Low Magnification Micrographs Research Articles

Antimicrobial susceptibility testing for Gram positive cocci towards vancomycin using scanning electron microscopy.

The rapid detection of resistant bacteria has become a challenge for microbiologists worldwide. Numerous pathogens that cause nosocomial infections are still being treated empirically and have developed resistance mechanisms against key antibiotics. Thus, one of the challenges for researchers has been to develop rapid antimicrobial susceptibility testing (AST) to detect resistant isolates, ensuring better antibiotic stewardship. In this study, we established a proof-of-concept for a new strategy of phenotypic AST on Gram-positive cocci towards vancomycin using scanning electron microscopy (SEM). Our study evaluated the profiling of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus after brief incubation with vancomycin. Sixteen isolates were analysed aiming to detect ultrastructural modifications at set timepoints, comparing bacteria with and without vancomycin. After optimising slides preparation and micrographs acquisition, two analytical strategies were used. The high magnification micrographs served to analyse the division of cocci based on the ratio of septa, along with the bacterial size. Susceptible strains with vancomycin showed a reduced septa percentage and the average surface area was consequently double that of the controls. The resistant bacteria revealed multiple septa occurring at advanced timepoints. Low magnification micrographs made it possible to quantify the pixels at different timepoints, confirming the profiling of cocci towards vancomycin. This new phenotypic AST strategy proved to be a promising tool to discriminate between resistant and susceptible cocci within an hour of contact with vancomycin. The analysis strategies applied here would potentially allow the creation of artificial intelligence algorithms for septa detection and bacterial quantification, subsequently creating a rapid automated SEM-AST assay.

Optical density analysis in autoradiographic images from BNCT protocols

In Boron Neutron Capture Therapy (BNCT) research, information on spatial distribution and concentration of boron in tissues is essential for the treatment planning and for dose determination. Neutron autoradiography technique offers the possibility to gain insight into boron biodistribution in tissues. The irradiation of tissue sections deposited on nuclear track detectors produces damages in the latter, which can be visualized by microscope imaging and that are spatially correlated with the boron localization in the biological section. We had previously developed a methodology for the quantification of boron concentration in tissue samples by nuclear track density determination (QTA).In this work we analyzed the optical density (OD) in autoradiographic images where track density is too high to be quantified by track counting. OD was determined from grey level measurements on low magnification micrographs and proved to be a suitable parameter to quantify boron concentration. The images were originated by placing polycarbonate detectors in contact with samples containing 10B atoms, irradiated with thermal neutrons and finally chemically attacked. Reference standards were developed from aqueous solutions prepared with known boron concentrations in order to construct calibration curves. The obtained optical density values were compared with curves proposed by other authors, showing similar tendencies. The calibration curve was used to make preliminary boron concentration determinations in histological tissue sections of animals infused with boron compounds, which agreed with measurements realized by the QTA approach.The methodology proposed in this work would allow a fast preliminary analysis of histological and autoradiographic images in samples of interest for BNCT.

Application of digital image correlation at the microscale in fiber-reinforced composites

Digital image correlation (DIC) is applied to analyzing the deformation mechanisms under transverse compression in a fiber-reinforced composite. To this end, compression tests in a direction perpendicular to the fibers were carried out inside a scanning electron microscope and secondary electron images obtained at different magnifications during the test. Optimum DIC parameters to resolve the displacement and strain field were computed from numerical simulations of a model composite and they were applied to micrographs obtained at different magnifications (250×, 2000×, and 6000×). It is shown that DIC of low-magnification micrographs was able to capture the long range fluctuations in strain due to the presence of matrix-rich and fiber-rich zones, responsible for the onset of damage. At higher magnification, the strain fields obtained with DIC qualitatively reproduce the non-homogeneous deformation pattern due to the presence of stiff fibers dispersed in a compliant matrix and provide accurate results of the average composite strain. However, comparison with finite element simulations revealed that DIC was not able to accurately capture the average strain in each phase.

In-situ examination and analysis of the gold jewellery from the Phoenician tomb of Kition (Cyprus)

The Phoenician tomb discovered in Kition in 1998 yielded some of the most impressive gold jewellery items recovered by archaeological excavations in Cyprus. Seal finger-rings, lunate earrings, necklace pendants, bracelets and a richly decorated elbow fibula exemplify the goldsmith’s skill in the 8th-7th century BC. The aim of this work is to describe and illustrate the goldsmith’s techniques with low magnification micrographs and to determine the variety in composition of the gold alloys used in the manufacture of 25 items. The study, integrated in the European project AUTHENTICO, was carried out at the National Museum of Archaeology of Nicosia with a porTable video camera equipped with a zoom and a handheld XRF spectrometer.

Regional Optic Nerve Damage in Experimental Mouse Glaucoma

To assess the relationship between regional variation of axon loss and optic nerve head anatomy in laser-induced experimental glaucoma in the mouse. Experimental glaucoma was induced unilaterally in eight NIH Swiss black mice. Intraocular pressure (IOP) was measured for 12 weeks, and the mice were killed. The eyes were enucleated, and both optic nerves were dissected and processed conventionally for electron microscopy. Low- and high-magnification images of the optic nerve cross sections 300 microm posterior to the globe were collected systematically and masked before analysis. For each nerve, cross-sectional area was measured in low-magnification micrographs. Axon number and density were determined in the high-magnification micrographs. Loss of axonal density was compared between the superior and inferior and nasal and temporal areas of the optic nerve cross section. Additional cross-section micrographs were collected at 10- or 20-microm intervals throughout the optic nerve head. In the treated (glaucoma) eyes, mean IOP was 44% higher than that in the control eyes. The optic nerve cross-sectional area, mean axonal density, and total axonal number were significantly less than those in the control eyes (P < 0.01 for each). Axon loss in the superior optic nerve was greater than in the inferior optic nerve in each glaucomatous eye (P = 0.012). The ratio of axonal density in the superior and inferior optic nerve (superior-to-inferior [S/I] ratio) in all treated eyes was <1.0 and significantly lower than that in the control eyes (P = 0.012). The central retinal vessels occupied approximately 20% of the central optic nerve head cross-sectional area, gradually shifted position ventrally as they progressed toward the scleral foramen (the mouse does not have a lamina cribrosa), and exited the inferior retrobulbar optic nerve adjacent to the posterior of the globe. Ocular hypertension in the mouse eye sufficient to cause optic nerve damage induces preferential loss of superior optic nerve axons. Optic nerve axon loss appeared less among the axons that were near the major optic nerve blood vessels at the scleral foramen. Topographic differences in optic nerve axon loss should be considered when evaluating optic nerve damage in experimental laser-induced glaucoma in the mouse.

Heterogeneity in the ultrastructure of the mucous (goblet) cells of the rabbit palpebral conjunctiva

Aim: The purpose of this study was to assess objectively the ultrastructure of the secretory granules in rabbit conjunctival mucin‐producing ‘goblet’ cells. Method: The upper eyelids from five young adult dioestrous female rabbits were dissected out, stretched onto a cardboard support and prepared for transmission electron microscopy by repeated application of an isotonic two per cent glutaraldehyde fixative at room temperature. Post‐fixation treatment included osmium tetroxide and staining with uranyl acetate and lead citrate. Low magnification micrographs were taken of the goblet cells of the conjunctiva, printed at a magnification of approximately 6,000 and the number, size and features of the secretory granules assessed. Results: Across the entire palpebral conjunctiva of ail five rabbits, the majority of mucous cells displayed a goblet shape and the secretory granules were uniformly pale in staining. The average width of the goblet cells was 10.8 ± 1.1 μm and the diameter of the secretory granules was 0.82 ± 0.16 μm. However, in localised regions across the palpebral conjunctiva of two of the rabbits, some goblet cells were different in that the secretory granules had either a denser‐staining core, in which some of the granules were densely staining (while others were pale) or most of the granules were densely staining. These mucous cells had an average diameter of 10.3 ± 1.7 μm and the granule diameters averaged 0.88 ± 0.01 μm. For these abnormal goblet cells, inflammatory cells were found in their immediate vicinity. Occasionally, goblet cells were seen to be in the process of degranulation with associated apparent cell necrosis and the mucin granule diameter was close to 1 μm. Conclusions: The ultrastructure of the mucin‐containing secretory granules of the conjunctival mucous cells is not necessarily homogeneous in character and further attention needs to be given to the effects of localised inflammation in the tissue and to possible hormonal influences.

Optic nerve damage in experimental mouse ocular hypertension.

To evaluate optic nerve damage in mice after laser-induced ocular hypertension. Ocular hypertension was induced unilaterally in 13 NIH Black Swiss mice by laser photocoagulation of the limbus. Over the following 12 weeks, intraocular pressure (IOP) was measured at regular intervals by the microneedle method. The optic nerves of these mice and of seven normal untreated mice were then processed conventionally for electron microscopy, and cross sections of the nerve 300 micro m posterior to the globe were collected. Low- and high-magnification images were collected systematically and masked before analysis. For each nerve, cross-sectional area was measured in low-magnification micrographs, and axon and glia numbers were counted in high-magnification micrographs. In normal untreated mice, the average number of axons was 59,597 +/- 3,112 (mean +/- SD). Variation among these measurements was 5.7% +/- 3.9%. After laser treatment, the duration of high IOP ranged from 2 to 12 weeks (6.2 +/- 3.6 weeks, mean +/- SD). The mean IOP in the treated eyes was 1.3 times greater than the mean IOP in the control eyes (P = 0.0012). The maximum IOP in the treated eyes was 1.6 times greater than that observed in the control eyes (P < 0.0001). The optic nerve cross-sectional area, mean axon density, and total number of axons in the treated eyes were significantly less than in the control eyes (28.5% +/- 23.4%, 57.8% +/- 37.8%, and 63.1% +/- 38.1%, respectively; P < 0.005 for each). The decrease in optic nerve cross-sectional area and the positive integral of elevated IOP and duration of IOP elevation correlated significantly with total axon loss (r(2) = 0.79, P < 0.0001 and r(2) = 0.36, P = 0.040, respectively). The number of astrocytes per cross section of optic nerve was significantly greater in the treated eyes than in the control eyes (P = 0.014). Laser-induced ocular hypertension in mouse eyes can induce optic nerve axon loss that correlates with the magnitude and duration of elevated IOP.

Electron microscopy and laser scanning microscopy of high-pressure frozen freeze-substituted samples of Sordaria humana

The process of spore formation (ascosporogenesis) has not been clearly defined in ascomycete fungi. Many conflicting reports exist concerning details of ascospore initial development. The relatively slow fixation (up to several minutes) of conventional chemical techniques does not adequately preserve many structures for which ultrarapid freezing techniques (within milliseconds) are particularly suited. High-pressure frozen freeze-substituted samples of the ascomycete Sordaria humana were thick-sectioned for laser scanning microscopy (LSM) and thin-sectioned for transmission electron microscopy (TEM). Perithecia were too large (up to 500μm in diameter) to freeze adequately using plunge or propane-jet freezing techniques. The LSM was used to examine portions of the perithecia which were too large for viewing by TEM without taking numerous low magnification micrographs.

Complementary scanning electron microscopy: technical notes and applications.

This report introduces practical techniques and applications of complementary scanning electron microscopy (SEM). To identify the complementary structures at high magnification, we first made a montage pair of low magnification micrographs as a guide map. Consulting the map, we took complementary micrographs at high magnification. When taking a picture at high magnification, we drew the outline of the most prominent structures on a transparent plastic plate attached to the cathode-ray tube of a SEM. Then another picture of the complementary structures was taken after adjusting the complementary structures to the reverse image of the plastic plate. We performed three applications of the complementary SEM: 1) complementary observation of the epithelial underside and lamina propria of the rat urinary bladder; 2) complementary observation of the fractured Golgi apparatus; and 3) evaluation of specimen drying methods. After proper digestion of the rat urinary bladder using strong alkali, the epithelium was detached from the underlying lamina propria. On the basal side of the epithelium, the meshwork of grooves were visible. The observation of the corresponding lamina propria confirmed that the grooves were occupied by blood capillaries located in the uppermost part of the lamina propria. The complementary observation of the Golgi apparatus was useful for understanding its three-dimensional architecture. The observation on both complementary fractured surfaces of the Golgi apparatus from the mouse lacrimal gland demonstrated the continuity of the Golgi stacks. It was also effective for observing both the cis and trans side of the same Golgi stack. Complementary SEM was also useful for evaluating the specimen drying method.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of prolactin in the pars distalis of the salamander Necturus maculosus

The prolactin cells in the anterior pituitary of Necutrus maculosus have been localized with anti-ovine prolactin using the peroxidase-labeled antibody technique. Two histological stains, carmoisine-L, orange G and hematolylin and eosin, were used to corroborate the immunocytochemical results. In low magnification micrographs, the peroxidase positive area corresponds to comparable acidophilic and carmoisine-L staining areas of other sections from the same part of the gland. Carmoisine-L stains the heavily granulated prolactin cells bright orange. These cells correspond in morphology and arrangement to those containing the dark brown precipitate which indicates the site where antigen bound peroxidase has reacted with its substrate. When tissue was stained with nonimmune serum or with immune serum adsorbed with prolactin instead of the first antibody, the results were negative.

Scanning Electron Microscopy of Trematodes Embedded for Transmission Electron Microscopy

Chipping plastic from the surface of trematodes embedded for transmission electron microscopy permits high resolution scanning electron microscopy of surface features. The surfaces of the trematodes Megalodiscus temperatus, Diclidophora merlangi, and Schistosoma mansoni are described and comparisons are made with previous studies using the transmission electron microscope. The structure of the integument of Schistosoma mansoni as visualized with the scanning electron microscope (SEM) has been reported in at least six publications to date (see Miller et al., 1972, for review). Occasional isolated scanning micrographs of other trematode species have also been published but in no instance has the information obtained been limited by instrument performance. Instead, results have been limited by methods of specimen preparation, usually involving fixation, dehydration, and air drying with a subsequent distortion of surface morphology. Critical-point drying techniques should improve specimen morphology although this technique has seen little use in studies of platyhelminth morphology. The purpose of this paper is to describe the results of examination with the SEM of fractured, plastic-embedded trematodes by the use of a technique recently described by Humphreys et al. (1973) as fractography. This technique makes it possible to utilize fully the resolution as well as depth of focus of the SEM in the examination of specimens prepared by routine methods for transmission electron microscopy. MATERIALS AND METHODS Specimens of Megalodiscus temperatus, Diclidophora merlangi, and Schistosoma mansoni which had been previously fixed, dehydrated, and embedded for transmission electron microscopy were used in this study. Any method of fixation which is satisfactory for transmission microscopy can be used. The embedding media used include Epon, Epon-Araldite mixture, and Maraglas. The blocks were mounted in vise-type holders and the plastic embedding medium removed with sharp razor blades. Excess plastic was first trimmed off and then the razor blade was sliced into the plastic close to, and parallel with, the specimen Received for publication 26 April 1973. surface. An outward flick of the razor blade caused substantial portions of plastic to separate from the worm surface. Entire worms could, in some instances, be separated from the surrounding embedding medium. The fractured specimens were oriented on specimen stubs and attached to the surface with a small drop of synthetic mounting medium. The specimens were then coated in a Denton DV-502 vacuum evaporator with a thin layer of gold-palladium and examined with an AMR-900 SEM. RESULTS AND DISCUSSION Low-magnification micrographs of the surface of Megalodiscus temperatus show the undulating surface to be covered with numerous, small protrusions (Fig. 1). Figure 2 shows the same surface at high magnification and demonstrates the surface detail which is visible in tissue prepared by this method. Although the spherical surface protrusions are seen in some transmission electron micrographs (see Bogitsh, 1968), their true contribution to the surface morphology of this worm is apparent only in high resolution scanning micrographs. Low-magnification scanning micrographs of the anterior dorsal surface of Diclidophora merlangi reveal not only the folded surface previously described by Morris and Halton (1971) but also numerous parallel rows of domelike surface protrusions, each of which has a diameter of about 6 to 8 /i (Figs. 3, 4). The microvilluslike structures described by Morris and Halton (1971) are numerous over the lateral surfaces of the protrusions and the intervening regions, but are absent from the apices (Fig. 5). In many instances the tips of the microvilli are broken off, leaving only small stubs behind. Figure 6 shows a low-magnification scanning micrograph of a partially fractured block containing male and female specimens of

Contrast in the Photoelectric Effect of Organic and Biochemical Surfaces: A First Step towards Selective Labeling in Photoelectron Microscopy

The photoelectric effect can provide the physical basis for a new method of mapping organic and biological surfaces. The technique, photoelectron microscopy, is similar to fluorescence microscopy using incident ultraviolet light except that photoejected electrons form the image of the specimen surface. In this work the minimum wavelengths of incident light required to produce an image were determined for the molecules 3,6-bis(dimethylamino)acridine (acridine orange) (I), benzo[a]pyrene (II), N,N,N',N'-tetraphenylbenzidine (III), and copper phthalocyanine (IV). The photoelectron image thresholds for these compounds are 220 (I), 215 (II), 220 (III), and 240 nm (IV), all +/-5 nm. Contrast of I-IV with respect to typical protein, lipid, nucleic acid, and polysaccharide surfaces was examined over the wavelength range 240-180 nm. The low magnification micrographs exhibited bright areas corresponding to I-IV but dark regions for the biochemical surfaces. The high contrast suggests the feasibility of performing extrinsic photoelectron microscopy experiments through selective labeling of sites on biological surfaces.