Low-temperature Scanning Electron Microscopy Research Articles

Morphological characteristics of sporangiospores of the tempe fungus Rhizopus oligosporus differentiate it from other taxa of the R. microsporus group

The fungus Rhizopus oligosporus ( R. microsporus var. oligosporus) is traditionally used to make tempe, a fermented food based on soybeans. Interest in the fungus has steadily increased, as it can also ferment other substrates, produce enzymes, and treat waste material. R. oligosporus belongs to the R. microsporus group consisting of morphologically similar taxa, which are associated with food fermentation, pathogenesis, or unwanted metabolite production (rhizonins and rhizoxins). The ornamentation pattern, shape, and size of sporangiospores of 26 R. microsporus group strains and two R. oryzae strains were studied using low-temperature SEM (LT-SEM) and LM. This study has shown that: (1) LT-SEM generates images from well-conserved sporangiophores, sporangia, and spores. (2) Robust spore ornamentation patterns can be linked to all different taxa of the R. microsporus group, some previously incorrectly characterized as smooth. Ornamentation included valleys and ridges running in parallel, granular plateaus, or smooth polar areas. Distribution of ornamentation patterns was related to spore shape, which either was regular, ranging from globose to ellipsoidal, or irregular. Specific differences in spore shape, size, and ornamentation were observed between Rhizopus taxa, and sometimes between strains. (3) R. oligosporus has a defect in the spore formation process, which may be related to the domesticated nature of this taxon. It had a high proportion, 10–31 %, of large and irregular spores, and was significantly differentiated from other, natural Rhizopus taxa as evaluated with partial least squares discriminant analysis. It is remarkable that the vehicle of distribution, the sporangiospore, is affected in the strains that are distributed by human activity. This provides information about the specificity and speed of changes that occur in fungal strains because of their use in (food) industry.

Changes in cohesive sediment properties associated with the growth of a diatom biofilm

Microalgal biofilms are associated with considerable variability in the properties of natural sediments, yet little effort has been made to isolate micro-scale spatial and temporal changes in sediment properties caused by the growth of a biofilm. Understanding the changes associated with biofilm growth and quantifying the time scales over which these changes occur is important for developing suitable experimental designs and for understanding how biofilms mediate sediment properties and processes. The development of a microphytobenthic biofilm and associated changes in the sediment was investigated over 45 days in the laboratory. The biogeochemical properties of the sediment: bulk density, water content, chlorophyll a concentration and colloidal carbohydrate concentration were measured on a sub-millimetre scale in the top 2 mm. The erosion threshold was measured with a Cohesive Strength Meter (CSM). Biofilm development was rapid, with changes in the properties occurring after 1 day and a visible film forming after just 3 days. The largest changes in sediment properties tended to occur in the surface 200 μm through time, with some variables also showing a differing response with depth. There were significant changes in water content, chlorophyll a concentration, colloidal carbohydrate concentration and erosion threshold in the surface 2 mm, with a general trend to increase with time. Bulk density was highly variable and did not show a consistent pattern of change with time. Erosion threshold was positively correlated with water content, chlorophyll a and colloidal carbohydrate in the surface 200 μm and these were also positively correlated with each other. Low Temperature Scanning Electron Microscopy (LTSEM) images revealed changes in the surface sediment structure and the formation of a thick multi-layer biofilm. The rapidity of biofilm growth and development and the associated changes to the sediment should be considered when designing experiments that investigate biofilms and properties of sediments and/or that involve biocide treatments or disturbance to the sediment.

Microstructural evolution in sintered ice particles containing NaCl observed by low-temperature scanning electron microscope

Ice particles containing NaCl were made by spraying 0.043 M salt solution into liquid nitrogen. The ice particles were packed into capsules and annealed at -8 °C for 168 h and -25 °C for 20 h. This material can be considered as a model material for sintered snow containing impurities. The capsules were fractured open inside the low-temperature scanning electron microscope, which minimized the artefacts caused by cryofixation. The morphology of the sintered structure was observed with low-temperature scanning electron microscope. The microstructure of the sintered material consists of ice grains with a liquid meniscus containing NaCl between the grains. This structure is similar to the equilibrium morphology of water-filled veins in polycrystalline ice and liquid phase sintered metallic materials. The combined effect of the surface energies between the solid, liquid, and vapour governs the morphology of the microstructure. A dihedral angle where the brine intersects a grain boundary in ice of 8.0 ± 2.6°, and a contact angle for brine on ice at the interface with vapour of 5.0 ± 1.3° were measured, for samples quenched from -8 °C. Using the dihedral angle measurement, a surface energy value for ice-brine of 32.6 ± 0.1 mJ/m2 was calculated.

Imaging of thermal domains in ultrathin NbN films for hot electron bolometers

The authors present low-temperature scanning electron microscopy (LTSEM) investigations of superconducting microbridges made from ultrathin NbN films as used for hot electron bolometers. LTSEM probes the thermal structure within the microbridges under various dc bias conditions, either via electron-beam-induced generation of an unstable hot spot or via the beam-induced growth of a stable hot spot. Such measurements reveal inhomogeneities on a micron scale, which may be due to spatial variations in the NbN film or film-interface properties. Comparison with model calculations for the stable hot spot regime confirms the basic features of common hot spot models.

Protein and water structural changes in fish surimi during gelation as revealed by isotopic H/D exchange and Raman spectroscopy

Structural changes of proteins and water during gelation of fish surimi, have been studied by isotopic H/D exchange of water and Raman spectroscopy assisted by monitoring of rheological characteristics, in order to get insights into the structural and functional properties of surimi gels. The results indicate the following: (i) Protein hydrogen bond rearrangements occur involving mainly α-helix to β-sheet transition, (ii) the relative intensity of the symmetric H 2O stretching band near 3220 cm −1 tends to decrease upon gelation, (iii) H/D exchange reveal a slower deuteration kinetics in the gels as compared to the surimi, (iv) the low temperature scanning electron microscopy shows a smaller pore size of the gel network as compared to the surimi, and suggests that water domains in gel are more inaccessible to D 2O, which is consistent with higher water holding capacity in the gel.

Lichens Survive in Space: Results from the 2005 LICHENS Experiment

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase–HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase–HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase–HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols–filipin signals were almost completely absent.

Desiccation-induced ultrastructural and biochemical changes in the leaves of the resurrection plant Myrothamnus flabellifolia

Light microscopy and low-temperature scanning electron microscopy were used to systematically compare the surface and internal ultrastructures of hydrated and desiccated leaves of the resurrection plant Myrothamnus flabellifolia (Welw.). This revealed that leaf tissue underwent considerable shrinkage and collapse on desiccation but was supported by a framework of vascular and sclerenchymous tissue, which is responsible for the fan-like shape of the leaves. In addition, the leaf ribs were covered with wax and an internal wax cuticle was observed. Biochemical analysis showed that the cyanidin 3-glucoside content increased on desiccation as did the trehalose and sucrose contents. Salt deposits were observed at the apices of desiccated leaves in the proximity of hydathode-like structures. We propose that this might regulate the leaf salt content since decreased intracellular cation concentration was observed in desiccated leaves. We believe that these unique adaptations contribute to the remarkable desiccation-tolerance properties of this plant.

Effect of calcium propionate on the microstructure and pectin methy‐ lesterase activity in the parenchyma of fresh‐cut Fuji apples

AbstractThere is an increasing demand for fresh‐cut fruits. One of the main problems with these products is their short shelf life. The use of additives such as calcium propionate may increase their shelf life, because calcium preserves the structural integrity of the cells and propionate acts as food preservative. This work analyses the microstructure of Fuji apple parenchyma treated with calcium propionate immediately after treatment and after storage for 1 and 2 weeks at 4 °C by using light microscopy (LM) and low‐temperature scanning electron microscopy (cryo‐SEM). This work also studies the effects of treatment and storage time on the enzymatic activity of pectin methylesterase (PME) by measuring the amount of acid released per unit of time at pH 7 and at 63 °C. The results show that calcium propionate treatment has consolidating and structuring effects on the parenchyma and partially minimises the degrading effects of fresh‐cut apples. In addition, treatment with calcium propionate decreases the activity of the PME both in non‐stored apples and in those stored for 1 and 2 weeks probably due to the inhibiting effect of propionate. Copyright © 2006 Society of Chemical Industry

Use of low-temperature scanning electron microscopy to compare and characterize three classes of snow cover

This study, which uses low-temperature scanning electron microscopy (LTSEM), systematically sampled and characterized snow crystals that were collected from three unique classes of snow cover: prairie, taiga, and alpine. These classes, which were defined in previous field studies, result from exposure to unique climatic variables relating to wind, precipitation, and air temperature. Snow samples were taken at 10 cm depth intervals from the walls of freshly excavated snow pits. The depth of the snow pits for the prairie, taiga, and alpine covers were 28, 81, and 110 cm, respectively. Visual examination revealed that the prairie snow cover consisted of two distinct layers whereas the taiga and alpine covers had four distinct layers. Visual measurements were able to establish the range of crystal sizes that occurred in each layer, the temperature within the pit, and the snow density. The LTSEM observations revealed the detailed structures of the types of crystals that occurred in the snow covers, and documented the metamorphosis that transpired in the descending layers. Briefly, the top layers from two of the snow covers consisted of freshly fallen snow crystals that could be readily distinguished as plates and columns (prairie) or graupel (taiga). Alternatively, the top layer in the alpine cover consisted of older dendritic crystal fragments that had undergone early metamorphosis, that is, they had lost their sharp edges and had begun to show signs of joining or bonding with neighboring crystals. A unique layer, known as sun crust, was found in the prairie snow cover; however, successive samplings from all three snow covers showed similar stages of metamorphism that led to the formation of depth hoar crystals. These changes included the gradual development of large, three-dimensional crystals having clearly defined flat faces, sharp edges, internal depressions, and facets. The study, which indicates that LTSEM can be used to enhance visual data by systematically characterizing snow crystals that are collected at remote locations, is important for understanding the physics of snowpacks and the metamorphosis that leads to potential avalanche situations. In addition, the metamorphosis of snow crystals must be considered when microwave radiometry is used to estimate the snow water equivalent in the winter snowpack, because large snow crystals more effectively scatter passive microwave radiation than small crystals.

Use of low-temperature field emission scanning electron microscopy to examine mites.

Partly because mites are microscopic in size and fragile in nature, acarologists estimate that less than five percent of extant species have been taxonomically described. Recently, data from conventional scanning electron microscopy (SEM) have been used to facilitate the descriptions and complement the information that has been historically obtained with the light microscope. However, the conventional preparation techniques associated with SEM frequently prevent or compromise the results. This study evaluated the use of low-temperature field emission SEM to image mites and their hosts. Results indicated that a modified cryofixation procedure, which was associated with this technique, retained the mites at their living/feeding sites in natural behavioral positions. Furthermore, the turgor of the specimens, even eggs and soft-bodied species, was also maintained. The structure and orientation of delicate structures such as setae, which would be subjected to mechanical damage during conventional chemical fixation, dehydration, and drying, were also preserved after cryofixation. Field emission SEM, which provided useful magnification beyond that attainable with a conventional SEM, also enabled resolution of ultrastructural features, such as tenent hairs on the empodium and pores on the dorsal surface that had not previously been observed. These advantages indicate that the low-temperature field emission SEM can provide important structural data that can be used to study the anatomy, morphology, and bioecology of mites.

Micromorphological characterization and lithification of microbial mats from the Ebro Delta (Spain).

The structural organization of microbial mats from the Ebro Delta (Spain) and their accretion and partial lithification processes were explored using scanning electron microscopy in back-scattered electron mode and low-temperature scanning electron microscopy. Two differentiated zones were distinguished in a transverse section of a fragment taken from the mat at a depth of 2.5 mm. The first consisted of an upper layer in which the dominant microorganisms, Microcoleus spp., actively grew in an embedded slack matrix of exopolysaccharides. Microcoleus filaments were oriented parallel to the surface and to each other, with filaments below arranged perpendicularly to one another but without crossing. Most of the minerals present were allochthonous grains of calcium phosphate biocorroded by cyanobacteria. The second zone was below a depth of 1 mm and made up of accretion layers with large deposits of calcium carbonate and smaller amounts of calcium phosphate of biological origin. The predominance of a particular type of mineral precipitation with a characteristic external shape and/or texture within a zone, e.g., sponge-like deposits of calcium phosphate, appears to depend on the taxa of the prevailing microorganisms.

Calorimetric and Microstructural Investigation of Frozen Hydrated Gluten

The thermal and microstructural properties of frozen hydrated gluten were studied by using differential scanning calorimetry (DSC), modulated DSC, and low-temperature scanning electron microscopy (cryo-SEM). This work was undertaken to investigate the thermal transitions observed in frozen hydrated gluten and relate them to its microstructure. The minor peak that is observed just before the major endotherm (melting of bulk ice) was assigned to the melting of ice that is confined to capillaries formed by gluten. The Defay–Prigogine theory for the depression of melting point of fluids confined in capillaries was put forward in order to explain the calorimetric results. The pore radius size of the capillaries was calculated by using four different empirical models. Kinetic analysis of the growth of the pore radius size revealed that it follows first-order kinetics. Cryo-SEM observations revealed that gluten forms a continuous homogeneous and not fibrous network. Results of the present investigation showed that is impossible to assign a Tg value for hydrated frozen gluten because of the wide temperature range over which the gluten matrix vitrifies, and therefore the construction of state diagrams is not feasible at subzero temperatures for this material. Furthermore, the gluten matrix is deteriorated with two different mechanisms from ice recrystallization, one that results from the growth of ice that is confined in capillaries and the other from the growth of bulk ice.

High Viscosity and Anisotropy Characterize the Cytoplasm of Fungal Dormant Stress-Resistant Spores

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.

Gelation under dynamic conditions: A strategy for in vitro cell ordering

Ordered gelation under spin-coating conditions, as reported here, is a suitable method to order cells in biogels. Cell ordering is of great importance for functional repair of central nervous system (CNS) injuries, because therapies must include strategies to bridge chystic gaps and facilitate axon growth towards its target. Organized biocompatible and biodegradable substrates may be used for this purpose, to supply trophic support and provide directional cues for neuronal process outgrowth. Atomic force microscopy (AFM) and low temperature scanning electron microscopy (LTSEM), confirmed that fibrils in kappa-carrageenan/chitosan and fibrin hydrogels prepared under spin-coating conditions, were longitudinally arranged. The cell model was conveniently tested using rat C6 glioma cells. C6 cells were distributed regularly in fibrin gels formed under centrifugal force. The ability of ordered fibrin scaffolds to promote uniform distribution of transplanted cells, was confirmed by fluorescence microscopy.

Biofilm Formation byStreptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial Accretion

Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 microm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

Electron-beam-induced shift in the apparent position of a pinned vortex in a thin superconducting film

When an electron beam strikes a superconducting thin film near a pinned vortex, it locally increases the temperature-dependent London penetration depth and perturbs the circulating supercurrent, thereby distorting the vortex's magnetic field toward the heated spot. This phenomenon has been used to visualize vortices pinned in SQUIDs using low-temperature scanning electron microscopy. In this paper I develop a quantitative theory to calculate the displacement of the vortex-generated magnetic-flux distribution as a function of the distance of the beam spot from the vortex core. The results are calculated using four different models for the spatial distribution of the thermal power deposited by the electron beam.

Sheet-current distribution in a dc SQUID washer probed by vortices

We present a novel method, based on vortex imaging by low-temperature scanning electron microscopy (LTSEM), to directly image the sheet-current distribution in YBa2Cu3O7 dc SQUID washers. We show that the LTSEM vortex signals are simply related to the scalar stream function describing the vortex-free circulating sheet-current distribution J. Unlike previous inversion methods that infer the current distribution from the measured magnetic field, our method uses pinned vortices as local detectors for J. Our experimental results are in very good agreement with numerical calculations of J.

Effects of whey protein aggregation on fat globule microstructure in whipped-frozen emulsions

Four oil-in-water emulsions, stabilized by native or pre-denatured whey proteins in the absence or presence of casein, were whipped at −5 °C and stored at −30 °C for several weeks. The microstructure of these whipped-frozen emulsions, which differed by their protein content, were observed by low-temperature scanning electron microscopy (LT-SEM), and transmission electron microscopy (TEM) following freeze-substitution and low-temperature embedding. Fat globule aggregation and adsorbed protein content in whipped-frozen emulsions were determined after application of thawing, dilution or centrifugation. Micrographs indicated that in aerated products, partial replacement of native whey proteins by pre-denatured whey proteins or casein introduced (i) more homogeneity in air bubble size, (ii) more attachment of fat globules to their air–serum interface, and (iii) fat globules in the continuous matrix that were in closer contact with each other. These differences in the microstructures of whipped-frozen emulsions were attributed to differing surface heterogeneity of adsorbed protein particles to fat globule interfaces.

Instrumental measurement of apple texture: A comparison of the single-edge notched bend test and the penetrometer

The single-edge notched bend (SENB) test is a technique for measuring fundamental properties of materials. This technique is an easily applied measure of fruit texture that is related to sensory characteristics such as ‘crunchiness’ [Vincent, J.F.V., Saunders, D.E.J., Beyts, P., 2002. The use of critical stress intensity factor to quantify “hardness” and “crunchiness” objectively. J. Texture Stud. 33, 149–159]. The present study focuses on the relationship between SENB and the penetrometer test routinely used for fruit quality assessment. Fruit from cultivars Cox's Orange Pippin, Sciros, Sciearly and Scifresh were removed from cold storage at regular intervals, and texture was assessed after 1 and 7 days at 20 °C. Texture was measured using a conventional puncture test with an Effegi probe and a SENB test. During the SENB test, a notched beam of apple tissue was subjected to three-point bending until it broke. Fracture toughness ( K c) and fracture energy ( G c) were calculated, and Low-Temperature Scanning Electron Microscopy (LTSEM) was used to examine the fracture surface following beam failure. Both K c and G c were correlated with puncture force, but the relationship was different for Sciros and Sciearly (two siblings from a Gala × Splendour cross) compared to the other cultivars. Sciros and Sciearly apples had higher K c and G c values than would be anticipated from measurements of puncture force. This supports the suggestion that these apples are crisper when eaten than might be expected from puncture (penetrometer) values. Comparison of the texture of shrivelled and non-shrivelled Sciros apples indicated that puncture measurements are unreliable when fruit are dehydrated. Puncture measurements suggested shrivelled apples were ‘crisper’ than non-shrivelled fruit, while SENB parameters indicated the reverse.