Scanning electron microscopy of plasma etched implant specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Quantitative analysis of sputtering due to ion beam bombardment of solids and biological specimens in high resolution electron microscopy

When a positively charged ion beam is used to bombard a solid target, most of the atoms are displaced and sputtered according to the atomic sputtering theory. In the case of biological specimens, most of the bond-breaking molecules in proteins are removed, when based on the molecular sputtering theory. It was found that the thinning rate for solids and the etching rate for biological specimens, when prepared by a normal double fixation and staining method, can be measured from the sputtering yield and density of the specimens. It was also found that the thinning and etching rates depend on the removal weight per sublimation energy and bonding energy, respectively. The angular distribution of sputtering yield, its dependence on incident angle and the secondary electron emission yield were measured, and the optimum etching condition of the incidence was obtained. Experiments showed that the in situ observation of intracellular structures of biological specimens prepared by ion beam etching can be a very effective method in electron microscopy.     

Negative staining studied by TEM and STEM mass measurements: Preliminary observations on collagen sheets negatively stained with uranyl acetate

Quantitative mass measurements by dark-field scanning transmission EM and conventional bright-field transmission EM have been used to determine the increase in mass brought about by negative staining. pN-collagen (which forms sheets of uniform thickness and known mass per unit area) was used as a test specimen; the negative stain was uranyl acetate (1%, pH 4.4). The mass increase corresponded to the addition of roughly 8 uranyl acetate molecules per nm² for lightly negatively stained specimens; for heavily stained specimens, it was 30 molecules or more. The appearance of the image was related to the mass increase. This preliminary study shows that mass measurements can provide a basis for the quantitative interpretation of images from negatively stained specimens.     

A contamination reducing method by ion beam bombardment of the specimen in high resolution electron microscopy

The theory of contamination and a contamination reducing method are discussed on the basis of time dependent micrograph series and their tilted images for determining contamination.For a high current density of an electron probe in the field emission scanning electron microscope, it is observed that contaminated cones are formed in proportion to the exposure time of an electron beam. From the measurement of the contamination layer thickness and its area, the contamination rate and time dependent shape are formulated, mainly depending on the cross-section and current density together with the average lifetime of adsorption molecules.It is found that the contamination rate and stray contamination of outgassing molecules forming part of the specimen are effectively reduced by a pre-bombardment of argon ions on the surfaces of specimens. The contamination rate is reduced to a small extent (5%) using the present method.     

The ups and downs of beam damage,contrast and noise in biological electron microscopy

A personal account of the early problems associated with contrast in images obtained by electron microscopy from biological specimens is presented, together with the effects of electron beam damage. The author's experiences with different types of electron microscope as well as problems of contrast enhancement is described. A short account is given of the physical effects occuring during specimen preparation and their relation to structural preservation when attempting to achieve atomic resolution. Recent developments in biological electron microscopy are also discussed with a view to future trends.     

Observations on charging effects of non-conductive and uncoated materials by ion beam pre-bombardment in scanning electron microscopy

The specimen charging defects of non-conductive materials in scanning electron microscopy are discussed with reference to the surface electric field generated by the illuminating electron beam dose. If the charge density depends on the relaxation time constant as defined by a product of the permittivity and resistivity when known or available, the electric field can be evaluated by the incident dose stored when illuminated by an electron scanning beam.It was found by observation that uncoated or non-conductive materials pre-bombarded by a positive ion beam, which contributes to the generated negative field, together with the charging effects, could be eliminated at the optimum time of neutralization.In the normal process of double fixation and staining of biological specimens, the local electric field produces increased contrast due to polarization effects. The dark and bright images of secondary and backscattered electrons, respectively, can be analysed by taking into account local polarization, in addition to voltage contrast.     

Radiation damage effects at four specimen temperatures from 4 to 100 K

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100 K. For each temperature, “exposure series” were collected consisting of consecutive images of the same area of sample, each with 10 e^?/Ų exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0–173.5 Å). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100 K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42 K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.     

Scanning Electron Microscopy of Plasma Etched Implant Specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 µl;m histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Scanning transmission electron microscopy of biological structures

The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen. Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies. Later the real applications have not followed the initial hopes. The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze-dried isolated macromolecules to unstained cryosections. Emphasis will be put on the mass-mapping, multi-signal and elemental mapping modes which are unique features of the STEM instruments.     

Applications of high voltage electron microscopy to botanical ultrastructure

Although high voltage electron microscopes have been in general use over the past decade microscopists have tended to ignore the contribution their use could make to the study of plant ultrastructure. The majority of biological high voltage research has been restricted to the fields of zoology and bio-medicine.The high voltage electron microscope (HVEM) has several advantages over the conventional transmission electron microscope (CTEM) when applied to biological specimens. These include increased penetrating power of the electron beam, reduced chromatic abberation in thick specimens, and both reduced beam heating and ionization damage. All these factors permit the observation of thick sections, whole cells and hydrated specimens. Most botanical HVEM research has been restricted to the study of thick sectioned material. Various staining techniques have been applied to overcome the decrease in image contrast at high accelerating voltages, but the commonest have been modifications of lead and uranium stains previously developed for thin sections. Selective staining can simplify the mass of information in a thick specimen thus specific structures may be studied against an unstained background. Acidified phosphotungstic acid can be used to stain the plasma membrane and osmium impregnation will selectively stain many of the cytoplasmic membranes in a variety of specimens. Other techniques for the selective localization of cell components, such as enzyme cytochemistry and autoradiography have yet to be fully exploited by high voltage electron microscopists.Interpretation of the great quantity of information in a thick specimen can be facilitated by tilting the specimen and producing stereo pairs. Quantitative depth information can be extracted from stereo pairs by the use of measuring mirror stereoscopes or by direct measurement from each member of a stereo pair. Serial thick sectioning has been employed as an alternative to prolonged serial thin sectioning to aid in the reconstruction of large specimens.Stereo images can be viewed in a variety of ways with lenticular pocket stereoscopes, reflecting mirror stereoscopes, prismatic spectacles, polarized spectacles when projected onto a non depolarizing screen or presented on TV monitors.     

A scanning electron microscope technique for restoring deformed fossils

Deformed fossils which originally had a regular geometric outline, such as crinoid columnals, can be restored using a scanning electron microscope technique called tilt correction. This magnifies the specimen in one direction only. It is unsuitable for specimens where deformation has occurred unevenly and can also distort small structures while restoring the whole specimen. D Crinoid columnals, scanning electron microscope, tilt correction.     

Use of confocal laser scanning microscopy in systematics of insects with a comparison of fluorescence from different stains

Abstract Confocal laser scanning microscopy has become a valuable tool for a wide range of investigations in the biological sciences, but its use in insect systematics has been neglected. Confocal microscopy depends on the degree of fluorescence of the examined specimens, which is aided either by fluorescent dyes or autofluorescence of the specimen. This study provides methods for using a combination of fluorescent dyes and autofluorescence to provide images that document the value of confocal microscopy for systematic research with insects. Fluorescence was compared from Lepidoptera genitalia dissections that were unstained or stained with merbromin (mercurochrome), safranine, chlorazol black E, eosin Y, eosin Y + chlorazol black E, and orange‐G. The unstained specimen showed that chitin autofluorescences to a small degree. The comparison of stains showed that use of eosin Y provides the best images, followed by safranine and mercurochrome. Orange‐G and chlorazol black are the least fluorescent and provide poor images, even when chlorazol black is combined with eosin.     

Individual filamentous phage imaged by electron holography

An in-line electron hologram of an individual f1.K phage was recorded with a purpose-built low energy electron point source (LEEPS) microscope. Cryo-microscopic methods were employed to prepare the specimen so that a single phage could be presented to the coherent low energy electrons: An aqueous phage suspension was applied to a thin carbon membrane with micro-machined slits. The membrane was rapidly cooled to freeze the remaining water as an amorphous ice sheet, which was then sublimated at low temperatures and pressures to leave individual free-standing phages suspended across slits. An image of a phage particle, depicted as the amplitude of the object wave, was reconstructed numerically from a digitized record of the hologram, obtained using 88 eV coherent electrons. The reconstructed image shows a single phage suspended across a slit in a supporting carbon membrane, magnified by a factor of 100,000. The width and shape in the reconstructed image compared well with a TEM image of the same filament. It is thus possible to record and reconstruct electron holograms of an individual phage. The challenge now is to improve the resolution of reconstructed images obtained by this method and to extend these structural studies to other biological molecules.     

High resolution scanning electron microscopy of the cell

The scanning electron microscope (SEM) has become a powerful tool for ultrastructural research with improvement of the instrument's resolution and progress in specimen preparation techniques. With regard to resolution, it has been improved step-by-step in this decade and, in 1985, an ultra-high resolution SEM (UHS-T1) was developed, with a resolution of 0.5 nm. Concerning specimen preparation, the osmium-DMSO-osmium method, which is effective for revealing intracellular structures, has come to be widely used. Techniques for observing smaller objects, such as bacteriophages, viruses, and biological macromolecules, have also been devised in recent years. As a result of these preparation techniques and the availability of the ultra-high resolution SEM, the application of SEM in biology is expanding rapidly. In this paper, an outline of the ultra-high resolution SEM, techniques for specimen preparation, findings of some biological materials by these techniques, and guidelines to making the specimens, are described.     

High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy

Scanning electron microscopy (SEM) is an important tool for the nanometre-scale analysis of the various samples. Imaging of biological specimens can be difficult for two reasons: (1) Samples must often be left unstained to observe detail of the biological structures; however, lack of staining significantly decreases image contrast. (2) Samples are prone to serious radiation damage from electron beam. Herein we report a novel method for sample preparation involving placement on a new metal-coated insulator film. This method enables obtaining high-contrast images from unstained proteins and viruses by scanning electron microscopy with minimal electron radiation damage. These images are similar to those obtained by transmission electron microscopy. In addition, the method can be easily used to observe specimens of proteins, viruses and other organic samples by using SEM.     

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM

It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST elastic scattering cross section database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy-metal clusters used to label intracellular proteins. In sectioned cells that have been stained only with osmium tetroxide, we find an average of 1.2+/-0.1 Os atom per nm(3), corresponding to an atomic ratio of Os:C atoms of approximately 0.02, which indicates that small heavy atom clusters of Undecagold and Nanogold can be detected in lightly stained specimens.     

Effect of specimen size on work-of-fracture measurements

It has been suggested that work-of-fracture, which quantifies the ability of a material to resist fracture, is dependent on specimen size. This experiment compared work-of-fracture, calculated as energy per unit area, for different specimen sizes of Plexiglas, bovine tibial bone and aluminum. Three different geometrically similar cross sections were tested for each material for a total of 54 specimens. Work-of-fracture was measured by loading a notched beam (triangular cross section) in three-point bending at a constant deformation rate. The energy necessary to cause fracture was measured from a load-deformation curve. Specimen fracture area was determined using macrophotography. Atomic absorption spectrophotometry was used to determine weight percent calcium of bone specimens and quantitative light microscopy was used to determine fractional void area. Analysis of variance showed no effect of specimen size on work-of-fracture for aluminum or Plexiglas specimens (p greater than 0.05). A significant difference was found, however, between the large (area = 11.7 +/- 1.9 mm2) and small (area = 3.48 +/- 0.68 mm2) bone specimens and between the medium (area = 5.89 +/- 0.69 mm2) and small (area = 3.48 +/- 0.68 mm2) bone specimens. No correlation was found between work-of-fracture and either calcium content (r2 = 0.128) or fractional void area (r2 = 0.0713). The mean work-of-fracture values found are as follows: aluminum, 59.8 +/- 13.7 kJ m-2; Plexiglas, 0.620 +/- 0.074 kJ m-2; bone (area 5.89 +/- 0.69 mm2-11.7 +/- 1.9 mm2), 9.72 +/- 1.93 kJ m-2 and bone (area 3.48 +/- 0.68 mm2), 5.48 +/- 1.79 kJ m-2.     

Tilted specimen in the electron microscope: A simple specimen holder and the calculation of tilt angles for crystalline specimens

A simple specimen holder is described for a Siemens electron microscope which will allow the specimen grid to be set at inclinations up to 75° to the electron beam in any azimuthal direction. This device is suitable for measuring tilted images for the three-dimensional reconstruction of crystalline specimens. A method is also described for calculating the tilt angles for such crystalline specimens by comparing the unit cell dimensions in tilted and untilted images.     

Scanning and transmission electron microscopic studies of jejunal microvilli of the rat, hamster and dog

Scanning and transmission electron microscopy were used to investigate the surface area and number per unit area of microvilli from jejunal villus epithelial cells in the rat, hamster and dog. The calculated mean microvillus surface area was 0.419 μ², 0.573 μ², 0.751 μ² for the rat, hamster and dog respectively. The largest number of microvilli per square micron freeze dried villus surface was measured in the rat with a mean value of 65. Hamster and dog freeze dried specimens had lower mean values of 54 and 34 microvilli respectively. The total microvilli surface area in square micron per square micron villus surface was more closely related for the three species with values of 27.23 for the rat, 30.94 for the hamster and 25.53 for the dog. These data indicate an inverse relationship between the mean microvillus surface area and population density in the species studied. However, the total microvilli surface area per unit villus surface is relatively similar for the three species. The observed number of microvilli per unit villus surface was shown to vary depending upon the dehydration technique employed for preparation of scanning electron microscopic specimens. This variation probably reflects shrinkage artifact and should be considered in soft tissue studies involving the scanning electron microscope.     

A sample holder for decomposing and processing delicate biological materials for scanning electron microscopy

SUMMARY. A simple, inexpensive sample holder was developed to permit delicate biological materials (faecal pellets) to be decomposed in aquatic environments and thereafter to be processed by dehydration and critical point drying procedures. Part of the holder itself is used to mount the sample onto specimen studs. Delicate materials are therefore never subjected to physical damage during handling at any stage of their processing for scanning electron microscopy.