Zeiss EM902 Research Articles

Electron energy loss spectroscopy microanalysis and imaging in the transmission electron microscope: example of biological applications

This paper reports original results obtained in our laboratory over the past few years in the application of both electron energy loss spectroscopy (EELS) and electron spectroscopy imaging (ESI) to biological samples, performed in two transmission electron microscopes (TEM) equipped with high-resolution electron filters and spectrometers: a Gatan model 607 single magnetic sector double focusing EEL serial spectrometer attached to a Philips 430 TEM and a Zeiss EM902 Energy Filtering TEM. The primary interest was on the possibility offered by the combined application of these spectroscopic techniques with those offered by the TEM. In particular, the electron beam focusing available in a TEM allowed us to perform EELS and ESI on very small sample volumes, where high-resolution imaging and electron diffraction techniques can provide important structural information. I show that ESI was able to improve TEM performance, due to the reduced chromatic aberration and the possibility of avoiding the sample staining procedure. Finally, the analysis of the oscillating extended energy loss fine structure (EXELFS) beyond the ionization edges characterizing the EELS spectra allowed me, in a manner very similar to the extended X-ray absorption fine structure (EXAFS) analysis of the X-ray absorption spectra, to obtain short-range structural information for such light elements of biological interest as O or Fe. The Philips EM430 (250–300 keV) TEM was used to perform EELS microanalysis on Ca, P, O, Fe, Al and Si. The assessment of the detection limits of this method was obtained working with well-characterized samples containing Ca and P, and mimicking the actual cellular matrix. I applied EELS microanalysis to Ca detection in bone tissue during the mineralization process and to P detection in the cellular membrane of erythrocytes treated with an anti-tumoral drug, demonstrating that the cellular membrane is a drug target. I applied EELS microanalysis and selected area electron diffraction (SAED) to study clay particle engulfed in the alveolar macrophages of human silicotic patients, demonstrating that the particle was surrounded by a Fe coating formed by ferritin. Moreover, the analysis of the EXELFS of Fe–L 2,3 edge allowed me to study the mineral core of the ferritin, which showed great similarity to the hematite structure. Finally, I performed ESI in a Zeiss EM902 (80 keV) TEM to study the subcellular localization of the anti-neoplastic drug 4′-deoxy-4′-iododoxorubicin, analogous to adriamycin, in treated human breast tumoral cell, showing that this technique is useful in the identification of the drug target.

Localization of Green Fluorescent Protein Absorbance by Energy Filtered Transmission Electron Microscopy

Abstract Energy filtered transmission electron microscopy has the potential to provide high resolution, spatially resolved, atomic and chemical information. However, aberrations generated by the electron spectrometer blur the energy resolution and limit the atomic or molecular distributions that can be studied. Energy absorptions corresponding to the visible light range fall below an energy loss of 5 eV. The selection of electrons that have lost an amount of energy corresponding to chromophore absorption by the sample thus requires a spectrometer with a high energy resolution over the full image plane. A corrected prism-mirror-prism filter that has a resolution of 1.1 eV, sufficient to select these low energy loss electrons, was developed and installed by us in a Zeiss EM902. Its imaging capability was verified for a number of different chromophores. The chromophore currently under study is that of the green fluorescent protein (GFP).

Localization of chromophore absorption signals in TEM with an improved prism-mirror-prism filter.

A corrected prism-mirror-prism electron energy filter with curved entrance and exit faces was designed and incorporated into a Zeiss EM902 transmission electron microscope. The installation of the new filter required little modification to the existing microscope geometry and electronics. The filter had an energy resolution of 1.1 eV over the full image plane (acceptance half angle 10 mradian). The improved energy resolution was applied in studies of the low electron energy loss region that includes molecular orbital excitations or chromophore energy absorptions. Chromophore signal behaviour under electron irradiation was characterized using embedded crystals of hematin and of the dye mercury orange. Images of these crystals confirmed the expected decrease in signal intensity on shifting the selected energy loss from the region of molecular orbital excitations (less than approximately 5 eV) to higher energy losses. Electron irradiation-induced fading of the chromophore signal from hematin and mercury orange yielded similar 1/e dose values of 1.1 x 10(5) e(-) nm(-2) and 1.4 x 10(5) e(-) nm(-2) respectively. In a cellular context, chromophore signals were obtained from energy-filtered images of RIF-1 cell sections containing the photosensitizer chlorin e6 and from sections of BS-C-1 cells with cytoskeletal labelling via FITC-conjugated antibodies. The respective signals were extracted using a dose-dependent method or a shift in selected energy. Chromophore signal distributions were in agreement with fluorescence light microscopic images, but provided detail at higher spatial resolution.

Enhanced Barrier Performance of SiOx-Modified Polymer Substrates: Some Morphological Considerations

Diffusive transport of environmental gases through the free volume inherent in commodity polymers renders these materials highly ineffective for use in a variety of packaging applications. Attempts to overcome this drawback have sought to either control polymer free volume using novel microstructured polymers [1, 2] polymer blends [3, 4], or to modify surface properties through the addition of thin inorganic (ceramic or metal) coatings [5, 6]. As an example of the latter, plasma-deposited SiOx ®lms on polymer substrates have been of considerable interest in the food packaging industry for the past decade [7]. Only recently have similar needs for high-barrier polymers emerged from the medical community, which is in the process of replacing brittle glass with tough polymeric materials in biomedical devices. Glass devices requiring a sustained vacuum exhibit excellent barrier properties but readily shatter upon impact, thereby endangering the safety of medical personnel. If glass is to be replaced by a commodity polymer in such devices, optical transparency (required for visual and spectroscopic analyses [8]) and impermeability cannot be compromised. Tranasparency is ensured by using amorphous (glassy) polymers or clari®ed (nucleated) semi-crystalline polymers [9, 10], while enhanced gas impermeability is most ef®ciently achieved through plasma deposition of a thin SiOx surface ®lm. Recent advances in plasma-enhanced chemical vapor deposition (PECVD) technology [11, 12] have greatly facilitated the surface modi®cation of polymers, especially those processed into complex shapes. Previous attempts to correlate the morphological characteristics of thin surface ®lms with corresponding barrier properties have relied extensively on thickness measurements acquired from interferometry [13] or scanning electron microscopy [14]. More recent efforts in this endeavor used [14±16] scanned probe microscopy techniques, such as atomic force microscopy (AFM). According to these previous studies, two morphology=barrier property correlations have been identi®ed: (1) gas permeability decreases with a reduction in ®lm roughness [17], and (2) surface roughness increases as the ®lm thickness increases [18]. It has been pointed out [16], however, that the presence of inhomogeneities in surface morphology over large areas severely hinders the establishment of relationships between morphology (e.g., surface roughness) and permeability. Moreover, while AFM is well-suited for highresolution topographical analysis, little (if any) information regarding sub-surface features in multilayered coatings have been extracted from AFM images. For this reason, planar transmission electron microscopy (TEM), which relies on electrons that penetrate through a specimen for image formation, constitutes an ideal complement to AFM in the characterization of such coatings. In this work, we employ both AFM and TEM to examine the morphological features of thin SiOx ®lms deposited on two polymeric substrates and provide preliminary evidence for additional morphology=permeability relationships. A series of SiOx ®lms was deposited onto extruded polystyrene (PS) and polycarbonate (PC) ®lms measuring ca. 75 im thick using Radio Frequency (RF) capacity-coupled glow discharge in conjunction with feed mixtures of oxygen and hexamethyldisiloxane (HMDSO). During the deposition process, conditions such as deposition time, power, pressure and O2=HMDSO ow rate were systematically varied to produce coatings differing in permeance, surface roughness, and ®lm thickness. Oxygen permeance was measured at 30 8C with an Oxtran 220 permeability testing instrument. Specimens examined by energy-®ltered TEM were prepared by arranging small pieces of the coated polymers on 400-mesh copper TEM grids. The grids were placed on a graphite screen in contact with a solvent capable of dissolving the polymer substrate (toluene for PS and chloroform for PC) so that after substrate dissolution, unsupported SiOx ®lms adhered to the grids. Planar, rather than cross-sectional [11] images of the ®lms were obtained with a Zeiss EM902 electron spectroscopic microscope operated at 80 kV and 0 eV energy-loss. Zero-loss imaging selectively removed inelastically scattered electrons

Determination of nucleic acid content of viruses by electron spectroscopic imaging and correlation averaging

Energy filtering offers the potential to form element-specific images, and thus to quantitate and map various elements in biological specimens. To assess the sensitivity and reproducibility with which we can detect phosphorus using current technology, we have performed ESI experiments in a model system comprising three well-characterized viruses - tobacco mosaic virus (TMV), LA virus of yeast, and bacteriophage T4 - that differ widely in their contents and spatial distributions of nucleic acid. Here we make a preliminary report of experiments in which the data were recorded in a Zeiss EM902 electron microscope equipped with a Henry-Castaing energy filter.An aliquot of the viral mixture was adsorbed to a thin, grid-mounted, carbon film, blotted to a thin film, rapidly frozen in liquid ethane, and transferred into the microscope in the frozen-hydrated state, where it was freeze-dried at 100°K. A SIT camera was used to record images at energy losses of 105, 120 and 150 eV respectively, with an energy window of 10eV.

Quantitative analysis of electron energy‐loss spectra from ultrathin‐sectioned biological material

A computer program for quantitative spectral analysis is proposed for the elemental analysis of biological material by electron energy-loss spectroscopy in a conventional transmission electron microscope, the Zeiss EM902. Bio-standards are used to test the performance of this program. The application of a simplex optimization method for curve-fitting is proposed to separate the ionization edge from the background. Making use of Ce-, Ca- and Fe-bio-standards, this method is compared with Egerton's well-known two-area method.

The use of low‐cost frame grabber boards on PC‐AT computers for ESI: possibilities and limitations

SUMMARYA Zeiss EM902 microscope was connected to a PC‐AT computer, equipped with a Data Translation DT2861 frame grabber board. Using this system, the three‐window background extrapolation algorithm was implemented to perform elemental mapping in the electron spectroscopic imaging mode of an energy filtering transmission electron microscope.

Advantage of Electron Spectroscopic Diffraction on Calcified Tissue Sections

Different stages in the mineralization of calcified tissues can be investigated by electron diffraction. A disadvantage is the strong background below the Debye—Scherrer rings caused by the large massthickness of calcified products and the high ratio (≃ 3) of the inelastic—to—elastic scattering cross—sections of the embedding material. Therefore, a large fraction of the background consists of inelastically scattered electrons with energy losses. The electron spectroscopic diffraction (ESD) mode of an energy—filtering microscope (ZEISS EM902) allows to record diffraction patterns using only the zero—loss electrons which consist of the primary beam, Bragg diffracted electrons and a smaller fraction of elastically scattered electrons between the Debye—Scherrer rings by thermal—diffuse scattering. Small—area diffraction patterns with different camera lengths are generated at the filter—entrance plane and the zero—loss electrons are selected by a slit in the energy—dispersive plane behind the Castaing—Henry filter lens.

Quantitative Energy-Filtered CRYO-EM of Hydrated Chromosome Fibers, Viruses, and Heavy Atoms

We have calculated and experimentally measured the extent to which energy filtration improves the S/N in phase- and scattering-contrast images of frozen-hydrated molecules. Multiple scattering calculations based on PW (partial wave) complex scattering amplitudes agree quantitatively with 80 KV image intensities of 50-500nm-thick ice. For an objective aperture of 2.5 nm-1 the PW elastic mean free path is 280 nm. From the ratio of filtered to unfiltered image intensities from 50-500 nm of ice at 80 KV, we calculate an empirical inelastic mean free path of 180 nm. The PW calculations show that Beer's Law should be obeyed for thicknesses up to five mean free paths, whether or not energy filtration is used. Thus the bright-field signal can be used to determine the absolute mass distribution of molecules embedded in thick ice layers.Energy filtration removes background intensity due to inelastic scattering (Fig.1a), and increases the scattering signal from molecules (becoming the sum of both elastic electrons excluded by the objective aperture and inelastic electrons excluded by the energy filter). As the result, the calculated contrast is 2-15 times greater in the Zeiss EM902 than in a TEM without energy filtration (Fig.1b). 80 KeV energy-filtered images of frozen-hydrated molecules should have a higher S/N than unfiltered images taken at intermediate or high voltage (Fig. 1c,d). TMV contrast and S/N is improved by filtration and agrees with calculations (Fig.1b,2). The advantages of energy-filtration are important to the microscopy of chromatin fibers, which are excluded from ice layers ≤200 nm and viruses, which are usually excluded from layers ≤100 nm. Radial density reconstructions from near-focus energy-filtered images of frozen-hydrated chromatin from Thyone sperm show that the centers are not hollow, but very dense, probably containing DNA (Fig.3). Energy-filtered images of icosahedral viruses in even the thinnest possible ice have a much improved S/N ratio, decreasing the dose required for analysis (Fig.2a,b).

Electron Spectroscopic Imaging and Diffraction in TEM

Energy-filtering electron microscopy at 80 keV (ZEISS EM902) offers the combination of electron spectroscopic imaging (ESI) and diffraction (ESD) and electron energy-loss spectroscopy (EELS). For details the reader is referred to a description of the different modes, applications of ESI to biological and crystalline specimens and of ESD. The very important mode of elemental mapping with the difference of ESI below and beyond an edge will not be discussed in this review.The ESI mode increases scattering contrast of stained and unstained biological sections and avoids chromatic aberration by zero-loss filtering. Filtering at ΔE=250 eV below the C edge increases the (structure-sensitive) contrast by non-carbon atoms of unstained sections (Fig.1). Phase contrast is also increased but inelastically scattered electrons show a faint phase contrast which can be explained by treating partial inelastic waves with different q as incoherent. Bragg contrast of crystalline specimens is enhanced due to avoiding chromatic aberration and a blurring by the spectrum of excitation errors of inelastically scattered electrons (Fig.2).

Comparison of Images of Crystalline Specimens by Energy-Filtering TEM at 80 keV and CTEM at 200 keV

One advantage of energy-filtering electron microscopy (EFEM) is to avoid the chromatic aberration of conventional transmission electron microscopy (CTEM) by the mode of electron spectroscopic imaging (ESI) using either zero-loss filtering of unscattered and elastically scattered electrons or a narrow selected energy window at the most probable loss of the electron-energy-loss spectrum (EELS). Chromatic aberration can also be reduced by high-voltage electron microscopy (HVEM). Comparisons of ESI at 80 keV and CTEM at 200 keV have already been reported for biological tissues. In this contribution we compare the imaging of evaporated crystalline films with ESI at 80 keV in a ZEISS EM902 and with CTEM at 200 keV in a Hitachi H800/NA.Zero-loss filtering at 80 keV can be applied for maximum mass-thicknesses of x=ρt≃150 μg/cm2 where the zero-loss transmission falls below 0.001 and an energy window at the most-probable energy loss can be used below ≃300 μg/cm2. Inelastic scattering preserves the Bragg contrast.

Localization of DNA in chromatin using ESI and osmium ammine staining

In the eukaryotic nucleus, DNA is packaged into nucleosomes, and the nucleosome chain folded into ‘30nm’ chromatin fibers. A number of different model structures, each with a specific location of nucleosomal and linker DNA have been proposed for the arrangment of nucleosomes within the fiber. We are exploring two strategies for testing the models by localizing DNA within chromatin: electron spectroscopic imaging (ESI) of phosphorus atoms, and osmium ammine (OSAM) staining, a method based on the DNA-specific Feulgen reaction.Sperm were obtained from Patiria miniata (starfish), fixed in 2% GA in 150mM NaCl, 15mM HEPES pH 8.0, and embedded In Lowiciyl K11M at -55C. For OSAM staining, sections 100nm to 150nm thick were treated as described, and stereo pairs recorded at 40,000x and 100KV using a Philips CM10 TEM. (The new osmium ammine-B stain is available from Polysciences Inc). Uranyl-lead (U-Pb) staining was as described. ESI was carried out on unstained, very thin (<30 nm) beveled sections at 80KV using a Zeiss EM902. Images were recorded at 20,000x and 30,000x with median energy losses of 110eV, 120eV and 160eV, and a window of 20eV.

Quantitative energy-filtered image analysis in cytochemistry: I. Morphometric analysis of contrast-related images

A combination of energy-filtered electron microscopy (EFEM) and an image-analyzing system (IBAS/2000) is used for morphometric analyses of cells and (reaction) products. Image contrast is objectively established and segmentation is based upon intrinsic contrasts, in ultrathin sections. Cross-sectioned platinum-stained erythrocytes are used as a model to determine optimal conditions for constant measuring results for contrast, area and perimeter. Results are related to changes in: (1) the objective-lens diaphragm diameter, (2) three most frequently used contrast modes obtainable by electron spectroscopical imaging (ESI) in a Zeiss EM902 transmission electron microscope (e.g., global, zero loss (or Δ E = 0 eV) and Δ E = 250 eV), and (3) the number of image integrations (1 − 250x) acquired by real-time video. A thresholding procedure is proposed for objective segmentation of such contrast-related images and applied to measure the area fraction of nuclear chromatin and the diameter of nominal 1 nm colloidal gold particles.

Electron spectroscopic diffraction at (111) silicon foils

An electron diffraction pattern (EDP) consists of an overlap of patterns of all energy losses in the electron energy-loss spectrum (EELS). Electron spectroscopic diffraction (ESD) in an energy filtering electron microscope (EFEM) allows to separate the contributions of different energy losses to the unfiltered diagram observed in conventional TEM. We report about diffraction experiments with a Zeiss EM902 on (111) silicon foils which show how the EDP of single-crystal foils changes with increasing energy loss and foil thickness. An EDP normally contains the Bragg spots, diffuse streaks by electron-phonon scattering, excess and defect Kikuchi lines when the number of electrons striking the lattice planes is different from opposite sites, a system of excess (bright) Kikuchi bands with an intensity proportional to the probability ψψ⋆ of the Bloch wave field at the nuclei, and defect Ki-kuchi bands when the number of diffusely scattered electrons is equal on both sides of the lattice plane and the intensity becomes proportional to ΣIg.EDPs of thin foils show an increase of contrast of the Bragg spots and the thermal diffuse streaks when comparing an unfiltered (Fig.1a) and zero-loss filtered EDP (Fig.1b). Because the streaks are caused by elastic scattering, they can not be ob served with the plasmon loss (Fig.1c). Bragg spots are also observed at higher energy losses because all delocalized inelastic scattering processes with energy losses less a few hundred eV show intraband transitions which preserve the type of excited Bloch waves.

Electron spectroscopic imaging of thick crystalline specimens

Electron spectroscopic imaging (ESI) by an energy-filtering electron microscope (EFEM, Zeiss EM902) shows the following advantages when compared with the unfiltered bright-field mode:1.The zero-loss image does not contain the contribution of inelastically scattered electrons. Though plasmon scattering shows a conversation of Bragg contrast - edge and bent contours and lattice defect images -, the angular distribution of inelastically scattered electrons results in a broader spectrum of excitation errors and a blurring of Bragg contrast.2.The zero-loss image avoids the chromatic aberration of inelastically scattered electrons for medium specimen thicknesses and can be applied so long as the intensity of the zero-loss peak in the electron energy-loss spectrum (EELS) is high enough for an exposure in a reasonable time (<100 s).3.Thick specimens with negligible zero-loss intensity can be imaged with an energy window at the highest multiple plasmon loss of the Poisson distribution or at the most probable energy of a Landau distribution. The angular distribution of electrons with these energy losses is so broad that the Bragg contrast is blurred, and the contrast is only caused by anomalous absorption effects similar to multi-beam images in the STEM mode when using a large probe aperture.

A temperature-jump technique for time-resolved cryo-transmission Electron Microscopy

Transmission electron microscopy of rapidly-frozen, hydrated specimens (cryo-TEM) is a powerful way of examining labile microstructures. This technique avoids some artifacts associated with conventional preparative methods. Use of a controlled environment vitrification system (CEVS) for specimen preparation reduces the risk of unwanted sample changes due to evaporation, and permits the examination of specimens vitrified from a defined temperature. Studies of dynamic processes with time resolution on the order of seconds, in which the process was initiated by changes in sample pH, have been conducted. We now report the development of an optical method for increasing specimen temperature immediately before vitrification. Using our method, processes that are regulated by temperature can be initiated in less than 500 msec on the specimen grid. The ensuing events can then be captured by plunge-freezing within an additional 200 msec.Dimyristoylphosphatidylcholine (DMPC) liposomes, produced by extrusion, were used as test specimens. DMPC undergoes a gel/liquid crystalline transition at 24°C, inducing a change in liposome morphology from polyhedral to spherical. Five-μl aliquots of DMPC dispersions were placed on holey-carbon-filmed copper grids mounted in the CEVS environmental chamber, and maintained at 6-8°C and 80% relative humidity. Immediately before the temperature jump most of the sample was blotted away with filter paper, leaving a thin specimen film on the grid. Upon pressing the trigger, an electronic control circuit generated this timed sequence of events. First, a solenoid-activated shutter was opened to heat the specimen by exposing it for a variable time to the focused beam of a 75W Xenon arc lamp. Simultaneously, a solenoid-activated cryogen shutter in the bottom of the CEVS was opened. Next, the lamp shutter was closed after the desired heating interval. Finally, a solenoid-activated cable release was used to trigger a spring-loaded plunger in the CEVS, propelling the sample into a reservoir of liquid ethane. Vitrified samples were subsequently transferred to a Zeiss EM902 TEM, operated in zero-loss brightfield mode, for examination at −163°C.

Tomographic reconstruction from energy-filtered images of thick biological sections.

Energy filtration makes it possible to image an approximately 0.5 microm biological section at 80 kV in the electron microscope. Based on spectra taken at different tilt angles, we chose the most probable energy loss, deltaEp +/- 10eV for each tilt angle, as the imaging energy window. A complete tilt series from +60 degrees to -60 degrees at 2.5 degree intervals was collected on the Zeiss EM902 and used in a tomographic reconstruction of transcriptionally active chromatin in the Balbiani ring of Chironomus tentans.

Removal of inelastically scattered electrons substantially increases phase contrast on frozen-hydrated molecules

Although electron diffraction indicates better than 0.3nm preservation of biological structure in vitreous ice, the imaging of molecules in ice is limited by low contrast. Thus, low-dose images of frozen-hydrated molecules have significantly more noise than images of air-dried or negatively-stained molecules. We have addressed the question of the origins of this loss of contrast. One unavoidable effect is the reduction in scattering contrast between a molecule and the background. In effect, the difference in scattering power between a molecule and its background is 2-5 times less in a layer of ice than in vacuum or negative stain. A second, previously unrecognized, effect is the large, incoherent background of inelastic scattering from the ice. This background reduces both scattering and phase contrast by an additional factor of about 3, as shown in this paper. We have used energy filtration on the Zeiss EM902 in order to eliminate this second effect, and also increase scattering contrast in bright-field and dark-field.

Zero-loss electron microscopy with the Zeiss EM902

The recently introduced Zeiss EM902 is the first commercially available electron microscope to incorporate a magnetic spectrometer capable of forming directly energy-filtered images and diffraction patterns. We have briefly assessed the potential usefulness of this microscope for structural studies of thin biological specimens, through removal of inelastically scattered electrons. The improvements we find in image contrast, without significant loss of resolution, suggest energy-filtering may prove particularly worthwhile with unstained specimens.

Ultrastructural localization of calcium in the CNS of vertebrates.

The ultrastructural localization of calcium in synaptic areas of the CNS of fish was investigated. Prefixation with phosphate-buffered glutaraldehyde followed by post-fixation with osmium/potassium-bichromate was used to precipitate and visualize endogenous calcium without the addition of external calcium. The presence of calcium in the electron-dense precipitates produced using this method was demonstrated by electron spectroscopic imaging using a Zeiss EM-902 transmission electron microscope, and in various control experiments using the calcium chelator EGTA. In the optic tectum of fish, electron dense precipitates containing calcium were found not only in intracellular compartments, e.g. the smooth endoplasmic reticulum, mitochondria and synaptic vesicles, but also at extracellular locations, particularly in synaptic clefts. In the extracellular sites, only chelate complexes of ionic calcium were found. This would seem to be in agreement with electrophysiological and biochemical data reported in earlier studies. Thus, using the present method, it should be possible to obtain further ultrastructural information concerning the mechanisms of synaptic transmission.