Scanning Transmission Mode Research Articles

Standard performance criteria for analytical electron microscopy

SummaryUsers of analytical electron microscopy lack easy‐to‐use standards for assessing the consistency and quality of analytical performance. We propose using a Cr thin film of known thickness to measure three important characteristics related to performance: the Cr Kα peak‐to‐background (P/B) ratio, the X‐ray spectrometer relative efficiency, and the spectrometer energy resolution. We used a Cr specimen to determine the instrumental factors which influence the P/B ratio, finding that the highest P/B ratios are achieved in scanning transmission mode at the highest available accelerating voltage. We present values of the P/B ratio, and the detector relative efficiency and energy resolution which can be used for comparison in other laboratories using the standard film.

Elemental analysis of non-living particles in picoplankton fractions from oligotrophic lake water

Electron microscopic (TEM) study of bacterial-sized planktonic fractions recovered from the water column samples of a small oligotrophic lake, consistently showed the presence of non-living organic and inorganic particles as prominent parts of the micro habitat. An all important objective of this work was to determine the composition and possible origins of the minute particles. They were of several morphological types, described by transmission electron microscopic (TEM) appearance as: spherical, oblong, cuboidal, plates and fibrillar. The TEM used in the scanning transmission mode (STEM) in conjunction with the Princeton Gamma Tech (PGT) System 4 energy dispersive X-ray spectrometer (EDX) allowed the determination of the elemental composition of individual particles. The particles referred to as spherical were electron dense and were less than 1 μm in diameter. They were mainly composed of either Fe or Ti but not both. Cuboidal iron particles 37–75 nm in diameter were found in about equal abundance, but they were arranged in chains of individuals. Oblong particles were from 0.18 to 0.2 nm in width to 0.2 to 0.45 μm in length. Most were composed mainly of Ca, S and O but other elemental compositions were observed also. Plates with jagged edges composed of Al and Si were somewhat electron transparent. Both were less than 1 μm in largest dimension. Fibrillar particles were most abundant and were composed of Fe, Si, Cl, K, Na, O and C. They ranged from 90 Å to 37 nm in thickness and 1 μm to several μm in length. Particles of planktonic origin were observed with regularity including fragments of diatom cell walls and scales. These particles were composed of Si and O. Hexagonally shaped virus-like particles of several sizes were observed also. The relationship between elements in micro particles and their possible origins are discussed.

Cryofixation combined with physical dehydration for quantitative immunoelectron cytochemistry.

Common methods for preparing samples for immunoelectron microscopy involve glutaraldehyde fixation (GA) followed by chemical dehydration (CD) or cryofixation (CF) succeeded by physical dehydration, i.e., freeze drying (FD) or freeze substitution (FS). The effects of these techniques have been evaluated with regard to the sizes of epoxy resin embedded rat somatotrophic secretory granules as well as the immunolabeling densities over these granules. The measurements were performed by computerized image analysis using electron microscopy in transmission (TEM) and scanning transmission (STEM) modes, which allowed us to define the immunolabeling in detail. The embedded secretory granules showed the same diameters after GA (2 hr) with CD and GA (15 min) with CF and FS, but were smaller after CF-FS, and smallest after GA (15 min) with CF and FD. The highest labeling density appeared after GA (15 min) and physical dehydration, in particular after freeze substitution. Based on our STEM pictures a new factor for evaluating and interpreting immunolabeling of granules is introduced; the "accessible immunogold labeling surface." It defines the fraction of the epoxy resin surface that is labeled and varies with the preparation methods. By using this factor, an order of labeling densities/micron 2 over the accessible areas could be established for the different techniques: GA-CF-FS > CF-FS > GA-CF-FD > GA-CD. The high labeling after GA-CF-FS may be due to the combination of a large accessible area and accurate preservation of the antigenicity of the hormones in the granules.

Ion mixing of thin ZrO2 films on α-Al2O3 by Cr+ or Kr+ ions

Analytical electron microscopy (AEM) has been used to study ion beam mixing in an oxide-oxide system: ZrO2 thin films on α-Al2O3. This mixing process is useful in applications such as adhesion enhancement.Films of ZrO2 (∼80 nm thick) were deposited on α-Al2O3 by radio frequency (rf) sputter deposition and annealed for 2 h at 500°C in 96%Ar+4%H2 to remove excess oxygen. Samples were bombarded at room temperature with either 340 keV Cr+ or 475 keV Kr+ ions to a fluence of 4×1012 ions/m2. An area of each specimen was masked during implantation to permit analysis of unimplanted interfaces. Specimens for AEM were mounted in cross-section, ground and dimpled to a thickness of ∼20 μm, and Ar+ ion milled to electron transparency. Specimens were sputter-coated with ∼5 nm of carbon to reduce charging effects. The AEM was performed in a Philips EM400T/FEG operated in the scanning transmission mode with a probe diameter of <2 nm (FWTM prior to beam spreading).

Performance measurements of a 1-keV electron-beam microcolumn

A complete 1-keV electron-beam microcolumn, measuring only 2.5 mm in length, has been assembled and tested. The microcolumn combines a scanning tunneling microscope aligned field-emission source with a miniaturized octupole scanner and a microfabricated einzel lens. Expected performance of this configuration, at a working distance of 1 mm and semiconvergent angle of 2.5 mrad, has been calculated to be a probe size of 8 nm with current density exceeding 104 A/cm2. The microcolumn has been successfully operated at 1 keV in scanning transmission mode using a 1-μm grid sample. Images were acquired with scan fields ranging from 10 to 100 μm and preliminary resolution measurements indicated Gaussian beam diameter of 200 nm. Significant improvements are expected in the near term.

Application of analytical electron microscopy to studies of equilibrium and non-equilibrium segregation in materials

Segregation of solute atoms to grain boundaries, dislocations, and other extended defects can occur under thermal equilibrium or non-equilibrium conditions, such as quenching, irradiation, or precipitation. Generally, equilibrium segregation is narrow (near monolayer coverage at planar defects), whereas non-equilibrium segregation exhibits profiles of larger spatial extent, associated with diffusion of point defects or solute atoms. Analytical electron microscopy provides tools both to measure the segregation and to characterize the defect at which the segregation occurs. This is especially true of instruments that can achieve fine (<2 nm width), high current probes and as such, provide high spatial resolution analysis and characterization capability. Analysis was performed in a Philips EM400T/FEG operated in the scanning transmission mode with a probe diameter of <2 nm (FWTM). The instrument is equipped with EDAX 9100/70 energy dispersive X-ray spectrometry (EDXS) and Gatan 666 parallel detection electron energy loss spectrometry (PEELS) systems. A double-tilt, liquid-nitrogen-cooled specimen holder was employed for microanalysis in order to minimize contamination under the focussed spot.

The new electron microscopy: imaging the chemistry of nature.

The electron microscope permits the direct observation of the microstructure of matter far beyond the resolving capabilities of the conventional light microscope. Consequently, the instrument has had a major, and often revolutionary, impact on many disciplines. During the last several years improvements in electron optics, vacuum systems, X-ray and electron spectrometers, specimens preparation techniques, and the development of powerful and inexpensive laboratory computers have extended the capability of the electron microscope to directly image and quantify the chemical elements which comprise a specimen. This added ability to provide compositional maps has substantially enhanced the importance of the electron microscope. This review will only cover compositional mapping utilizing electron microscopes (> 80 kV) operating in the scanning transmission mode (STEM) since this is currently the only mode to permit simultaneous detection of all the various signals. References will include both biological and materials science applications since the subject matter is germane to both. The period covered by this review is essentially the last 10 years.

Elemental imaging by EELS and EDXS in the analytical electron microscope

Electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS) can be used to obtain elemental maps from thin biological samples in the analytical electron microscope. The EELS is particularly sensitive for the low-atomic-number elements, including C, N, and O, as well as other elements with favorable ionization cross-sections, such as Fe. The EDXS is useful for a complementary range of atoms, such as P, S, K, and Ca. A system is described for obtaining elemental distributions in an analytical electron microscope operated in the scanning transmission mode at 100-200 keV beam energy. The spatial resolution is typically limited to 10-20 nm when a conventional source is used. A satellite microcomputer controls acquisition of EELS and EDXS data from successive pixels in an image. These data are processed "on-the-fly" by a host computer to remove the noncharacteristic background intensity. Resulting images are stored on disk and can be analyzed by means of an image display system controlled by interactive software. The technique is demonstrated with elemental maps from two samples: alveolar macrophages containing respirable particles; and pancreatic beta cells that secrete insulin.

Changes in elemental composition of single muscle fibers following tenotomy of the rat soleus muscle.

Cores were produced in type 1 muscle fibers by tenotomy of rat soleus muscle. The morphology and histochemistry of the muscle fibers was established by light microscopy on cryostat sections stained for hematoxylin-eosin or myofibrillar ATPase and on semithin plastic sections. The ultrastructure was visualized on thin plastic sections. On 6 micrometers of freeze-dried cryosections, energy dispersive X-ray microanalysis was performed on muscle fibers visualized in the scanning-transmission mode of electron microscopy. This procedure permitted quantification of different intracellular elements such as sodium (Na), chlorine (Cl), potassium (K), magnesium (Mg), sulphur (S), and phosphorus (P). Spectra from core fibers could easily be compared with those of normal fibers. A conspicuous finding was an increased Na and Cl content and a decreased K content in core fibers compared to normal fibers. It is known that core fibers produced after tenotomy exhibit distinct changes in plasma membrane morphology similar to that found in Duchenne muscular dystrophy (DMD). The results in this study point to a change in normal intracellular ion composition which could be a result of a deficiency of mechanisms maintaining normal membrane ion gradients.

Diagnostic Virology Using Electron Microscopic Techniques

Publisher SummaryThis chapter illustrates the development of the use of electron microscopy in viral diagnosis. The field covered is confined to medical viral diagnosis, but parallel developments have taken place in both veterinary and botanical fields and techniques derived from both these sources are also included where relevant. It is reported that the scanning transmission mode of operation, which can induce image contrast changes electronically, may enhance studies with unstained sections and perhaps facilitate thin section immune electron microscopy (IEM). The application of negative stain IEM has been particularly useful for the study of the antigenic nature of some of the newly discovered noncultivable viruses. Viral antigens can also be detected in thin sections of infected cells by IEM with suitably labeled specific antibodies. Confirmation of viral infection by electron microscopy on tissues originally processed for light microscopy is also frequently useful.

Electron-microscopic microprobe analysis on the initial stages of mineral formation in the epiphyseal growth plate.

Dry thin sections (300-500 nm thick) of shock-frozen, freeze-dried and embedded epiphyseal growth plates from the proximal tibia of guinea pigs were cut longitudinally from the plate. Dark round bodies (phi less than 0.5 micron) were observed using the scanning transmission mode of the electron microscope initially directly in the vicinity of the chondrocytes. They gradually spread out in the direction of the metaphysis to the center of the longitudinal septum and represent most probably the matrix vesicles. By use of a microscan of 0.25 X 0.25 micron the element-concentrations of these bodies were measured. The measurements started on those bodies that could be clearly recognized and were extended to a length of 30-40 micron in the metaphyseal direction. To obtain approximate quantitative results the registered CaK alpha and PK alpha x-ray counts were directly compared with counts of fully mineralized regions, the Ca and P contents of which are known. Ca as well as p could be detected in the first visible vesicle-like structures (Ca approximately 0.2%, P approximately 0.4%) and increased steeply in the metaphyseal direction, amounting to approximately 6% Ca and 3% P. These results may lead to the conclusion that Pi becomes split from phosphate esters and transformed into the matrix vesicles already in a very early stage of enrichment. Incorporation of Ca may be coupled with this process.

Sectioning and imaging hard mineral fibres in biological tissues.

A technique is described which overcomes the problems associated with sectioning biological tissue containing hard mineral fibres. 0.2--0.5 micrometer thick sections were cut with a diamond knife, placed in a folding grid, conventionally stained with uranyl acetate and lead citrate and viewed at an accelerating voltage of 200 kV in the scanning transmission mode.

Sarcoplasmic bodies in distal myopathy compared with nemaline rods: X-ray microanalytical and histochemical observations

Sarcoplasmic bodies in a late onset distal myopathy and rods in nemaline myopathy have been investigated by electron probe X-ray microanalysis correlated to light microscopy. Sarcoplasmic bodies were glassy, drop-shaped structures up to 10 μm of length and easily distinguished in the scanning mode of electron microscopy performed on thick freeze-dried cryosections. They were found to be source of X-ray spectra characterized by a high sulphur peak. On semithin epoxy sections the sarcoplasmic bodies were observed in the scanning transmission mode as electron-dense structures which, according to X-ray microanalysis, exhibited a high sulphur and osmium content, possibly indicating the presence of sulphydryl groups. The nemaline rods were of 3 different types as judged by their light-microscopical appearance. They were, however, not visible in the scanning mode of electron microscopy performed on freeze-dried unstained cryosections. There were small differences in the elemental composition between different rods within the same biopsy but these were not systematically related to the differences in morphological appearance.

Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. III. Elemental content of cells in the rat renal papillary tip.

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5-micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at -175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.

Ultrastructural autoradiography of 0.5 μm sections of tritium-labelled specimens using scanning transmission electron microscopy (STEM)

Thick (0.5 μm) sections of epoxide-embedded tritium-labelled specimens may be used for ultrastructural autoradiography if the electron microscope (EM) is operated in the scanning transmission (STEM) mode. The advantage of using thick sections is that substantially shorter exposures are possible than with thin sections. EM autoradiography can thus be performed on specimens which were hitherto too feebly labelled for the technique to be practicable. The proposed procedure is illustrated with sections of cultured cells labelled with [ 3H]uridine.

High-resolution scanning electron microscopy of bacteriophages 3C and T4.

An account is presented of the design and operation of a new scanning electron microscopic, and its first application to the study of biological samples. Bacteriophages were chosen because much of their ultrastructure is beyond the resolution of the conventional scanning electron microscope. The new instrument permits examination of bulk samples with a resolution that exceeds, by at least a factor of 2.5, the resolution obtained in the best secondary electron scanning electron microscopes using high brightness guns, and exceeds by an order of magnitude the resolution of standard scanning electron microscopes using tungsten filament guns. It also permits examination of biological samples in scanning transmission mode at resolutions similar to conventional transmission electron microscopes.

Quantitative Electron Microprobe Studies of Human-Platelet Dense Bodies

As seen in air-dried whole mounts, human-platelet dense bodies (DB) are discrete, electron-opaque structures ranging in size from approximately 50 nm to 1000 nm in diameter (Fig. 1). We have examined DB elemental composition and peak-to-background (p/bg) ratios using a Hitachi HU-12A microscope in the transmission-scanning mode and a Kevex energy-dispersive X-ray detector and analysis system. Qualitatively, spectra from probed DB showed strong signals for calcium and phosphorous, and a less prominent peak for potassium. No magnesium peak was detected (Fig. 2).Quantitatively, probing 11 randomly-selected DB with a beam spot approximately 10 nm in diameter (75 kv accelerating voltage) gave phosphorous p/bg ratios with a coefficient of variation (CV) of 71.32, calcium p/bg ratios with a CV of 75.34, and elemental phosphorous/calcium (P/Ca) ratios with a CV of 18.10 (Fig. 3). Peak-to-background ratios for both elements demonstrated low correlations with the area of the DB probed (r = 0.12 and 0. 39 for phosphorous and calcium, respectively, based on a sample size of 67 DB's), suggesting that only a small fraction of the total DB area was being sampled.

Resolution Enhancement in Electron Microscopy by A Posteriori Holographic Image Deblurring

A new refinement of the holographic image deblurring method originated by Stroke in 1967 has now permitted us to demonstrate an increase of resolution to less than 70Å in a scanning electron(SEM) microcraph taken with a commercial microscope(MAC SX-II) under the best normal operating conditions which had been considered to be “realistic” for “average specimens” and the type of instrument used, according to generally accepted criteria, e.g. according to C.W.Oatley, W.C.Nixon and R.F.Pease, A.N.Broers and others. We have obtained the same increase of resolution, by factors certainly in excess of 2 and more, as a part of yet unpublished collaborative work of us with Prof. Albert V. Crewe, in which we have succeeded in enhancing micrographs obtained at much higher magnifications (in excess of 800,000) in the much higher resolution transmission scanning mode, indicating an enhancement of resolution from about 5 Å to probably much better than 2.5Å. The principle of holographic resolution and contrast enhancement is theoretically equally applicable to high-resolution transmission electron microscopy of the “convential” type, and the theory has been unquestionably confirmed by the results obtained at the time of this writing.

A high resolution electron spectrometer for use in transmission scanning electron microscopy.

The design and construction of a double focusing uniform field magnetic electron spectrometer are described. The pole pieces of the magnet are curved in an effort to correct all median plane aberrations and thereby increase the collecting power. The spectrometer has been installed on a scanning microscope which uses a field emission gun to produce a 100 Å probe. Energy analysis of the transmitted electrons as well as filtered diffraction patterns can be obtained from areas as small as 100 Å in diameter. In addition, the area under study can be observed directly in the scanning transmission mode, using electrons of any selected energy loss. A resolution of the spectrometer of 2 ppm at an electron energy of 20 keV is shown and the measured second order aberration coefficient is <5% of that of an equivalent straight edge sector magnet. Also, the solid angle for collection of electrons is about an order of magnitude greater than that of the equivalent straight edge magnet at any given resolution. Experimental results in all modes of operation are presented.

The use of agar gel electrophoresis for the electron microscopic analysis of virus particles

This chapter illustrates the development of the use of electron microscopy in viral diagnosis. The field covered is confined to medical viral diagnosis, but parallel developments have taken place in both veterinary and botanical fields and techniques derived from both these sources are also included where relevant. It is reported that the scanning transmission mode of operation, which can induce image contrast changes electronically, may enhance studies with unstained sections and perhaps facilitate thin section immune electron microscopy (IEM). The application of negative stain IEM has been particularly useful for the study of the antigenic nature of some of the newly discovered noncultivable viruses. Viral antigens can also be detected in thin sections of infected cells by IEM with suitably labeled specific antibodies. Confirmation of viral infection by electron microscopy on tissues originally processed for light microscopy is also frequently useful.