Double-layer coating for high-resolution low-temperature scanning electron microscopy

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g. 100 000x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e^— nm^—2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5–10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.     

Design of an aberration corrected low-voltage SEM

The low-voltage foil corrector is a novel type of foil aberration corrector that can correct for both the spherical and chromatic aberration simultaneously. In order to give a realistic example of the capabilities of this corrector, a design for a low-voltage scanning electron microscope with the low-voltage foil corrector is presented. A fully electrostatic column has been designed and characterised by using aberration integrals and ray tracing calculations. The amount of aberration correction can be adjusted relatively easy. The third order spherical and the first order chromatic aberration can be completely cancelled. In the zero current limit, a FW50 probe size of 1.0 nm at 1 kV can be obtained. This probe size is mainly limited by diffraction and by the fifth order spherical aberration.     

High-resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens

Intracellular structures of rapidly frozen biological tissues were observed in 3-D under a low-temperature scanning electron microscope using a newly developed side-entry type cryo-holder. The present low-temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo-holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo-holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3-D configuration of lipid droplets which had been difficult to preserve by chemical fixation.     

A portable cryo-storage system for low-temperature scanning electron microscopy,suitable for international transport of cryo-specimens

A cryo-specimen storage system for low-temperature scanning electron microscopy (LTSEM) specimens is described, which: liberates multi-specimen experiments from sampling restrictions imposed by the rate at which LTSEM specimens can be examined in the SEM; provides security against experiment loss resulting from breakdown of the SEM or cryo-system; enables collection of specimens in the field or in laboratories remote from the SEM laboratory; and facilitates international air transport of LTSEM specimens. The components of the system, which has a capacity of 98 stub-mounted specimens, are readily made in a laboratory workshop. The details of the design may be altered to suit particular specimen types or experimental approaches.     

Recent progress in freeze-fracturing of high-pressure frozen samples

Pancreatic tissue, bacteria and lipid vesicles were high‐pressure frozen and freeze‐fractured. In addition to the normal holder, a new type of high‐pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low‐mass copper platelets, as used for propane‐jet freezing. Both the standard and the new suspension holder allowed us to make cryo‐fractures without visible ice crystal damage. High‐pressure frozen rat pancreas tissue samples were cryo‐fractured and cryo‐sectioned with a new type diamond knife in the microtome of a freeze‐etching device. The bulk fracture faces and blockfaces were investigated in the frozen‐hydrated state by use of a cryo‐stage in an in‐lens SEM. Additional structures can be made visible by controlled sublimation of ice (‘etching’), leading to a better understanding of the three‐dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life‐like state, preventing the artefacts associated with conventional fixation techniques.     

Contrast effects using a two-detector system in low-voltage scanning electron microscopy

A system of two opposite Everhart–Thornley detectors A and B has formerly been applied in conventional SEM for electron energies between 5 and 20 keV to separate material, topographic and other types of contrast by sum and difference signals. This technique can also be used successfully for low-voltage scanning electron microscopy. The decreasing information depth with decreasing electron energy shows differences in the surface composition and contamination which cannot be observed beyond 5 keV. Also below 5 keV material and topographic contrast can be separated and increased by the A + B and A − B signals, respectively.     

Determination of local deep-levels homogeneity using SEM

A method to study microhomogeneity of deep centres in high resistivity and low resistivity semiconductors is described herein. It uses measurements of current decay induced in Schottky diodes and p-n junctions by a probing electron beam of an SEM and is closely related to a method of photoelectron relaxation spectroscopy. The advantages of this method are demonstrated for an example of dislocation-related inhomogeneity in semi-insulating GaAs.     

Imaging of cytoskeletal elements by low-temperature high-resolution scanning electron microscopy

Actin filaments and microtubules, both in situ and in vitro, were imaged using high-resolution scanning electron microscopy (HRSEM) at low temperature. For visualization of cytoskeletal elements in situ, fibroblasts were first extracted and fixed; for cytoskeletal elements in vitro, purified proteins were polymerized and fixed. Both types of specimen were then subjected to plunge freezing, controlled freeze-drying, cryo-sputter coating with a thin chromium layer, cryo- transferring and cryo-observation in an FESEM. The three-dimensional architecture of the cytoskeleton was well preserved, permitting examination of the structural relationships among cytoskeletal elements. Actin filaments and microtubules were identified by their characteristic helical features. Two periodicities of actin filaments, the short pitch of the left-handed helix measured at 5·5 nm and the 37-nm-long pitch helix, were revealed. Individual protofilaments were seen in microtubules as well as the characteristic 4-nm repeat of tubulin subunits along the protofilament. Clathrin cages were also observed. This technique provides a powerful approach for direct imaging of macromolecular structures with high contrast and high signal-to-noise ratio at a resolution of 2–3 nm.     

Observations of snow crystals using low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy (SEM) was used to observe precipitation particles commonly known as “snowflakes.” The snowflakes were collected in Beltsville, Maryland, at temperatures ranging from ?6° to +1°C, mounted on SEM stubs, frozen in liquid nitrogen (LN₂), and then transferred to a cryosystem mounted on a field-emission SEM. Neither sputter coating with platinum nor irradiation by the electron beam affected their delicate fine structure. SEM observations revealed that snowflakes consisted of aggregations of snow crystals that occurred as hexagonal plates, prismatic columns, needles, and dendrites. In some cases, the snow crystals contained minute surface structures that consisted of rime, microdroplets, short prismatic columns, and amorphous films. Snow crystals from wet snow, which were collected at temperatures of 0° and +1°C, exhibited varying degrees of metamorphism or melting. The discrete crystalline faces and their sharp intersecting angles were gradually replaced by sinuous surfaces that tended to exhibit more spherical shapes. This study indicates that low-temperature SEM is a valuable technique for studying the formation and metamorphosis of snow crystals. The results suggest that combining low-temperature SEM and x-ray analysis could also provide qualitative elemental information on the nucleation particles of snow crystals as well as on the composition of acid snow.     

An on-pole-piece-tip deflector for high-resolution,low-voltage scanning electron microscopes

A proposal to enlarge the field of view of low-voltage, high-resolution scanning electron microscopes is presented in this paper. The proposal is based upon mounting primary beam deflectors onto the pole tip of the objective lens. Simulation results show that this compact arrangement of beam deflection should widen the present attainable field of view at probe resolutions < 5 nm by over an order of magnitude.     

Artefacts arising during preparation of hydrated paper pulp samples for low-temperature SEM

Beating, a pulp treatment widely used in the paper industry, causes disruption of cell wall layers and fibrillation. Previous studies of the effects of beating on fibre morphology have used conventional methods of specimen preparation, with all the attendant problems of shrinkage and distortion during dehydration. Low-temperature scanning electron microscopy (LTSEM) therefore seemed to offer an ideal method for examining fully hydrated wood pulp fibres. Cryofixation of pulp followed by sublimation of superficial ice, however, is shown to generate artefacts indistinguishable from structures present in the samples. Fibrillar and membranous structures were generated in LTSEM-prepared sugar solutions; their presence in pulp samples was therefore attributed to the dissolved carbohydrates inherent in pulp suspensions. Since artefact and fact are currently impossible to distinguish in LTSEM-prepared pulp samples, it seems that the technique should be applied to wet paper or pulp samples with considerable circumspection.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

An improved low-voltage ride-through performance of DFIG based wind plant using stator dynamic composite fault current limiter

In this paper, an enhanced low-voltage ride-through (LVRT) performance of a grid connected doubly fed induction generator (DFIG) has been presented with the usage of stator dynamic composite fault current limiter (SDCFCL). This protection circuit comprises of a suitable series resistor–inductor combination and parallel bidirectional semiconductor switch. The SDCFCL facilitates double benefits such as reduction of rotor induced open circuit voltage due to increased value of stator total inductance and concurrent increase of rotor impedance. Both effects will limit rotor circuit over current and over voltage situation more secured way in comparison to the conventional scheme like the dynamic rotor current limiter (RCL) during any type of fault situation. The proposed concept is validated through the simulation study of the grid integrated 2.0 MW DFIG.     

Evaluation of the effects of biodegradable nanoparticles on a vaccine delivery system using AFM, SEM, and TEM

Hepatitis B is a deadly disease, and is carried by 30% of the world's population. Antibodies are produced through a series of three manual vaccinations during infancy and childhood. However, the current needle vaccination not only induces pain in patients, but also can be inconvenient to administer. This is particularly true for the case of newborn babies. Intranasal vaccination is emerging as an alternative parenteral drug delivery method that facilitates drug delivery without causing pain. Chitosan, which is obtained through the deacetylation of chitin from crustacea, is a cationic polymer that is biodegradable, avirulent, and highly absorptive. In this study, ionic gelation between chitosan and TPP was conducted to synthesize chitosan nanoparticles with sizes of 200-400nm and a surface potential of 55-60mV, and which can be used as Hepatitis B vaccine carriers. Then, Heptatitis B antigen protein was impregnated to manufacture chitosan-recombinant gene vaccine protein (RGVP) nanoparticles. AFM, SEM, TEM, and STEM were used to analyze the manufactured nanoparticles, whose function as drug carriers and whose usefulness for intranasal vaccination were confirmed through in vivo tests with SD rats.     

3D analysis of SEM images of corrosion casting using adaptive stereo matching

The corrosion casting method represents one of the most widely used technique to study the 3D microvascularization of many tissues, both in their normal and pathological conditions. For a long time this technique was used only to perform a qualitative evaluation of the images obtained by scanning electron microscopy (SEM). A quantitative evaluation of vascular parameters (e.g., interbranching and intervascular distances, angle measurements, lengths and diameters) was lacking, mainly because of the difficulties found in the measurement performed on 2D SEM images. Then, some authors reported a quantitative method based on the analyses of stereo-pair images that allowed precise morphometric measurements. To visualize the specimens in 3D, it was necessary to use red-green glasses. In this article we describe a new approach by which we can automatically obtain a 3D reconstruction of vascular cast specimen's surface directly from stereo-images. Moreover, we developed a software that performed micrometric measurements on the 3D construct generated from the stereo-pictures. In conclusion, implementing together these two softwares and applying them to corrosion casting samples made it possible to render in 3D the surface of corrosion cast as well as make quantitative measurements on the corrosion casts.     

Three‐dimensional analysis of intermediate filament networks using SEM tomography

We identified tomographic reconstruction of a scanning electron microscopy tilt series recording the secondary electron signal as a well‐suited method to generate high‐contrast three‐dimensional data of intermediate filament (IF) networks in pancreatic cancer cells. Although the tilt series does not strictly conform to the projection requirement of tomographic reconstruction, this approach is possible due to specific properties of the detergent‐extracted samples. We introduce an algorithm to extract the graph structure of the IF networks from the tomograms based on image analysis tools. This allows a high‐resolution analysis of network morphology, which is known to control the mechanical response of the cells to large‐scale deformations. Statistical analysis of the extracted network graphs is used to investigate principles of structural network organization which can be linked to the regulation of cell elasticity.     

Enhanced EDX images by fusion of multimodal SEM images using pansharpening techniques

The goal of this paper is to explore the potential interest of image fusion in the context of multimodal scanning electron microscope (SEM) imaging. In particular, we aim at merging the backscattered electron images that usually have a high spatial resolution but do not provide enough discriminative information to physically classify the nature of the sample, with energy‐dispersive X‐ray spectroscopy (EDX) images that have discriminative information but a lower spatial resolution. The produced images are named enhanced EDX. To achieve this goal, we have compared the results obtained with classical pansharpening techniques for image fusion with an original approach tailored for multimodal SEM fusion of information. Quantitative assessment is obtained by means of two SEM images and a simulated dataset produced by a software based on PENELOPE.     

Controlled dehydration of a biological sample using an alternative form of environmental SEM

In this study a non-conductive biological sample is observed free of charging artefacts when placed on a cooled Peltier stage in the specimen chamber of an alternative form of the environmental scanning electron microscope, equipped with a specially designed hydration system. This system was used to create dynamically changing surrounding conditions leading to controlled dehydration of the sample enabling us to visualize the topographical structure of a rat tongue in the transition region between the liquid and the gas state of water in the microscope specimen chamber.     

Systematic significance of pollen morphology and foliar epidermal anatomy of medicinal plants using SEM and LM techniques

Alternative medicinal source is a hot issue within medicine community now a days due to various side effects of allotropic drugs; most importantly the multidrug resistance phenomena. Medicinal plants provide an attractive and effective alternative to modern synthetic drugs as they contain different types of bioactive compounds, having great potential in treatment of various diseases. The present study deals with identification of micromorphological characteristics of 10 ethnomedicinally important plants of Northern Pakistan, that is, Aster himalaicus C. B. Clarke, Artemisia maritima (Berg.) Wellkomm, Isodon rugosus (Wall. ex Benth.), Myricaria squamosal Desv, Pedicularis groenlandica Retz. Polygonum aviculare L, Salvia himmelbauer E. Peter, Swertia kingie Hook. f., Tanacetum dolichophyllum (Kitam) Kitam, Verbascum epixanthinum Boiss. & Heldr. as potential source for pharmaceutical industry. The characters studied were shape and size of pollen, polar diameter and equatorial diameter, epidermal cell shape and size, stomata width and length, trichome shapes, and type. Most of the species had tricolporate pollen type but hexacolporate pollen was also observed in Isodon rugosus, Polygonum aviculare, Salvia himmelbauer. Different types of exine sculpturing observed were foveolate, scabrate, lophate, regular, steriate, and alveolate. Minimum equatorial diameter was noted in Tanacetum dolichophyllum (12.4 μm) and maximum in Myricaria squamosal (20.9 μm). Shape of epidermis cell ranged from rectangular and irregular, polygonal and pentagonal. Different types of stomata like anisocytic, actinocytic, paracytic, and diacytic were noticed. Variations in microscopic characters observed in the current study have great potential to be used as identification tools by the taxonomists for further studies.