Imaging the cell surface: argon sputtering to expose inner cell structures

Established microscopies such as Scanning Electron Microscopy (SEM) and more recent developments such as Atomic Force Microscopy (AFM) and X-ray Photo-Electron Emission spectroMicroscopy (X-PEEM) can only image the sample surface. We present an argon sputtering method able to progressively expose inner cell structures without apparent damage. By varying the sputtering time, the structure of cell cytoskeleton, vesicles, mitochondria, nuclear membrane, and nucleoli can be imaged. We compared images obtained with confocal fluorescence microscopy, transmission electron microscopy (TEM), SEM, and X-PEEM on similar samples after argon sputtering, then confirmed the similarity of reference intracellular structures, including cytoskeleton fibers, cell-cell and cell-substrate adhesion structures, and secretory vesicles. We conclude that the sputtering method is a new valuable tool for surface sensitive microscopies.     

Confocal microscopy and image processing in the study of plant nuclear structure

We describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide-field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three-dimensional (3-D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3-D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.     

Electron microscopy of squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck carries a bad prognosis. In order to achieve cure, the most important thing to attain is local tumour control. The main therapy available is external radiotherapy, which can be supplemented when necessary, with interstitial radiotherapy, chemotherapy and surgery. In this paper we have evaluated specimens, taken before therapy, from 35 patients with squamous cell carcinoma of the head and neck. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were made. With SEM, the parameters analysed were the amount and appearance of microvilli, filaments, and blood vessels. From TEM, scoring was made of the filaments, desmosomes, nuclei, nucleoli, mitochondria, and blood vessels. Scoring of the samples showed a difference between the group with recurrent disease (n = 10, Group 1) and the group with local tumor control (n = 25, Group 2) in regard to both blood vessels and intracellular filaments. No differences of the nuclei, nucleoli, or the mitochondria were observed.     

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.     

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.     

Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning electron microscopy

Scanning electron microscopy (SEM) has produced a wealth of novel images that have significantly complemented our perception of biological structure and function, derived initially from transmission electron microscopy (TEM) information. SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by reduced resolution in comparison with TEM. Recently, SEM resolution has been considerably improved by the advent of high-brightness sources used in field-emission instruments (FEISEM) which have produced resolution of around 1 nm, virtually equivalent to TEM “working resolution.” Here we review our findings in the use of FEISEM in the imaging of nuclear envelopes and their associated structures, such as nuclear pore complexes, and the relationships of structure and function. FEISEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance.     

A rapid and simple technique for correlating light microscopy,transmission and scanning electron microscopy of fixed tissues in Epon blocks

A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33μm/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.     

Human chromatid ultrastructure: new observations with scanning and transmission electron microscopy

Two new observations have been made on human chromatid/chromosome ultrastructure using both scanning and transmission electron microscopy (SEM, TEM). A bipartite, apparently half-chromatid-like structure was observed in whole human chromosomes studied with SEM and in longitudinally sectioned chromosomes analyzed with TEM. In addition, we also observed a zipper-like configuration as the parallel sister chromatids separated likely due to the supercoiled structure of the chromosome and chromatid. It is possible that either or both of these new observations resulted from our (improved) method of preparing the chromosomes for SEM and TEM.     

Computer reconstruction of nucleolar architecture by interactive three-dimensional color display

The first complete three dimensional ultrastructural reconstruction of pancreatic cell nucleoli, was done using EM and computer 3D-assisted reconstruction of serial sections with interactive 3D back-to-front and color display methods based on voxel representation. The purpose of the study was to depict the architecture of the nucleolar components. We obtained information about the location of the nucleolus within the nuclear volume and about the shape and polarity of the 3 main nucleolar territories.     

Glassy cholesteric structure: thickness variation induced by electron radiation in transmission electron microscopy investigated by atomic force microscopy.

During the observation of glassy cholesteric liquid crystals in transmission electron microscopy (TEM), a new contrast is created or enhanced by electron radiation which has a direct relationship with the periodic microstructure of the specimen. In this paper, we investigate the variations of the sample thickness and mass density as possible causes of this irradiation contrast. By means of observations in atomic force microscopy (AFM) coupled to TEM, we compared the surface corrugations of non-irradiated and irradiated specimens. It is shown that the final contrast is the result of several processes. including fracture during ultramicrotomy and mass loss during irradiation. Mass loss acts as an etching, and hence results in a decrease of the sample thickness. The etching depends on the initial molecular orientation, thus evidencing the latent structure. An electron channelling mechanism is suggested to explain this behaviour.     

Rapid diagnosis of fungal infection of intravascular catheters in newborns by scanning electron microscopy.

Intravascular catheters carry a significant risk of becoming colonized with bacteria and fungi and are important risk factors of septicemia in premature neonates. The study was undertaken to evaluate whether scanning electron microscopy (SEM) examination of removed catheters can be useful in early diagnosis of plastic infection by Candida, providing information useful for initiation of an eventual therapy. The evolution of biofilms in 28 catheters (umbilical or central) implanted in 24 newborns for prematurity was studied by SEM and transmission electron microscopy (TEM). In 4 of 24 patients, SEM examination revealed the presence of Candida in form of yeast or hyphae. In one of these patients, TEM confirmed the presence of organisms. In each case, hemoculture and culture of the catheter itself confirmed the diagnosis. The study demonstrates that SEM can identify fungi in the biomaterials covering the catheter surface in a few hours, allowing an early diagnosis of plastic infection.     

Scanning electron microscopy of rust haustoria

The three-dimensional structure of rust haustoria has been studied by scanning electron microscopy (SEM). Rust infected leaves were fixed and embedded in Spurr's resin as for transmission electron microscopy (TEM). The cut surface of blocks containing embedded material was etched with sodium ethoxide and examined in the SEM.     

Wave field restoration using three-dimensional Fourier filtering method.

A wave field restoration method in transmission electron microscopy (TEM) was mathematically derived based on a three-dimensional (3D) image formation theory. Wave field restoration using this method together with spherical aberration correction was experimentally confirmed in through-focus images of amorphous tungsten thin film, and the resolution of the reconstructed phase image was successfully improved from the Scherzer resolution limit to the information limit. In an application of this method to a crystalline sample, the surface structure of Au(110) was observed in a profile-imaging mode. The processed phase image showed quantitatively the atomic relaxation of the topmost layer.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Scanning electron microscopy observation of the substructural evolution of aluminium alloys during cold rolling and partial annealing

Classical etching techniques for revealing cold deformation and partial recrystallisation in metals have been optimised for optical microscopy, which is limited by its resolution. Detailed studies of the mechanisms involved in recovery and recrystallisation during heat treatment are generally made by transmission electron microscopy. The limitation of this technique, with a few exceptions, is its small field of view and the small fraction of the sample available for inspection. The present article departs from the statement that etching, which is a surface alteration technique, must have effects that are detectable by scanning electron microscope (SEM). It was found that carefully adapted polishing and etching procedures allow for substructural investigations by SEM, resulting in various advantages compared with both optical microscopy and TEM.     

Correlative scanning-transmission electron microscopy of the primate infundibular recess.

This report describes a basic techinque for the analysis of subhuman primate brains with correlative scanning (SEM) and transmission electron microscopy (TEM) of the same tissue sample. The methodology is described in detail and this report characterizes the type II supraependymal histiocyte.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy,scanning electron microscopy,and transmission electron microscopy

The polyethylene glycol (PEG) embedding and subsequent deembedding method was applied to the observation of general tissues in scanning electron microscopy (SEM). Resulting SEM images were of high quality. It was demonstrated that intermicroscopic correlation of images between light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) is easily and reliably done by means of the PEG method. In particular, the exact correlation of immuno-LM with SEM is shown to be of potential value.     

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM – SEM)

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.