Schwann cells. Proliferation was indicated by the higher incidence of Schwann cell nuclei in cross-sections of diseased nerves than in controls. The earliest sign of involvement was an increase in number of profiles and of small isolated Schwann cell projections, observed in 33 of 37 diseased nerves, as the only abnormality in 7 nerves. The number of unmyelinated nerve fibres by itself was of little value to indicate loss of fibres, since regeneration often replaced or more than replaced degenerated fibres. Regeneration was indicated by an increase in number or incidence of small unmyelinated fibres, present in nearly half of 45 diseased nerves; and by an increased in the total number, present in a third of the nerves. An increase in the number of collagen pockets and of fibres undergoing degeneration (loss of organelles) and a decrease in the number of unmyelinated fibres per Schwann cell sub-units was present in only a quarter to a third of diseased nerves and was not related to other criteria of loss of fibres or of regneration.

The materials released from myofibrils by Ca2+ -activated factor (CAF) were fractionated by using a combination of Sepharose 6B and Bio-gel A 50m columns. To investigate the exact constituents of Z-disks on the basis of success or failure of reconstitution of Z-disks, reconstitution of Z-disks by incubating individual fractions with Z-disk-extracted fiber bundles was carried out, and the proteins in each fraction were analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis.From the electron micrographs of the reconstituted Z-disks, SDS-polyacrylamide gel electrophoretic analysis and amino acid analysis of the isolated proteins from the gels, it was clear that the proteins required for the reconstitution of the Z-disk, in other words, the principal constituents of the Z-disk, were Z-nin, a new high molecular weight protein having a subunit molecular weight of 300,000 ~ 400,000 dalton mentioned in this report, α-actinin and tropomyosin.

Si deposits in the endodermis of the seminal roots of sorghum following culture in nutrient solution containing 100 ppm SiO2 for 7 days were investigated by transmission electron microscopy. Si microassay was carried out by means of the EMMA-4 system. Endodermal ultrastructure is discussed. Electron micrographs of the Si deposits provided information on formative processes and indicated a considerable involvement with the cellulosic structure of the inner tangential wall. The initial deposits were believed to be composed structurally of particles designated as primary spherical units. The EMMA-4 conclusively indicated that Si was localized and confined to the inner tangential wall; implications of this result for previous studies are discussed. Tentative mechanisms for silicic acid transport and subsequent Si deposition on the inner tangential wall were proposed

Vesicles were prepared on hydration of a mixture of a single or double alkyl-chain, non-ionic surfactant with cholesterol. These vesicles, or ‘niosomes’, are capable of entrapping and retaining water soluble solutes such as carboxyfluorescein, are osmotically active and can be formulated to release entrapped solute slowly. The physical characteristics of the vesicles were found to be dependent on the method of production and three such methods, based on liposome technology, are described. The vesicles have been characterized by photon correlation spectroscopy, freeze fracture electron micrography, measurement of solute entrapment efficiency, and solute release rates. Vesicular forms of the single chain surfactant which could be formed under certain conditions in the absence of cholesterol are also described.

It is argued that a knowledge of the extent of individual cell membrane systems is of crucial importance to the understanding of results obtained from lipid analysis of tissues and isolated membrane systems. In this preliminary study stereological methods have been applied to electron micrographs of cucumber leaves at different stages of expansion, to assess the area of each membrane system present. The equivalent phospholipid-protein membrane area for the largest leaf was found to be 500 x 109 pm2 compared with a value of 961 x 109pm2 calculated from published data on the phospholipid content of a similar leaf. Possible applications of quantitative electron microscopy to certain lipid studies are reviewed and it is concluded that this approach should be more widely adopted.

Electron micrographs of copper-deficient oat leaves showed only minor structural differences from normal oat leaves in that there were reduced numbers of compartments in the chloroplast stacks with no differences in stromacentres, plastoglobuli, mitochondria, nuclei, or cell walls. The cells of the white withered tip characteristic of copper-deficient oat leaves were found to be full of fibrous masses.

Electron micrographs of transverse sections of the distal portion of the hypopharynx of female yellow-fever mosquitoes, Aedes aegypti (L.), showed a structural articulation between this stylet and a lacinia within 1 μ, of their distal tips, and an interdigitation of 3 edges that produced a functional closure of the dorsal wall of the salivary duct. Two regions in the cuticle can be distinguished on the basis of their electron density and surface topography.

Three strains of Mycoplasma mycoides var. mycoides were studied. Dark-ground microscopy of broth cultures showed some filamentous forms, but single cells predominated. The filaments disintegrated on agitation. Single cells passed through 0.3-[tm pores but not 0.22-[tm pores of Millipore membranes. They were destroyed by shaking, sonication, storage at 2-4 C, and freezing and thawing. Ultravioletinactivation curves showed that single-cell suspensions contained both mononucleate and binucleate cells that could not be cloned into pure cultures of either type. They are considered part of a life cycle in which filaments are a characteristic but not a fundamental part. Electron micrographs showed that replicating cells varied in size from 0.15 [tm to 4.5 jtm in diameter. All multiplied by budding. Most synchronized cells produced one bud, but some produced two simultaneously, which confirmed that these cells were multinucleate and have an alternative mode of division in addition to binary fission.

Electron micrographs of transverse and saggital sections of the opisthosoma of the two-spotted spider mite, Tetranychus telarius (L.), showed a layered cuticle and a hypodermal layer with a basement membrane. The entire cuticle measured 0.70 μ–0.91 μ thick, the epicuticle being 0.01 μ, exocuticle 0.09 μ–0.11 μ, and endocuticle 0.16 μ–0.78 μ. Cuticular extensions, lobed at the apices, were 1.37 μ–1.74 μ high and about 0.09 μ thick at the narrowest point. The hypodermal layer was about 1.19 μ–1.71 μ and delineated by the basement membrane.

Numerous white lesions in the fat body of the alfalfa leafcutter bee, M. rotundata (F.), are composed of proteins, DNA and RNA. Electron micrographs indicate that the lesions are groups of inclusion bodies, 4—10 µ in diameter. Larvae containing high concentrations of lesions have a higher mortality rate. The relationship with mortality structure, and chemical components suggests that that the lesions may be viral in origin.

The types, numbers, and distribution of antennal sense organs of female Culex tarsalis Coquillett are reported. There are about 1025 sense organs on the flagellum which are classified as 2 types of sensilla chaetica, sensilla basiconica, 3 types of sensilla trichodea, and sensilla campaniformia. Except for the first few basal segments, the pointed-tipped sensilla trichodea are fairly evenly distributed on the flagellum. The number of type I blunttipped sensilla trichodea decrease distally while the sensilla basiconica increases. Type II blunt-tipped sensilla trichodea occur 1 per segment. Electron micrographs show a light tip on the sensilla basiconica indicating a decrease in thickness of the cuticle. A central canal in the basiconica is visible with the light microscope. Using the crystal violet staining technique for detecting permeable sense organs, it was found that the sensilla basiconica took up the stain as well as the type I blunt-tipped sensilla trichodea.

A few years ago Anderson & Richards (1942) reported that the brilliant iridescent blue scales of the tropical butterfly Morpho cypris contain no chitin. The report, incidental to a study of scale architecture, was based on the usual test of solubility in hot concentrated alkali but also included the fact that electron micrographs of less drastically treated scales showed intermediate stages of dissolution. Being desirous of knowing whether or not this is generally true of insect scales, the present author performed tests on a series of specimens representing 109 species of 103 genera and 45 families of Lepidoptera in addition to scales of a mosquito and silverfish.[3][1] Results were highly variable from one species to another and not always easy to duplicate. We were forced to conclude that lepidopterous scales may contain either large amounts or small amounts or no demonstrable chitin. [1]: #fn-3

Protoplasts have been prepared from the mycelium of Aspergillus funigatus, using a lytic enzyme mixture from Trichoderma harzianum. A variety of experimental conditions were investigated in order to achieve optimal conditions for protoplast production. Electron micrographs showed the preparations to be free of cell-wall. Material obtained by this procedure can be used as a source of cytoplasmic antigens for further analysis.