Transmission And Scanning Electron Microscope Preparations Of The Same Cell Culture

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

Modification of a scanning transmission electron microscope specimen holder for large section viewing.

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter "giant" grids is explained and the procedure for sample preparation is outlined. The modification aids the microscopist in his evaluation of tissue structural relationships by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm2 electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.     

Ultrastructure of cells cultured on polycarbonate membranes.

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 micrometer or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Sequential scanning electron microscopy of a growing plant meristem

Summary A two-step replica technique has been developed for sequential study of the epidermal cell pattern of a living plant by scanning electron microscopy. This method is nondestructive, allows periodic high resolution observation of the same developing tissue, and can precede use of any destructive technique, such as transmission electron microscopy. The replicas can be trimmed allowing observation of occluded surfaces, such as the areas between leaves, which are inaccessible in continuousin vivo studies. Here we study the developing leaf primordium ofGraptopetalum and discuss potential uses of the technique.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Scanning probe recognition microscopy investigation of tissue scaffold properties

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.     

A note on the development of a non-aldehyde fixation technique for examining spores of Bacillus subtilis under the electron microscope

Various techniques were studied for fixing spores of Bacillus subtilis prior to examining them by transmission or scanning electron microscopy. A non-aldehyde technique employing carbodiimide in cacodylate buffer produced excellent results and could be of value in studying the cytological changes produced in spores exposed to inimical treatments.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Colloidal gold, a useful marker for transmission and scanning electron microscopy.

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.     

Correlation of Scanning and Transmission Electron Microscopy on the Same Tissue Sample

Tissue processed for scanning electron microscopy (SEM) in a critical point bomb utilizing Freon-13 showed excellent subsurface preservation when prepared for transmission electron microscopy (TEM). The critical point method is the only commercially available SEM preparation technique in which the quality of preservation will not limit microscopists in efforts to correlate SEM and TEM observations.     

Bovine olfactory and nasal respiratory epithelium surfaces

Summary High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 m using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12–13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.     

Back-Scattered Electron Imaging of Sections Through the Cochlea: A New Technique for Studying Cochlear Morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Back-scattered electron imaging of sections through the cochlea: a new technique for studying cochlear morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Ponceau-Fuchsin as a Routine Countebstain

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Ultrastructural changes in murine lymphocytes induced by aflatoxin B1

This investigation sought to determine whether splenic lymphocytes obtained from Balb/C mice exposed to aflatoxin B1 (AFB1) showed any ultrastructural changes which could account for the immunodysfunction attributable to aflatoxins. Lymphocytes obtained from Balb/C mice administered aflatoxin B1 in olive oil daily for three weeks were studied using both transmission and scanning electron microscopy. The lymphocytes demonstrated ultrastructural changes primarily in the mitochondria where marked internal dissociation of the cristae was revealed by transmission electron microscopy. All other cellular organelles were unaffected. No significant alterations in external structure were observed under scanning electron microscopy. The findings of this study indicate that AFB1 administration does not affect the surface topography of lymphocytes, but AFB1, by causing extensive mitochondrial damage, may affect the way in which these cells function. This could be a possible explanation for the immunodysfunction associated with AFB1.Abbreviations AFB1 Aflatoxin B1 - SEM scanning electron microscopy - TEM transmission electron microscopy     

Versatile Method for Evaluating a Cell Culture by Various Morphological Techniques

Cellulose acetate is a versatile material for evaluating cells grown under identical conditions by various morphological techniques. This inexpensive material is transparent, easily cut to size and shape, nontoxic to cell cultures, and resistant to most chemicals used in histochemistry and in scanning and transmission electron microscopy. Samples may be obtained during and after the culture process. Cellulose acetate slides can be mounted directly over glass slides for direct observation and are easily peeled off plastic blocks for electron microscopy, leaving the cells behind. Relative disadvantages include its autofluorescence and a tendency to soften in strong acids or pure solutions of organic solvents such as xylene and propylene oxide.     

Preparation of Fossil Bone Specimens for Scanning Electron Microscopy

A method is described for preparing fossil bone specimens for scanning electron microscopy. To obtain bone surfaces suitable for study, material was embedded in Epon 812 and selected faces exposed by grinding were subjected to controlled etching with a 4:1 mixture of 5% HNO₃ and 1% OsO₄, Surfaces thus prepared were further processed by the so-called clearing replicas technique. As a result of this procedure the bone surfaces revealed a network of anastomosing vascular canals the inner surface of whose walls could be examined in the scanning electron microscope. By etching extremely thin ground sections of bone stuck to plastic tape the contents of vascular canals as well as osteocytes can be isolated. This method ensures the good preservation of spatial relations between bone elements essential for studies of fossil bones, which an sometimes very brittle.