Low-temperature Scanning Electron Microscopy Research Articles

Cryo and Cryo-Etch Methods for Quality Preservation of Hydrogels Imaged at High Magnification by Low Temperature SEM

Cryo and Cryo-Etch Methods for Quality Preservation of Hydrogels Imaged at High Magnification by Low Temperature SEM

Imaging of trapped vortices in YBCO coated conductor by scanning SQUID microscope

We investigated trapped vortices in Y1Ba2Cu3O7−δ (YBCO) coated conductor by using a scanning SQUID microscope (SSM). Several peculiar properties of the vortices were observed such as a large bundle of vortices at a certain position and a coexistence of positive- and negative-sign vortices. Comparing the SSM images with that of low temperature scanning laser microscopy (LTSLM) and SEM, we identified the relationship between local dissipation and position of current blocking defect. Furthermore, regular vortex images were also observed by the SSM in the region where higher Jc has been identified by the LTSLM.

Observations on the honey bee tracheal mite Acarapis woodi (Acari: Tarsonemidae) using low-temperature scanning electron microscopy

Observations were made of cryo-preserved honey bee tracheal mites Acarapis woodi (Rennie) using scanning electron microscopy. We describe various new morphological attributes of A. woodi based on the ability of the cryo-technique to capture live mites in natural positions and observe the Low-Temperature Scanning Electron Microscopy (LT-SEM) photographs under a 3-D viewer. Most striking was the observation that each leg has the ability to independently twist its segments with the ambulacrum rotating a minimum of 180 degrees during locomotion; this is a more sophisticated form of locomotion than has been proposed for the Acari. Adult daughter mites are known to be the dispersal instar moving from the tracheal tube to the thoracic hairs of the bee and then transferring to a new bee. We hypothesize that adult tarsal claws and setae on the legs play a role in attachment to hairs during dispersal. However, our evidence is that none of the life stases use their tarsal claws within the tracheal tubes. Larvae were observed to be 'freely moving' within the tracheal system, their tarsal claws rendered inoperative due to an enlarged swollen pulvillar pad. The solenidia of leg I are now known to have striations and the famulus is bifurcated. The bifurcated famulus, solenidial striations, and segmentation of leg IV of females may have taxonomic implications in the family Tarsonemidae. The body and leg setae of adults appear to be used as a tactile tool to sense the amount of space within the tracheal tubes; most of the setae are oriented distally and may help the mite to measure the space or radius of the tracheal tubes. The modified caudal region of the male revealed remnants of the h1 and h2 setae and a smooth clean surface, void of a film, supporting that pharate nymphs are not attached in this species.

Cryo-SEM and subsequent TEM examinations of identical neural tissue specimen

Low temperature scanning electron microscopy of frozen-fractured specimens under cryo-protecting, non-dehydrating, and non-etching “wet” conditions, that is, direct cryo-SEM, was followed by transmission electron microscopy (TEM) with the same neural tissue specimens. In comparison to replica TEM, direct cryo-SEM can obtain images with a smooth gradation of contrast. The major advantage of direct cryo-SEM combined with TEM was that time was saved in SEM preparation. It had a high potentiality at a wide-range survey of multi-dimensional specimen structures with less-artifacts. Because the specimens were prepared as quickly as possible under “wet” conditions, the target structures could be examined under lower through higher magnifications. In the present study, neuronal and glial elements, such as plasma membranes and cell organelles that include the synaptic vesicles, were localized on the fractured surface. In subsequent TEM examination, it was confirmed that the underlying internal structures could be further characterized from cytological as well as molecular biological aspects. In addition, direct cryo-SEM distinctively demonstrated small intra-membrane particles (ca. 10 nm in diameter). However, due to electron lucency, they could not be confirmed in the re-processed TEM specimens. Applying the present protocol, stereological and internal architectural examinations of the neural tissues have been simultaneously conducted at ultra-fine levels.

Ice friction, wear features and their dependence on sliding velocity and temperature

AbstractFriction processes for ice samples sliding on steel have been determined by examining wear and debris morphology with low-temperature scanning electron microscopy and relating the processes to the velocity and temperature of formation. Friction experiments were carried out over a temperature range of −27 to −0.5°C and velocity range of 0.008–0.37 m s−1. Data were used to develop a friction map. Low friction (µ < 0.1) at high temperature (–3.4ºC)–low velocity (0.02 ms−1), and low temperature (–25.1ºC)–high velocity (0.30 ms−1) is due to the presence of liquid water which lubricates the sliding interface. Diagnostic morphologies for lubricated sliding include the presence of residual liquid in wear grooves and the development of a consolidated mass of debris on the trailing side of the wear surface with distinct grain boundaries and spheroidal air bubbles. High friction (µ > 0.15) at low temperature (−24.5ºC)–low velocity (0.03 m s−1) results from insufficient lubrication at the sliding interface, leading to plastic deformation. Diagnostic morphologies of plastic deformation include scuffing features on the wear surface and the accumulation of sheets of unconsolidated debris on the trailing edge of the wear surface.

Microscopic analysis of low-frequency flux noise in YBa2Cu3O7 direct current superconducting quantum interference devices

We use low-temperature scanning electron microscopy combined with superconducting quantum interference device (SQUID) detection of magnetic flux to image vortices and to investigate low-frequency flux noise in YBa2Cu3O7 thin film SQUIDs. The low-frequency flux noise shows a nonlinear increase with magnetic cooling field up to 60μT. This effect is explained by the surface potential barrier at the SQUID hole. By correlating flux noise data with the spatial distribution of vortices, we obtain information on spatial fluctuations of vortices on a microscopic scale, e.g., an average vortex hopping length of ∼10nm.

Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms ( Phoenicococcus marlatti Ckll., 1899)

We have evaluated the parasitism of the red scale insect of the date palm ( Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves ( Phoenix dactylifera L.) and did not parasite the scale insects.

Imaging of μm wavelength collective cavity resonances in Bi2Sr2CaCu2O8+x intrinsic Josephson junction stacks under microwave irradiation

We have used low-temperature scanning electron microscopy to image the electromagnetic field distribution in microstructured Bi2Sr2CaCu2O8+x intrinsic Josephson junction stacks under microwave irradiation. Even for microwave frequencies below 10GHz, standing waves along the stack with wavelengths of order 10μm can be observed. The data, together with numerical simulations based on coupled sine-Gordon equations, strongly indicate that the standing waves are due to collective Josephson plasma cavity resonances, with a large number of junctions oscillating in phase. The resonance frequencies are estimated to be of order 100–500GHz, that is, much higher than the drive frequency. We suggest that, under the low-frequency microwave irradiation, fluxon∕antifluxon pairs form, in turn exciting the high-frequency collective cavity resonances.

The effect of calcium and cellular permeabilization on the structure of the parenchyma of osmotic dehydrated ‘Granny Smith’ apple

AbstractCalcium plays a very important role in the maintenance of quality in vegetable foods and it is used as a texturing agent to preserve the structure of minimally processed vegetables. The osmotic dehydration (OD) of ‘Granny Smith’ apple parenchyma by immersion in a sucrose solution allows us to obtain products with sensorial characteristics similar to those of fresh products, but it produces a loss of cellular integrity. Here we study the use of calcium as a possible preservative for the microstructural integrity of the product during OD, as well as to test whether cellular permeabilization improves the impact of calcium on the edible parenchyma of the ‘Granny Smith’ apple. Sodium dodecyl sulfate (SDS) has been used for convenience as a permeating agent although we recognize this may not be used in practice. The microstructure of the OD apple treated with calcium chloride and with SDS has been compared with fresh apple by means of light microscopy, transmission electron microscopy and low temperature scanning electron microscopy. The results show that calcium has a protective effect on the structural integrity during OD: it strengthens the cellular walls cements, avoids cellular collapse and protects protoplasts from plasmolysis. Although SDS favours calcium penetration, its use is not advisable since it encourages the destructuring effect of the OD and counteracts the consolidating effect of calcium. Copyright © 2004 Society of Chemical Industry

Investigating entrained air voids and Portland cement hydration with low-temperature scanning electron microscopy

This paper describes the application of low temperature scanning electron microscopy to the materials science of Portland cement. The details of low-temperature scanning electron microscopy are described, along with a number of specimen preparation techniques. There are three main research topics presented in this paper: (1) ice morphology in entrained air voids, (2) development of air voids during early hydration and (3) progression of hydration in Portland cement. The first research focus examines ice in air voids at freezing temperatures, and various cement paste ages. The second research focus tracks the development of the air voids during the first hour of hydration. In the third research focus, the progression of hydration with and without accelerating and retarding admixtures is described. Each of these research programs demonstrates how low-temperature scanning electron microscopy can be an effective tool in Portland cement research.

An unusual infection mechanism and nodule morphogenesis in white lupin (Lupinus albus).

• The infection of white lupin (Lupinus albus) roots and the early stages in organogenesis of the lupinoid nodule are characterized in detail in this work. • Immunolabelling of Bradyrhizobium sp. (Lupinus) ISLU16 and green fluorescent protein labelling of Mesorhizobium loti NZP2037, two strains that induce nodulation in L.albus, allowed us to monitor the infection and morphogenesis process. Light and transmission electron microscopy, low-temperature scanning electron microscopy, fluorescence and confocal microscopy were employed. • Rhizobia penetrated the root intercellularly at the junction between the root hair base and an adjacent epidermal cell. Bacteria invaded the subepidermal cortical cell immediately beneath the root hair through structurally altered cell wall regions. The newly infected cell divided repeatedly to form the central infected zone of the young nodule. Bacteria seemed to be equally distributed between the daughter cells. • A new mode of direct epidermal infection and an unusual morphogenesis for indeterminate nodules lead to the formation of the lupinoid nodule with unique characteristics.

Magnetic domain structure of a La0.7Sr0.3MnO3 (001) surface observed by a spin-polarized scanning electron microscope

The magnetization vector distribution at a cleaved surface of La0.7Sr0.3MnO3 (001) crystal has been quantitatively analyzed by using a newly developed low-temperature spin-polarized scanning electron microscope. The magnetic structure essentially consists of two kinds of domains, where magnetizations are parallel or antiparallel to the [110] direction with no surface-normal component. The rhombus-shaped domains range from a few to several tens of micrometers across. The domain structure can be considered to be made by laying down the magnetization from the out-of-surface-plane easy axis to the surface plane to reduce the magnetostatic energy without forming closure domains.

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum–carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.

Alteration of biogenic structure and physical properties by tube-building chironomid larvae in cohesive sediments

From earlier research it has been suggested that chironomid larvae may significantly affect the physical properties of cohesive sediments, leading to chemical alteration in lake ecosystems. The present study was aimed to evaluate the effect of Tanytarsini larvae on the physical properties of cohesive sediments, both in controlled environments and in natural environments. For this the sediment surface was examined by low temperature scanning electron microscopy (LTSEM) and direct measurement of the shear strength. The larvae are able to modify the surface sediment significantly, e.g., by secretion of silk, which forms a matrix at the sediment surface in the vicinity of the larval tubes. The shear strength increased with greater sediment depth in all the experimental treatments, except where the densities were high. Considerably higher shear strength was observed in the natural sediment than in the experimental set up. A very significant density-dependent effect was observed within the top 1-cm by tube building Tanytarsini larvae. The relationship between larval densities and shear strength was not significant for 2 deeper sections (1–3 cm). The tube length was significantly greater in experimental containers with the low density (4960 ind. m−2) than in containers with high larval densities (19840 and 39680 ind. m−2).

Microstructural characterization of cyanobacterial mats from the McMurdo Ice Shelf, Antarctica.

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.

NEW APPLICATIONS OF LIGHT AND ELECTRON MICROSCOPIC TECHNIQUES FOR THE STUDY OF MICROBIOLOGICAL INCLUSIONS IN AMBER

Amber is a superb medium for the fossilization of delicate organisms. Besides light microscopy techniques for the study of insects in amber, scanning electron microscopy (SEM) in backscattered electron mode (SEM-BSE), low temperature SEM (LTSEM) and also confocal laser scanning microscopy (CLSM) were used to examine microcenosis and particulate plant remains (microdebris). We applied these techniques to such inclusions in amber Alava, northern Spain (Allaian: Early Cretaceous). Confocal microscopy provides a 3D image of partial microcenosis showing bifurcate fungal hyphae. The huge potential of SEM-BSE yields high resolution images, in which the relationship between protozoa and fungal hyphae may be observed and the characterization of further ultrastructural details in flagellates. According to the SEM-BSE images, food and pulsatile vacuoles appear better preserved than mitochondria and lipids in amber-embedded protozoa. A process of protozoan mineralization has led to the deposition of S and Fe in peripheral areas, and the Fe is also present in the core of surrounding fungal hyphae. Application of LTSEM for the study of protozoan inclusions produces images of their exteriors showing many vacuoles. Plant tissues under SEM-BSE show mummified cell walls, while the cytoplasm exhibits a bright appearance and is very rich in Fe and S. SEM in secondary electron mode (SEM-SE) also reveals a microbiota trapped in gas bubbles.

Collecting, shipping, storing, and imaging snow crystals and ice grains with low-temperature scanning electron microscopy.

Methods to collect, transport, and store samples of snow and ice have been developed that enable detailed observations of these samples with a technique known as low-temperature scanning electron microscopy (LTSEM). This technique increases the resolution and ease with which samples of snow and ice can be observed, studied, and photographed. Samples are easily collected in the field and have been shipped to the electron microscopy laboratory by common air carrier from distances as far as 5,000 miles. Delicate specimens of snow crystals and ice grains survive the shipment procedures and have been stored for as long as 3 years without undergoing any structural changes. The samples are not subjected to the melting or sublimation artifacts. LTSEM allows individual crystals to be observed for several hours with no detectable changes. Furthermore, the instrument permits recording of photographs containing the parallax information necessary for three-dimensional imaging of the true shapes of snowflakes, snow crystals, snow clusters, ice grains, and interspersed air spaces. This study presents detailed descriptions of the procedures that have been used successfully in the field and the laboratory to collect, ship, store, and image snow crystals and ice grains.

Diatom migration and sediment armouring – an example from the Tagus Estuary, Portugal

This study measured sediment stability, colloidal and total carbohydrate and chlorophyll a in the upper 2 mm of the sediment over a whole emersion period (0800–1140) in the Tagus estuary, Portugal on the 18th April 2000. Low-temperature scanning electron microscopy (LTSEM) time series images revealed the migration of microalgae throughout the emersion period, including their appearance at the sediment surface at the beginning of emersion and their subsequent return below the surface at the end of emersion. Different species arrived at the sediment surface at different times and there was a slight lag between the appearance of the first cells and the subsequent increase in sediment stability. Increased chlorophyll a concentration in the surface sediments preceded the increase in sediment stability, whilst colloidal and total carbohydrate concentrations increased afterwards. Sediment water content decreased during the emersion period. Erosion threshold increased shortly after the microalgal cells appeared at the sediment surface, suggesting that the cells themselves act to `armour' the sediment surface, retarding erosion. Lack of correlation between sediment stability and factors traditionally considered to control sediment stability (e.g., water content and carbohydrate content) indicates that an important variable or interaction has yet to be identified. One possibility is that the carbohydrate fraction extracted does not measure accurately the binding effectiveness of the carbohydrates in the sediment. We propose feedback and `critical point' models to explain how the various sediment properties determine sediment stability. The implication is that sediment stability varies in an apparently idiosyncratic and site-specific fashion due to the complex interaction of physical and biological variables. Given the importance of ecological processes in intertidal sediments, the measurement, understanding and modelling of sediment erosion would benefit greatly from the application of ecological methods of experimental design and sampling.

Rime and graupel: description and characterization as revealed by low-temperature scanning electron microscopy.

Snow crystals, which form by vapor deposition, occasionally come in contact with supercooled cloud droplets during their formation and descent. When this occurs, the droplets adhere and freeze to the snow crystals in a process known as accretion. During the early stages of accretion, discrete snow crystals exhibiting frozen cloud droplets are referred to as rime. If this process continues, the snow crystal may become completely engulfed in frozen cloud droplets. The resulting particle is known as graupel. Light microscopic investigations have studied rime and graupel for nearly 100 years. However, the limiting resolution and depth of field associated with the light microscope have prevented detailed descriptions of the microscopic cloud droplets and the three-dimensional topography of the rime and graupel particles. This study uses low-temperature scanning electron microscopy to characterize the frozen precipitates that are commonly known as rime and graupel. Rime, consisting of frozen cloud droplets, is observed on all types of snow crystals including needles, columns, plates, and dendrites. The droplets, which vary in size from 10 to 100 microm, frequently accumulate along one face of a single snow crystal, but are found more randomly distributed on aggregations consisting of two or more snow crystals (snowflakes). The early stages of riming are characterized by the presence of frozen cloud droplets that appear as a layer of flattened hemispheres on the surface of the snow crystal. As this process continues, the cloud droplets appear more sinuous and elongate as they contact and freeze to the rimed crystals. The advanced stages of this process result in graupel, a particle 1 to 3 mm across, composed of hundreds of frozen cloud droplets interspersed with considerable air spaces; the original snow crystal is no longer discernible. This study increases our knowledge about the process and characteristics of riming and suggests that the initial appearance of the flattened hemispheres may result from impact of the leading face of the snow crystal with cloud droplets. The elongated and sinuous configurations of frozen cloud droplets that are encountered on the more advanced stages suggest that aerodynamic forces propel cloud droplets to the trailing face of the descending crystal where they make contact and freeze.

Morphological Observations on Brevipalpus phoenicis (Acari: Tenuipalpidae) Including Comparisons with B. californicus and B. obovatus

The genus Brevipalpus has over 300 species worldwide. The three most important agricultural pest species in the genus, Brevipalpus californicus (Banks), B. obovatus Donnadieu, and B. phoenicis (Geijskes), have been consistently confused and misidentified for more than 50 years. The present study provides a discussion of the characters and character states used to separate these mites. Low-temperature scanning electron microscopy and traditional light microscopy techniques were used to illustrate the subtle morphological differences between these three species. Morphology of the dorsal propodosoma, opisthosoma, and leg chaetotaxy of all three species was examined and compared. The number of dorsal setae, the number of solenidia (omega) on tarsus II, and dorsal cuticular patterns were the most important characters in the identification of Brevipalpus species. B. phoenicis is similar to B. californicus in having two omega on tarsus leg II and different from B. obovatus which has only one omega on tarsus leg II and similar to B. obovatus in having only one pair of F setae (f3), but differing from B. californicus which has two pairs of F setae (f2-3). The dorsal opisthosomal and propodisomal cuticular patterns frequently used to distinguish between these three species are useful but one must be aware that age, feeding, and mounting techniques can affect the appearance of these characters.