Freeze-drying process of biological specimens observed with a scanning electron microscope

A pit membrane was observed with a cryo-SEM during the course of dehydration at low temperatures. The freeze-drying process of sea-urchin eggs and parenchyma cells of higher plants was also examined with this microscope. Conditions for observation of frozen specimens in the native state were discussed on the basis of morphological studies of alterations such as shrinkage or deformation which appeared during the freeze-drying process.     

Double-layer coating for field-emission cryo-scanning electron microscopy—present state and applications

Imaging of fast-frozen samples is the most direct approach for electron microscopy of organic material. It prevents chemical fixation and drying artifacts. Frozen samples can be replicated and imaged in the transmission electron microscope (TEM), or they can be directly visualized in the cryo-scanning electron microscope (cryo-SEM). Double-layer coating combines these two techniques and many of their advantages. With this method, the frozen bulk sample is coated similar to the TEM-replica technique with, for example, a shadow of platinum (at an angle of 45°) and an additional layer of carbon. Then, the sample is cryo-transferred to an SEM equipped with a cold stage and imaged with the material-dependent backscattered electron signal that shows the platinum distribution. With this method, charging artifacts and the effects of beam damage are significantly reduced. Although currently the resolution of the replica technique cannot be surpassed, the method greatly facilitates the processing of brittle, rapidly frozen samples because no replica cleaning is necessary. This makes the method especially suitable for high-pressure frozen samples.     

Cryoscanning electron microscopy of microbial extracellular polysaccharides and their association with minerals

The morphology of pure microbial extracellular polysaccharides (EPS) and of their association with a mineral was studied by scanning electron microscopy. Several methods were compared: cryo-SEM, critical point dehydration (CPD), and TEM observation after solvent exchanges and inclusion in a resin. The EPS were observed as fibrils, interconnected into a network. Their dimensions were compared with literature data on the conformation and shape of the given EPS. It was deduced that these EPS have an undoubtably fibrillar structure when hydrated, and that some aggregation or collapse artifacts took place in both CPD or cryo-SEM samples but were quite limited. Cryo-SEM appears then as a relevant tool for the study of the secretion of EPS by microorganisms in soils.     

Light,scanning, and transmission electron microscopy of a single population of detergent-extracted cardiac myocytes in vitro

A technique for performing light, scanning, and transverse transmission electron microscopy on cultured cells grown within a single tissue culture flask is described. Permanent light microscopy slides are obtained by removing selected portions of the plastic tissue culture vessel and mounting them on glass slides with an aqueous mounting solution. The images obtained from these slides are superior to viewing through the bottom of the flask with an inverted stage microscope. For scanning electron microscopy, selected areas are also cut from the remainder of the vessel and prepared for viewing. The final portion of the culture container is transferred and attached to a new tissue culture vessel and prepared for transmission electron microscopy using alcohol instead of acetone and propylene oxide during dehydration, infiltration, and embedding.     

Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs

The ultrastructures of novel threadlike structures (NTSs) and corpuscles on the surfaces of internal organs of rats were investigated using electron microscopy. The samples were studied in situ by using a stereomicroscope and were taken for further morphological analysis. Scanning electron microscope (SEM) images revealed a bundle structure of threadlike tissue, which was composed of several 10-micro m-thick subducts. The surfaces of the corpuscles were rather coarse and fenestrated. The corpuscles had cucumber-like shapes with an average length of about 2 mm and a thickness of about 400 micro m. Transmission electron microscope (TEM) images disclosed disordered collagen fibers, which formed the extracellular matrix of the threadlike tissue, and immune-function cells, like macrophages, mast cells, and eosinophils. Sinuses of various diameters, which were thought to be cross-sections of the lumens of the subducts, were observed in the TEM, cryo-SEM and focused-ion-beam SEM images. These SEM images were obtained for the first time to reveal the detailed structure of the NTSs that were only recently discovered.     

Examination of occlusions in xylem vessels of cut rose flowers,using cryoscanning electron microscopy and cryoultramilling cross-sectioning

The occlusions occurring in xylem vessels of cut rose flowers (Rosa hybrida L.) were examined using freeze fracturing and conventional SEM, or cryoultramilling cross-sectioning followed by cryo-SEM. The study provides an excellent example of the utility of the latter procedure. The stems contained bacteria surrounded by amorphous material. The amorphous material, which was likely bacterial slime, was more abundant in cryo-SEM. The ultramilling method resulted in smooth cross-sectioning of the xylem walls. The results show that bacteria do not degrade the xylem walls.     

Visualisation of microalgal lipid bodies through electron microscopy

In this study, transmission electron microscopy (TEM) and cryo-scanning electron microscopy (cryo-SEM) were evaluated for their ability to detect lipid bodies in microalgae. To do so, Phaeodactylum tricornutum and Nannochloropsis oculata cells were harvested in both the mid-exponential and early stationary growth phase. Two different cryo-SEM cutting methods were compared: cryo-planing and freeze-fracturing. The results showed that, despite the longer preparation time, TEM visualisation preceded by cryo-immobilisation allows a clear detection of lipid bodies and is preferable to cryo-SEM. Using freeze-fracturing, lipid bodies were rarely detected. This was only feasible if crystalline layers in the internal structure, most likely related to sterol esters or di-saturated triacylglycerols, were revealed. Furthermore, lipid bodies could not be detected using cryo-planing. Cryo-SEM is also not the preferred technique to recognise other organelles besides lipid bodies, yet it did reveal chloroplasts in both species and filament-containing organelles in cryo-planed Nannochloropsis oculata samples.     

Localized grounding, excavation, and dissection using in-situ probe techniques for focused ion beam and scanning electron microscopy: experiments with rock varnish

While investigating rock varnish, we explored novel uses for an in‐situ micromanipulator, including charge collection, sample manipulation, as well as digging and dissection at the micron level. Dual‐beam focused ion beam microscopes (DB‐FIB or FIBSEM) equipped with micromanipulators have proven to be valuable tools for material science, semiconductor research, and product failure analysis. Researchers in many other disciplines utilize the DB‐FIB and micromanipulator for site‐specific transmission electron microscope (TEM) foil preparation. We have demonstrated additional applications for in‐situ micromanipulators. SCANNING 34: 279–283, 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of polyox ® granules using environmental scanning electron microscopy

The scanning electron microscope (SEM) has been frequently used in the pharmaceutical industry for studying pharmaceutical products. However, the technique does not allow for the continuous characterization of a product in both dry and hydrated states without processing the product. Through the recent advent of the environmental scanning electron microscope (ESEM), it is now possible to observe a sample in both the dry and hydrated states without extensive product preparation. The ESEM also allows for continual observation during the hydration process from the dry state until the sample is dissolved. In this study, the ESEM was used to characterize the morphologic differences and hydration patterns of granules formulated with a water-soluble hydrophilic swelling polymer, Polyoxy. Two molecular weights (1, 000, 000 and 7, 000, 000) of the polymer were used in concentrations ranging from 10 to 25% w/w. Visual differences in granule surface morphology and differences in hydration patterns were seen in granules prepared from different polymer concentrations. The morphologic data were corroborated by surface area measurements taken on a surface area analyzer. The rate at which the granule dissolved in the ESEM also correlated with the drug dissolution times determined by the standard USP dissolution method.     

A simple cryo-holder facilitates specimen observation under a conventional scanning electron microscope

A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories.     

Cryo-SEM method for the observation of entrapped bubbles and degree of water filling in large wet powder compacts

There are generally two problems associated with cryogenic scanning electron microscopy (cryo-SEM) observations of large wet powder compacts. First, because water cannot be vitrified in such samples, formation of artefacts is unavoidable. Second, large frozen samples are difficult to fracture but also to machine into regular pieces which fit in standard holders, especially if made of hard materials like ceramics. In this article, we first describe a simple method for planning hard cryo-samples and a low-cost technique for cryo-fracture and transfer of large specimens. Subsequently, after applying the entire procedure to green pellets of iron ore produced by balling, we compare the influence of plunge- and unidirectional freezing on large entrapped bubbles throughout the samples as well as the degree of water filling at the outer surface of the pellets. By carefully investigating the presence of artefacts in large areas of the samples and by controlling the orientation of the sample during freezing and preparation, we demonstrate that unidirectional freezing enables the observation of large entrapped bubbles with minimum formation of artefacts, whereas plunge freezing is preferable for the characterization of the degree of water filling at the outer surface of wet powder compacts. The minimum formation of artefacts was due to the high packing density of the iron ore particles in the matrix.     

Combining Ar ion milling with FIB lift-out techniques to prepare high quality site-specific TEM samples

Focused ion beam (FIB) techniques can prepare site‐specific transmission electron microscopy (TEM) cross‐section samples very quickly but they suffer from beam damage by the high energy Ga⁺ ion beam. An amorphous layer about 20–30 nm thick on each side of the TEM lamella and the supporting carbon film makes FIB‐prepared samples inferior to the traditional Ar⁺ thinned samples for some investigations such as high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). We have developed techniques to combine broad argon ion milling with focused ion beam lift‐out methods to prepare high‐quality site‐specific TEM cross‐section samples. Site‐specific TEM cross‐sections were prepared by FIB and lifted out using a Narishige micromanipulator onto a half copper‐grid coated with carbon film. Pt deposition by FIB was used to bond the lamellae to the Cu grid, then the coating carbon film was removed and the sample on the bare Cu grid was polished by the usual broad beam Ar⁺ milling. By doing so, the thickness of the surface amorphous layers is reduced substantially and the sample quality for TEM observation is as good as the traditional Ar⁺ milled samples.     

The correlation of light and electron microscope observations

A technique is described to allow electron microscopic investigation of a specific feature of a section on a glass slide. A section on a glass slide (previously treated with a silicone release agent) is processed as required for light microscopy. The section is then impregnated with Araldite and cured with an epoxy resin block on top of it. The section and block are removed from the slide and viewed with a light microscope. The selected area for ultrastructural study remains under continuous observation while the block is trimmed. Semi-thin (1 μm) sections retain the original staining for light microscopy and ultra-thin sections are stained with heavy metals in the normal manner. We show how an inflammatory lesion in a large area of muscle in a case of polymyositis may be quickly located and studied at the ultrastructural level.     

Ultramicrotomy in the ESEM, a versatile method for materials and life sciences

We here present the results of the first materials science analyses obtained with the prototype of a serial block-face sectioning and imaging tool, 3View™ of Gatan, Inc (Pleasanton, CA, U.S.A.). It is a specially designed ultramicrotome operating in situ within an environmental scanning electron microscope originally developed for life science research. The microtome removes thin slices from the sample and the environmental scanning electron microscope images each new block surface of the specimen (serial block-face scanning electron microscopy). The Schottky emitter (FEG) of the microscope delivers high spatial resolution and has the advantage of stable performance and high durability. The slice thickness can typically be selected between 50 and 100 nm. It is possible to cut hundreds of slices and simultaneously acquire images with Digital Micrograph™ Model 700 (Gatan, Inc.). This article outlines the set-up and describes the automated process. The preparation of specimens for in situ ultramicrotomy is explained and the parameters for good image quality are discussed. In addition, special operative and analytic features of the controlling software are presented. Three different technical materials and one botanical specimen were analyzed delivering first results of this method for materials science and for botany.     

Cryo-planing for cryo-scanning electron microscopy

In the past decade, investigators of cryo-planing for low-temperature scanning electron microscopy (cryo-SEM) have developed techniques that enable observations of flat sample surfaces. This study reviews these sample preparation techniques, compares and contrasts their results, and introduces modifications that improve results from cryo-planing. A prerequisite for all successful cryo-planing required a stable attachment of the specimen to a holder. In most cases, clamping with a screw mechanism and using indium as space-filler sufficed. Once this problem was solved, any of three existing cryo-planing methods could be used to provide successful results: cryo-milling, microtomy in a cold room, and cryo-ultramicrotomy. This study introduces modifications to the cryo-planing technique that produces flat surfaces of any desired plane through a specimen. These flat surfaces of frozen, fully hydrated samples can be used to improve observations from cryo-SEM as well as to enhance results from x-ray microanalysis and (digital) image analysis. Cryo-planing results of chrysanthemum (Dendranthema x grandiflorum Tzvelev) stems, hazel (Corylus avelane L.) stems, and repeseed (Brassica napus L.) pistils are presented to illustrate the use of the planing method on fibrous, hard, and delicate materials, respectively.     

Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy

We report a simple methodology to provide complete pulse characterization at the sample plane of a two-photon excited fluorescence (TPEF) microscope. This is achieved by using backward propagating second-harmonic generation (SHG) from starch granules. Without any modification to the microscope, SHG-autocorrelation traces were obtained by using a single starch granule that was placed alongside the biological specimen being imaged. A spectrally resolved SHG autocorrelation was acquired by placing a spectrometer at the output port of the microscope. Complete in situ pulse information is then directly retrieved in an analytical way using the measurement of electric filed by interferometric spectral trace observation (MEFISTO) technique.     

Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

Development of high-resolution field emission scanning electron microscopy with multifunctions for chargeless observation of nonconducting surface

We have developed a fully digital field emission scanning electron microscope (FE-SEM) with multifunctions to compensate the charging up of nonconducting surfaces. High-voltage observation, minimum electron dose, variable scanning speed, averaging, integration, tuning of surface potential, and cyclotron movements of secondary electrons have been achieved. This FE-SEM was successfully applied to observe resist, diatomaceous earth, aluminum oxide, and zeolite surfaces. The accelerating voltage is changeable in a range from 0.5 to 30 kV, and the probe current on the sample can be varied from 2×10^-9 to l×10^-13A to supply optimum electron dose. By using a snorkel- type, strongly excited objective lens (OL) immersing the samples in the magnetic field, the secondary electrons are extracted from the sample. For guiding electrons into the built-in lens-type secondary electron detector (SED), newly developed accelerating and retarding electrodes are installed in the OL to tune the surface potential. Furthermore, this FE-SEM can select 10 scan speeds, and the averaging and integration of secondary electron image signals are possible under every selected scan speed.