Low-temperature scanning electron microscopy of birch leaves after exposure to ozone

Comparison of different harvesting and preparation pathways showed that low-temperature SEM is an adequate method to conserve the stomatal aperture for SEM. Both critical point drying and freeze drying cause considerable artefacts. Exposure to site-relevant concentrations of ozone led to reduced width of the stomatal aperture. Moreover, unetchable droplet-like exudates were found on the outer face of mesophyll cells of leaves where the trees had been exposed to ozone. These exudates were later followed by collapsed mesophyll cells and ended in necrotic zones before premature leaf loss.     

Low-temperature scanning electron microscopy of particle-exposed mouse lung

Inflated frozen mouse lungs were examined using low-temperature scanning electron microscopy (LTSEM) following bulk fracture under vacuum. Various aspects of pulmonary architecture were identified and correlated with structures revealed by SEM following conventional fixation and preparation techniques. Surface etching of selected samples was performed by radiant heating, revealing characteristic cytoplasmic, nuclear and extracellular lattice patterns resulting from ice crystal formation during freezing. These patterns aided in distinguishing between intra- and extracellular spaces. Pulmonary fluids such as mucus and surfactant were identified. Iron oxide particles were introduced into the lungs of some animals by intratracheal instillation and were subsequently identified in frozen-hydrated lung tissue using characteristic X-ray identification and mapping techniques. Particles were observed both intra-and extracellularly and were commonly found in large deposits. These observations confirm the utility of LTSEM techniques for examination of particles within pulmonary tissue. Particle exposure by intratracheal instillation was found to result in a non-uniform distributional pattern.     

Freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit SCU 020

A dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low-temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high-vacuum preparation chamber equipped with a cold-stage, motor-driven fracturing microtome, planar magnetron (PM) sputter source, quartz-crystal thin-film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high-vacuum valve) residing in the SEM chamber. This is equipped with an anti-contamination cold trap, a fully movable goniometer cold stage (having motor drives for x, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice. LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze-dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (-150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on-line, high-vacuum, specimen cryo transfer between them. In-situ etching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high-resolution LTSEM in a field emission microscope.     

A cassette system for systematic cryostorage of low-temperature scanning electron microscopy stubs

A system is described for the storage of cylindrical (10 × 3.5 mm) stubs for low-temperature scanning electron microscopy. The system facilitates rapid retrieval of mounted specimens, maximizes the capacity of the low-temperature (liquid nitrogen) specimen store, locates each stub exactly in a protected well, and eliminates the possibility of specimen damage from conventional hazards during transport between the storage facility and microscope.     

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.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

A study of living plant specimens by low-temperature scanning electron microscopy

Young fresh Tradescantia reflexa stamen hair cells were used to clarify the optimal conditions for direct viewing and taking photographs with a scanning electron microscope (SEM) equipped with a cryo-system. The rate of protoplasmic streaming in the cells was measured under an optical microscope after examining and photographing them in the SEM over a period of a few minutes. Almost the same rate of streaming (5.5 μm/second, 20°C) was observed in nonirradiated control cells and irradiated cells photographed in the SEM using an accelerating voltage of 10 kV with the cryo-stage at a temperature of – 15°C. (The specimen holder and specimen were not at this temperature, but, rather, probably somewhat higher.) Fresh plant organs, tissues, and cells were also tested under the same conditions. The fine structure was well preserved in detail. The procedures were as follows: (1) prompt attachment of fresh plant materials on an aluminum specimen holder with double-faced adhesive Scotch tape or a small amount of plastic adhesive for woodcraft; (2) setting the holder on the cryo-stage cooled to –15°C in advance and rapid evacuation; and (3) quick SEM examination and photography (within several minutes). The advantages of this method are summarized as follows: (1) high possibility of viewing living materials; (2) minimal artifacts: freedom from chemical fixation and additional procedures utilized in ordinary SEM specimen preparation; and (3) simplicity, speediness, and economy in preparation for viewing. Since the specimens were not likely to be frozen during quick examination and photography, this method might well be called “low-temperature SEM” (LT-SEM) as distinguished from “cryo-SEM”.     

Low-temperature scanning electron microscopy in fungus-nematode interaction

A comparative study has been made of different preparation techniques used for the scanning electron microscope (SEM), with regard to their application to fungus-nematode interaction. The preparation of frozen-hydrated specimens of both healthy and Arthrobotrys-oli-gospora-infected second-stage larvae of the root-knot nematode (Meloidogyne sp.) is described, and the results are compared with those obtained by critical point-drying and freeze-drying. In all cases the frozen-hydrated specimens consistently showed the best preservation.     

Digital imaging for scanning electron microscopy

The development and application of digital imaging technology has been one of the major advancements in scanning electron microscopy (SEM) during the past several years. This digital revolution has been brought about by significant progress in semiconductor technology, notably the availability of less expensive, high-density memory chips and the development of inexpensive, high-speed, analog-to-digital and digital-to-analog converters, mass storage, and high-performance central processing units. This paper reviews a number of the advantages presented by digital imaging as applied to the SEM and describes a system developed at the National Institute of Standards and Technology for this purpose.     

Variations in fungal spore dimensions in relation to preparatory techniques for light microscopy and scanning electron microscopy

The volumes of urediospores of Uromyces viciae-fabae and ascospores of Sordaria humana in the critical point-dried, freeze-dried, osmium vapour-fixed/freeze-dried, frozen-hydrated and ‘wet’ states were measured. Dried spores shrank and were significantly smaller than frozenhydrated and ‘wet’ spores. Urediospores exhibited a greater range of dimensional changes in relation to the preparatory treatment than did ascospores. Ascospores, dried in different ways, were not significantly different from each other but were significantly smaller than frozenhydrated ones. Both urediospores and ascospores dehydrate naturally and collapse. On wetting they imbibe water rapidly and swell. Low temperature scanning electron microscopy may be suitable for investigating fungal spores in their ‘natural states' on the leaf surface.     

A natural color scanning electron microscopy image

A method for obtaining a scanning electron microscopy (SEM) image with natural color information (NCSEM image) has been developed by using an SEM operated under low vacuum or low voltage, a video microscope (VM), and a high-performance personal computer. In order to produce an NCSEM image with both the fine structures of the SEM image and the color information included in the VM image, an improvement of the depth of focus in VM image is required. This can be successfully performed by some digital image processing techniques. A difference in resolution between the SEM image and the VM image does not disturb our study as shown in the text. In addition, the well-known hue, saturation, intensity (HSI) color model, which is frequently utilized for color image manipulation, was originally altered for the present purpose.     

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.     

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.     

Deconvolution of scanning electron microscopy images

This paper describes a method of removing blurs in scanning electron microscopy (SEM) images caused by the existence of a finite beam size. Although the resolution of electron microscopy images has been dramatically improved by the use of high-brightness electron guns and low-aberration electron lenses, it is still limited by lens aberration and electron diffraction. Both are inevitable in practical electron optics. Therefore, a further reduction in resolution by improving SEM hardware seems difficult. In order to overcome this difficulty, computer deconvolution has been proposed for SEM images. In the present work, the SEM image is deconvoluted using the electron beam profile estimated from beam optics calculation. The results show that the resolution of the deconvoluted image is improved to one half of the resolution of the original SEM image.     

High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Low-temperature scanning electron microscopy of frozen hydrated mouse lung

A method is presented by which water is preserved as ice during examination of the lung in the scanning electron microscope (SEM). The lung need only be inflated, frozen, transferred to the microscope and examined with the electron beam. Chemical fixation, solvent dehydration, and drying are not necessary. The low-temperature SEM of Pawley and Norton [11] maintains lung at ?180° C, nearly liquid nitrogen temperature, for extended periods with a Joule-Thomson refrigerator built into the stage. It has an integral high-vacuum preparation chamber attached to the microscope column which allows serial fracture, low-magnification stereo light microscopy, radiant etching, and evaporative coating with gold or carbon. The stage can be tilted from 0° to 45° and rotated a full 360°. It is demonstrated that the air-liquid interface in the lung can be examined and that low-temperature SEM can be used to investigate the shape of alveoli, the patency of the pores of Kohn in the hydrated state, and the shrinkage and distortion of lung with drying.     

Electron-specimen interactions in low-voltage scanning electron microscopy

Measurements of the electron range R, and the backscattering coefficient η and the secondary electron yield δ at normal and tilted incidence for different elements show characteristic differences for electron energies in the range of 0.5 to 5 keV, compared with energies larger than 5 keV. The backscattering coefficient does not increase monotonically with increasing atomic number; for example, the secondary electron yield shows a lesser increase with increasing tilt angle. This can be confirmed in back-scattered electron (BSE) and secondary electron (SE) micrographs of test specimens. The results are in rather good agreement with Monte Carlo simulations using elastic Mott cross-sections and a continuous-slowing-down model with a Rao Sahib-Wittry approach for the stopping power at low electron energies. Therefore, this method can be used to calculate quantities of BSE and SE emission, which need a larger experimental effort. Calculations of the angular distribution of BSEs show an increasing intensity with increasing atomic number at high takeoff angles than expected from a cosine law that describes the angular characteristics at high electron energies. When simulating the energy distribution of BSEs, the continuous-slowing-down model should be substituted by using an electron energy-loss spectrum (EELS) that considers plasmon losses and inner-shell ionizations individually (single-scattering-function model). The EELS can be approached via the theory for aluminium or from EELS spectra recorded in a transmission electron microscope for other elements. Measurements of electron range Rα Eⁿ of 1 to 10 keV electrons are obtained from transmission experiments with thin films of known mass thickness. In agreement with other authors the exponent n is lower than at higher electron energies.     

Charging control using pulsed scanning electron microscopy

This paper describes a novel method to observe highly charging specimens at high-beam voltages without specimen preparation. It is found that the technique greatly reduces charging artifacts such as image shift, astigmatism, and defocussing without sacrificing image quality. Images obtained of uncoated specimens are found to be comparable to gold-coated specimens and without exhibiting charging effects. The technique also allows the study of charge distribution effects in specimen charging of which very little understanding exists, particularly as far as the spatial and time-dependent properties of charging are concerned.     

Cryogenic‐temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids

Cryogenic electron microscopy (cryo‐EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo‐TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo‐SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo‐EM involve either water or organic components. In this paper, we introduce the use of novel cryo‐TEM and cryo‐SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single‐walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo‐TEM and cryo‐SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid‐crystalline CNT phases, used as the ‘dope’ for all‐carbon‐fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self‐assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.